( H ) Increase in ubiquitinated form of HuR in Starved Huh7 . of ubiquitinated HuR in Fed Starved ( 5h ) HA - Ub expressing Huh7 treated either with ethanol or MG132 ( 20µM ) for same duration . lysates were immunoprecipitated with HuR western blotted for HA HuR . Position of ubiquitinated bands were marked by arrows . of ubiquitinated HuR were measured by densitometry relative increase in ubiquitinated HuR against non - ubiquitinated form were measured plotted ( mean + / - s . e . m . , n = 3 ) .

O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Increase in ubiquitinated form of HuR in Starved Huh7 cells. Levels of ubiquitinated HuR in Fed and Starved (5h) HA-Ub expressing Huh7 cells treated either with ethanol or MG132 (20µM) for same duration. The lysates were immunoprecipitated with HuR and western blotted for HA and HuR. Position of ubiquitinated bands were marked by arrows. The levels of ubiquitinated HuR were measured by densitometry and relative increase in ubiquitinated HuR levels against the non-ubiquitinated form were measured and plotted (mean+/- s.e.m., n=3).


( I ) Full a truncated version of HA - HuR without hinge region ( graphical scheme in upper panel ) was used to score effect of deletion of hinge region on EV associated miR - 122 content measured by qRT - PCR ( bottom panel ) . Western blots to check expression of HuR its truncated variants are also shown . " * " denotes HA - HuR band ( left lower panel ) . Data represented as mean + / - s . e . m . , n = 3 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Full length and a truncated version of HA-HuR without the hinge region (graphical scheme in upper panel) was used to score the effect of deletion of hinge region on EV associated miR-122 content measured by qRT-PCR (bottom panel). Western blots to check expression of HuR and its truncated variants are also shown." * " denotes the HA-HuR band (left lower panel). Data represented as mean+/- s.e.m., n=3.


( J ) Ubiquitination status of full a truncated version of HA - HuR without hinge region between RRMII RRMIII in Huh7 . HuR ( full lenght or truncated version ) were immunoprecipitated from lysates of Huh7 expressing these immunoprecipitated materials were western blotted for HA - HuR ubiquitination . Ubiquinitated HuR bands are marked by arrows . postion of non - ubiquitinated were marked by arrowheads ( upper panel ) . qRT - PCR estimation of associated miRNA per unit of HA - HuR immunoprecipitated materials were estimated normalized against respective quantified by densitometric of western blots ( lower panel ) . Data represented as mean + / - s . e . m . , n = 3 . PCIneo transfected were used as control in immunoprecipitation reaction but no amplification of miR - 122 were detected for HA immunoprecipitated materials from pCIneo tranfected .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

(J) Ubiquitination status of full length and a truncated version of HA-HuR without the hinge region between RRMII and RRMIII in Huh7 cells. HuR (full lenght or truncated version) were immunoprecipitated from cell lysates of Huh7 cells expressing these proteins and the immunoprecipitated materials were western blotted for HA-HuR and ubiquitination. Ubiquinitated HuR bands are marked by arrows. postion of non-ubiquitinated proteins were marked by arrowheads (upper panel). qRT-PCR based estimation of associated miRNA per unit of HA-HuR immunoprecipitated materials were estimated and normalized against respective protein levels quantified by densitometric quantification of western blots (lower panel). Data represented as mean+/- s.e.m., n=3. PCIneo transfected cells were used as control in immunoprecipitation reaction but no amplification of miR-122 were detected for HA immunoprecipitated materials from pCIneo tranfected cells.


Bar graph represents IL - 2 secretion by NKT hybridoma ( 2C12 ) co - cultured with ( A ) a murine macrophage ( J774 . 2 ) treated either with thapsigargin ( black bars ) , tunicamycin ( red bars ) or DMSO ( bars ) . Graphs show mean ± from n = 3biological replicates . * * * * P < 0 . 0001 ( Unpaired t ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with (A) a murine macrophage cell line (J774.2) treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).


Bar graph represents IL - 2 secretion by NKT hybridoma ( 2C12 ) co - cultured with a human macrophage ( U937 ) , treated either with thapsigargin ( black bars ) , tunicamycin ( red bars ) or DMSO ( bars ) . Graphs show mean ± from n = 3biological replicates . * * * * P < 0 . 0001 ( Unpaired t ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with a human macrophage cell line (U937), treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).


Heat map represents cytokine secretion by in vitro expanded ( C ) murine splenic iNKT co - cultured either with thapsigargin - or tunicamycin - or DMSO - treated murine BMDMs . heat map shows average result of two pooled biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heat map represents cytokine secretion by in vitro expanded (C) murine splenic iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.


Heat map represents cytokine secretion by in vitro expanded human peripheral blood iNKT co - cultured either with thapsigargin - or tunicamycin - or DMSO - treated murine BMDMs . heat map shows average result of two pooled biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heat map represents cytokine secretion by in vitro expanded human peripheral blood iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.


Wild type C57BL / 6 mice were injected intravenously with thapsigargin - loaded PLGA nanoparticles or vehicle , 12 h later , hepatic iNKT were analysed by flow cytometry . Histograms represent expression of intracellular IL - 4 IFN - γ in hepatic iNKT from mice injected with thapsigargin - loaded PLGA nanoparticles ( red histogram ) or vehicle - treated ( grey histogram ) mice . Dot plots represents MFI for intracellular IL - 4 IFN - γ expression in hepatic iNKT from thapsigargin - loaded PLGA nanoparticles ( red dots ) or vehicle - treated ( black dots ) mice . Data plots show mean ± from n = 5 mice per group . * * P ≤ 0 . 001 ( Mann - Whitney U ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild type C57BL/6 mice were injected intravenously with thapsigargin-loaded PLGA nanoparticles or vehicle, and 12 h later, hepatic iNKT cells were analysed by flow cytometry. Histograms represent expression of intracellular IL-4 and IFN-γ levels in hepatic iNKT cells from mice injected with thapsigargin-loaded PLGA nanoparticles (red histogram) or vehicle-treated (grey histogram) mice. Dot plots represents MFI for intracellular IL-4 and IFN-γ expression in hepatic iNKT cells from thapsigargin-loaded PLGA nanoparticles (red dots) or vehicle-treated (black dots) mice. Data plots show mean ± SEM from n = 5 mice per group. **P≤ 0.001 (Mann-Whitney U test).


Thapsigargin or αGalCer treated J774 . 2 were cocultured with 2C12 in presence or absence of murine CD1d - specific antibody ( black circles ) or isotype ( red squares ) for 16 hours . addition of an anti - CD1d antibody blocked activation of 2C12 demonstrating that NKT - activation was CD1d - specific . Graphs show mean ± from n = 2 biological replicates .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

Thapsigargin or αGalCer treated J774.2 cells were cocultured with 2C12 in the presence or absence of the murine CD1d-specific antibody (black circles) or isotype (red squares) for 16 hours. The addition of an anti-CD1d antibody blocked the activation of 2C12 demonstrating that the NKT-cell activation was CD1d-specific. Graphs show mean ± SEM from n=2 biological replicates.


Bar graph represents IL - 2 secretion by NKT hybridoma ( 2C12 ) co - cultured murine macrophage ( J774 . 2 ) treated either with thapsigargin - or tunicamycin - treated CD1d knockdown ( bars ) or untransfected ( black bars ) . Knockdown of CD1d blocked IL - 2 secretion by 2C12 . In total , n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± of each biological replicate .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured murine macrophage cell line (J774.2) treated either with thapsigargin- or tunicamycin-treated CD1d knockdown (white bars) or untransfected (black bars). Knockdown of CD1d blocked IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Bar graph represents IL - 2 secretion by NKT hybridoma ( 2C12 ) co - cultured either with thapsigargin - or tunicamycin - treated BMDM isolated from CD1d - / - ( bars ) or wild type ( black bars ) mice . 2C12 cocultured with CD1d - / - BMDMs resulted in reduced IL - 2 secretion . Graphs show mean ± from n = 3 biological replicates . * * * P < 0 . 001 ( Unpaired t ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured either with thapsigargin- or tunicamycin-treated primary BMDM isolated from CD1d-/- (white bars) or wild type (black bars) mice. 2C12 cocultured with CD1d-/- BMDMs resulted in reduced IL-2 secretion. Graphs show mean ± SEM from n=3 biological replicates. ***P < 0.001 (Unpaired t test).


qPCR analyses showing expression ( fold induction ) of UPR signature genes compared to DMSO in murine macrophage lines J774 . 2 stimulated either with thapsigargin , tunicamycin or DMSO for 4 or 8 hours . Graphs show mean ± from n = 2 biological replicates ( two technical replicates per biological replicate ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).


qPCR analyses showing expression ( fold induction ) of UPR signature genes compared to DMSO in human macrophage lines U937 stimulated either with thapsigargin , tunicamycin or DMSO for 4 or 8 hours . Graphs show mean ± from n = 2 biological replicates ( two technical replicates per biological replicate ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in human macrophage cell lines U937 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).


Bar graph represents of IL - 2 secretion by NKT hybridoma ( 2C12 ) co - cultured with murine macrophage ( J774 . 2 ) treated either with thapsigargin or tunicamycin in presence of IRE1α inhibitor ( 4μ8c ) ( black bar ) or PERK inhibitor ( GSK ) ( grey bar ) or ATF6α inhibitor ( Ceapin - a7 ) ( red bar ) respectively . Inhibition of IRE1α or PERK resulted in significant reduction in IL - 2 secretion by iNKT hybridoma compared to controls . In total , n = 2 biological replicates were performed ( two four technical replicates per biological replicate respectively ) . Graphs show mean ± of each biological replicate .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with murine macrophage cell line (J774.2) treated either with thapsigargin or tunicamycin in the presence of IRE1α inhibitor (4μ8c) (black bar) or PERK inhibitor (GSK) (grey bar) or ATF6α inhibitor (Ceapin-a7) (red bar) respectively. Inhibition of IRE1α or PERK resulted in significant reduction in IL-2 secretion by iNKT hybridoma compared to controls. In total, n=2 biological replicates were performed (two and four technical replicates per biological replicate respectively). Graphs show mean ± SEM of each biological replicate.


Immunoblots represents knockdown efficiency for IRE1α PERK in J774 . 2 transduced with lentiviral particles carrying gRNA against IRE1α or PERK gene .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O

Immunoblots represents the knockdown efficiency for IRE1α and PERK protein in J774.2 cell line transduced with lentiviral particles carrying gRNA against the IRE1α or PERK gene.


Bar graph represents of IL - 2 secretion by NKT hybridoma ( 2C12 ) co - cultured with thapsigargin - treated murine IRE1α or PERK knockdown macrophage ( J774 . 2 ) . Knockdown of IRE1α ( black bar ) or PERK ( grey bar ) resulted in significant reduction in IL - 2 secretion by iNKT hybridoma . In total n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± of each biological replicate .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with thapsigargin-treated murine IRE1α or PERK knockdown macrophage cell line (J774.2). Knockdown of IRE1α (black bar) or PERK (grey bar) resulted in significant reduction in IL-2 secretion by iNKT hybridoma. In total n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Neutral ( Fractions 1 - 3 ) polar lipids ( Fractions 4 - 6 ) were isolated from J774 . 2 macrophages stimulated with thapsigargin , tunicamycin or DMSO fractionated using solid - phase extraction ( SPE ) cartridges . Bar graph represents NKT activation by CD1d bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or tunicamycin - treated ( red bars ) J774 . 2 . Polar lipid fractions induce very little IL - 2 secretion by 2C12 . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs show mean ± of each biological replicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutral (Fractions 1-3) and polar lipids (Fractions 4-6) were isolated from J774.2 macrophages stimulated with thapsigargin, tunicamycin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents NKT cell activation by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or tunicamycin-treated (red bars) J774.2 cells. Polar lipid fractions induce very little IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin - treated J774 . 2 at acidic pH reduced mCD1d loading at neutral pH . Graphs show mean ± from n = 2 biological replicates ( two technical replicates per biological replicate ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin-treated J774.2 at acidic pH and reduced mCD1d loading at neutral pH. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).


Neutral ( Fractions 1 - 3 ) polar lipids ( Fractions 4 - 5 ) , isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO fractionated using solid - phase extraction ( SPE ) cartridges , were co - cultured with purified NKT1 subsets . Bar graphs represent IFN - γ release by NKT1 induced by CD1d bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or DMSO - treated ( bars ) murine macrophages . Polar lipid fractions induce a minimal amount of IFN - γ secretion by NKT1 sublineage . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs shows mean ± of each biological replicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutral (Fractions 1-3) and polar lipids (Fractions 4-5), isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges, were co-cultured with purified NKT1 subsets. Bar graphs represent IFN-γ release by NKT1 cells induced by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) primary murine macrophages. Polar lipid fractions induce a minimal amount of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.


Neutral ( Fractions 1 - 3 ) polar lipids ( Fractions 4 - 5 ) were isolated from macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO fractionated using solid - phase extraction ( SPE ) cartridges . Bar graph represents IFN - γ release by NKT1 by CD1d bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or DMSO - treated ( bars ) human macrophages . Polar lipid fractions induce only minimal amounts of IFN - γ secretion by NKT1 sublineage . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs shows mean ± of each biological replicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutral (Fractions 1-3) and polar lipids (Fractions 4-5) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents IFN-γ release by NKT1 cells by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) human primary macrophages. Polar lipid fractions induce only minimal amounts of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.


Bar graph represents that recycling of CD1d between various endomembrane compartments surface is required for optimal IL - 2 secretion by NKT . In total , n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± of each biological replicate .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents that recycling of CD1d between various endomembrane compartments and the cell surface is required for optimal IL-2 secretion by NKT cells. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


qPCR analyses showing expression ( fold induction ) of genes encoding for glycosphingolipid degrading enzymes compared to DMSO in murine macrophage lines J774 . 2 stimulated either with thapsigargin or DMSO for 8 hours . In total n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs show mean ± of each biological replicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR analyses showing the expression (fold induction) of genes encoding for glycosphingolipid degrading enzymes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin or DMSO for 8 hours. In total n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Glycolipid processing in intracellular compartments is not required prior to presentation to NKT . IL - 2 secretion in UPR - induced NKT activation in presence or absence of castanospermine or deoxygalactonojirimycin . Graphs show mean ± from n = 3 biological replicates . n . s ( non - significant ) ( One - way ANOVA ) biological replicates .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Glycolipid processing in intracellular compartments is not required prior to presentation to NKT cells. IL-2 secretion in UPR-induced NKT cell activation in the presence or absence of castanospermine or deoxygalactonojirimycin. Graphs show mean ± SEM from n = 3 biological replicates. n.s (non-significant) (One-way ANOVA) biological replicates.


L363 antibody does not impact UPR - induced NKT activation . 2C12 NKT were co - cultured with ER - stressed macrophages J774 . 2 treated with L363 antibody or isotype control . α - GalCer induced responses served as positive control . In total , n = 2 biological replicates were performed ( Four technical replicates per biological replicate ) . Graphs show mean ± of each biological replicate .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L363 antibody does not impact UPR-induced NKT cell activation. 2C12 NKT cells were co-cultured with ER-stressed macrophages J774.2 cells and treated with L363 antibody or isotype control. α-GalCer induced responses served as positive control. In total, n=2 biological replicates were performed (Four technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Representative confocal 3D of LysoTracker ( red ) , DAPI ( blue ) , BODIPY ( green ) stained ATM subsets obtained from eWAT of HFD ( 16 ) treated mice ( n = 8 mice from two independent experiments ) . merged figures are result of overlap of three fluorochromes . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal 3D images of LysoTracker (red), DAPI (blue), and BODIPY (green) stained ATM subsets obtained from eWAT of HFD (16 weeks) treated mice (n = 8 mice from two independent experiments). The merged figures are the result of the overlap of all three fluorochromes. Scale bar, 10 μm.


Mito Stress of sorted ATM subsets ( red , MHCIIlow ; green , MHCIIhi ; blue , CD11c + ) from 16 - week - HFD obese mice showing overall oxygen consumption rate ( OCR ) , maximal respiration , spare capacity respiration , basal respiration , ATP production , non - mitochondrial respiration . OCR data shown are representative of four independent experiments expressed as mean ± SD ( n = 4 MHCIIlow ; n = 7 MHCIIhi ; n = 5 CD11c + ) , with statistical significance determined using one - way ANOVA ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : no significant difference ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mito Stress assay of sorted ATM subsets (red, MHCIIlow; green, MHCIIhi; and blue, CD11c+) from 16-week-HFD obese mice showing overall oxygen consumption rate (OCR), maximal respiration, spare capacity respiration, basal respiration, ATP production, and non-mitochondrial respiration. OCR data shown are representative of four independent experiments and expressed as mean ± SD (n= 4 MHCIIlow; n= 7 MHCIIhi; n=5 CD11c+), with statistical significance determined using one-way ANOVA test (*P < 0.05; **P < 0.01; ***P < 0.001; ns: no significant difference).


Representative flow cytometric showing specific depletion efficiency of ATM subsets from eWAT of CD169 - DTR ND - ( A ) or HFD - treated ( 16 ) ( B ) mice . A representative of three independent experiments is shown .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative flow cytometric showing the specific depletion efficiency of ATM subsets from eWAT of CD169-DTR ND- (A) or HFD-treated (16 weeks) (B) mice. A representative analysis of three independent experiments is shown.


eWAT was collected from lean ( C ) obese ( HFD 16 ) ( D ) WT or CD169 - DTR mice after 12 days DT - mediated ATM depletion . Representative pictures of dissected eWAT are shown on left , corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in right , with statistical significance calculated using Student ' s t - ( * * P < 0 . 01 * * * * P < 0 . 0001 ) . zoomed - in shows clear , oily lipid accumulation in absence of ATMs ( lower left part ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

eWAT was collected from lean (C) and obese (HFD 16 weeks) (D) WT or CD169-DTR mice after 12 days DT-mediated ATM depletion. Representative pictures of dissected eWAT are shown on the left, the corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in the right, with statistical significance calculated using the Student's t-test (**P < 0.01 and ****P < 0.0001). The zoomed-in area shows clear, oily lipid accumulation in the absence of ATMs (lower left part).


Adipocyte hypertrophy is visible in absence of ATMs . Representative of hematoxylin eosin ( H & E ) ( E ) corresponding adipocyte diameters ( F ) of lean obese ( HFD 16 ) eWAT from WT or CD169 - DTR mice treated for 7 or 12 days with DT . Scale bar , 50 μm . Each group comprised 3 - 5 mice . Statistical significance was determined using one - way ANOVA . * * P < 0 . 01 ; * * * * P < 0 . 0001 ; ns : no significant difference . box whisker plots show median value 10 - 90 percentiles of adipocyte diameters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Adipocyte hypertrophy is visible in the absence of ATMs. Representative images of hematoxylin and eosin staining (H&E) (E) and corresponding adipocyte diameters (F) of lean and obese (HFD 16 weeks) eWAT from WT or CD169-DTR mice treated for 7 or 12 days with DT. Scale bar, 50 μm. Each group comprised 3-5 mice. Statistical significance was determined using one-way ANOVA test. **P < 0.01; ****P < 0.0001; ns: no significant difference. The box and whisker plots show the median value and 10-90 percentiles of adipocyte diameters.


qPCR showing relative expression of genes for adipogenesis ( Pparγ ) , lipolysis ( Lpl ) , lipogenesis ( Dgat1 ) , plus other lipid receptor / transporters ( Cd36 , Abca1 ) or chaperones ( Fabp4 ) in obese ( HFD 16 ) eWAT of CD169 - DTR ( red bars ) mice DT - injected WT controls ( blue bars ) ( upper panel ) obese eWAT of anti - CSF1R antibody - injected mice ( green bars ) mice isotype control - injected WT controls ( bars ) ( lower panel ) . Mice were under DT or anti - CSF1R antibody treatment for 12 days . ( n = 5 pooled mice per group , mean ± SD ) . Statistical significance was determined using an unpaired Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : no significant difference .

O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR showing the relative expression of genes for adipogenesis (Pparγ), lipolysis (Lpl), and lipogenesis (Dgat1), plus other lipid receptor/transporters (Cd36, Abca1) or chaperones (Fabp4) in obese (HFD 16 weeks) eWAT of CD169-DTR (red bars) mice and DT-injected WT controls (blue bars) (upper panel) and obese eWAT of anti-CSF1R antibody-injected mice (green bars) mice and isotype control-injected WT controls (white bars) (lower panel). Mice were under DT or anti-CSF1R antibody treatment for 12 days. (n = 5 pooled mice per group, mean ± SD). Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference.


Violin plots depicting expression of representative marker genes for each CD45 - cluster : ASC1 ( Gsn , Pdgfrb , Eln ) ( red ) , ASC2 ( Pi16 , Pdgfra , Cebpd ) ( yellow ) , stromal ( Col1a1 , Col1a2 , Myl9 ) ( green ) , VEC1 / VEC2 ( Pecam - 1 , Cldn5 , Aqp1 ) ( purple blue ) , mesothelial - like ( MLCs ) ( Upk3b , Msln , Acta2 ) ( orange ) .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O

Violin plots depicting the expression of representative marker genes for each CD45- cell cluster: ASC1 (Gsn, Pdgfrb, Eln) (red), ASC2 (Pi16, Pdgfra, Cebpd) (yellow), stromal cells (Col1a1, Col1a2, Myl9) (green), VEC1/VEC2 (Pecam-1, Cldn5, Aqp1) (purple and blue), and mesothelial-like cells (MLCs) (Upk3b, Msln, Acta2) (orange).


Representative 3D imaging of lean obese ( HFD 16 ) eWAT samples stained with anti - MHCII ( green in lean samples ) , anti - CD11c ( green in obese samples ) , anti - CD31 ( blue ) , anti - CD169 ( pink ) , DAPI ( ) . two at bottom show merged for lean obese conditions without DAPI . Scale bar : 100 μm ( lean ) 70 μm ( obese ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative 3D fluorescence imaging of lean and obese (HFD 16 weeks) eWAT samples stained with anti-MHCII (green in lean samples), anti-CD11c (green in obese samples), anti-CD31 (blue), anti-CD169 (pink), and DAPI (white). The two images at the bottom show the images merged for the lean and obese conditions without DAPI. Scale bar: 100 μm (lean) and 70 μm (obese).


Higher magnification of specific eWAT areas . Scale bar : 30 μm ( lean ) 50 μm ( obese ) . ( left panels ) of different ATM subsets to blood vessels . Distances ( in μm ) were calculated using Imaris software , as described in Materials Methods section . box whisker plots show median value 10 - 90 percentiles of values . right panels show distribution of ATM subsets to blood vessels within a certain ( ≤ 1 μm , 1 - 10 μm , ≥ 10 μm ) . C : CD169 + MHCIIlow CD169 + MHChi ATMs in lean eWAT . D : CD169 + CD11c - CD169 + CD11c + ATMs in obese ( HFD 16 ) eWAT . Four mice from two independent experiments were analyzed ( n = 4 - 5 ; mean ± SD ) . Statistical significance was determined using an unpaired Student ' s t - . * P < 0 . 05 , * * * * P < 0 . 0001 , bars with no asterisks showed no significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Higher magnification of specific eWAT areas. Scale bar: 30 μm (lean) and 50 μm (obese). (left panels) Distance of different ATM subsets to blood vessels. Distances (in μm) were calculated using the Imaris software, as described in the Materials and Methods section. The box and whisker plots show the median value and 10-90 percentiles of distance values. The right panels show the distribution of ATM subsets to blood vessels within a certain distance (≤1 μm, 1-10 μm, ≥10 μm). C: CD169+MHCIIlow and CD169+MHChi ATMs in lean eWAT. D: CD169+CD11c- and CD169+CD11c+ ATMs in obese (HFD 16 weeks) eWAT. Four mice from two independent experiments were analyzed (n = 4-5; mean ± SD). Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, ****P < 0.0001, bars with no asterisks showed no significant difference.


Model construction on 3D reconstructed showing relative of CD11c + ATMs from traced large blood vessels . Blood vessels larger than 10µm diameter in image were marked out on image by tracing ( filaments ) . CD11c + ATMs are close ( < 10µm ) to traced large blood vessels ( red ellipsoids ) or far ( > 10 µm ) ( grey ellipsoids ) from traced blood vessel . Blood vessels are shown in green . Scale bar : 100 μm . graph to right shows of CD11chi ATMs showing a proximity of ≤ 10 or > 10 μm to blood vessels quantified from 3 biological replicates from 1 experiment , data was expressed as mean ± SD . Statistical significance was determined using an unpaired Student ' s t - . * * * P < 0 . 001 .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Model construction based on 3D reconstructed images showing the relative distance of CD11c+ ATMs from traced large blood vessels. Blood vessels larger than 10µm diameter in the image were marked out on the image by tracing (white filaments). CD11c+ ATMs are close (< 10µm) to the traced large blood vessels (red ellipsoids) or far (>10 µm) (grey ellipsoids) from the traced blood vessel. Blood vessels are shown in green. Scale bar: 100 μm. The graph to the right shows the percentage of CD11chi ATMs showing a proximity of ≤10 or >10 μm to blood vessels quantified from 3 biological replicates from 1 experiment, data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. ***P < 0.001.


Two representative of eWAT dissected from Evans blue - injected obese ( HFD 16 ) WT CD169 - DTR mice treated for 7 days with DT . Evans blue dye was extracted from eWAT using formamide absorption of extract was measured at 620 nm ( absorption values were normalized to tissue in grams ) . A chart showing normalized mean optical density ± SD ( n = 5 - 7 mice per group ) . Statistical significance was determined using an unpaired Student ' s t - . * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Two representative images of eWAT dissected from Evans blue-injected obese (HFD 16 weeks) WT and CD169-DTR mice treated for 7 days with DT. Evans blue dye was extracted from eWAT using formamide and the absorption of the extract was measured at 620 nm (the absorption values were normalized to the tissue weight in grams). A chart showing the normalized mean optical density ± SD (n = 5-7 mice per group). Statistical significance was determined using an unpaired Student's t-test. ***P < 0.001.


Mmp12 ( H ) Vegfα ( F ) are highly expressed in MHCIIhi CD11c + ATMs purified from obese ( HFD 16 ) eWAT . qPCR showed relative expression in three ATM subsets ( n = 3 ) , data was expressed as mean ± SD . Statistical significance between three ATM subpopulations was determined using one - way ANOVA . * P < 0 . 05 , * * P < 0 . 01 . qPCR showing relative expression of Vegfα in obese ( HFD 16 ) eWAT of WT ( n = 3 ) or CD169 - DTR mice ( n = 5 ) treated for 7 days with DT , data was expressed as mean ± SD . Statistical significance was determined using an unpaired Student ' s t - . * * * * P < 0 . 0001 .

B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mmp12 (H) and Vegfα (F) are highly expressed in MHCIIhi and CD11c+ ATMs purified from obese (HFD 16 weeks) eWAT. qPCR analysis showed the relative expression in all three ATM subsets (n=3), data was expressed as mean ± SD . Statistical significance between the three ATM subpopulations was determined using one-way ANOVA test. *P < 0.05, **P < 0.01. qPCR analysis showing the relative expression of Vegfα in obese (HFD 16 weeks) eWAT of WT (n=3) or CD169-DTR mice (n=5) treated for 7 days with DT, data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. ****P < 0.0001.


Elastin showing more pronounced ECM accumulation in eWAT of CD169 - DTR ( HFD 16 ) when compared with ECM accumulation in WT obese ( HFD 16 ) control ( DAPI , blue ; elastin , red ; CD31 , green ) . fourth image in each row shows three channels merged . Scale bar : 50 μm . Picrosirius red - depicts excessive collagen deposits in obese ( HFD 16 ) eWAT of CD169 - DTR mice when compared with collagen deposits in WT controls . Scale bar : 250 μm . Upper panels : brightfield microscopy ; lower panels : microscopy .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Elastin staining showing more pronounced ECM accumulation in eWAT of CD169-DTR (HFD 16 weeks) when compared with ECM accumulation in the WT obese (HFD 16 weeks) control (DAPI, blue; elastin, red; CD31, green). The fourth image in each row shows the three channels merged. Scale bar: 50 μm. Picrosirius red-staining depicts excessive collagen deposits in obese (HFD 16 weeks) eWAT of CD169-DTR mice when compared with collagen deposits in WT controls. Scale bar: 250 μm. Upper panels: brightfield microscopy; lower panels: fluorescence microscopy.


Violin plots showing pro - inflammatory cytokine profile ( Il - 6 , Tnf - α , Il - 1β ) of 23 clusters obtained from obese ( HFD 16 ) WT CD169 - DTR eWAT . Cluster 0 : ASC1 ; Cluster 1 : Stromal ; Cluster 2 : VEC1 ; Cluster 3 : VEC2 ; Cluster 4 : NK1 ; Cluster 5 : MAC / Mono1 ; Cluster 6 : ASC2 ; Cluster 7 : B ; Cluster 8 : cDC1 ; Cluster 9 : mono - derived cDC ; Cluster 10 : Th2 / ILC2 / Treg ; Cluster 11 : MAC / Mono3 ; Cluster 12 : Th17 ; Cluster 13 : VEC2 ; Cluster 14 : CD8 ; Cluster 15 : MLCs ; Cluster 16 : CD11b + DC ; Cluster 17 : Neutrophils ; Cluster 18 : cDC2 ; Cluster 19 : cDC ; Cluster 20 : NKT ; Cluster 21 : NK2 ; Cluster 22 : Mast / Basophils . ASC : adipocyte ; VEC : vascular endothelial ; MLC : mesothelial - like ; NK : natural killer ; MAC : macrophage ; Mono : monocyte ; DC : dendritic ; NKT : natural killer T .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Violin plots showing the pro-inflammatory cytokine profile (Il-6, Tnf-α, and Il-1β) of all 23 cell clusters obtained from obese (HFD 16 weeks) WT and CD169-DTR eWAT. Cluster 0: ASC1; Cluster 1: Stromal cells; Cluster 2: VEC1; Cluster 3: VEC2; Cluster 4: NK1; Cluster 5: MAC/Mono1; Cluster 6: ASC2; Cluster 7: B; Cluster 8: cDC1; Cluster 9: mono-derived cDC; Cluster 10: Th2/ILC2/Treg; Cluster 11: MAC/Mono3; Cluster 12: Th17; Cluster 13: VEC2; Cluster 14: CD8; Cluster 15: MLCs; Cluster 16: CD11b+ DC; Cluster 17: Neutrophils; Cluster 18: cDC2; Cluster 19: cDC; Cluster 20: NKT; Cluster 21: NK2; Cluster 22: Mast cells/Basophils. ASC: adipocyte stem cell; VEC: vascular endothelial cell; MLC: mesothelial-like cell; NK: natural killer; MAC: macrophage; Mono: monocyte; DC: dendritic cell; NKT: natural killer T cells.


( A ) Confocal scanning showing localization of a functional CDKA ; 1 : mVenus fusion in wildtype ( WT ) cartoons on top highlighting changes in abundance of CDKA ; 1 : mVenus in nucleus cytoplasm during course of meiosis . region colored in beige represents cytoplasm , in green nucleoplasm , in nucleolus . Scale bar : 10 μm .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Confocal laser scanning micrographs showing the localization of a functional CDKA;1:mVenus fusion protein in the wildtype (WT) and cartoons on top highlighting the changes in abundance of CDKA;1:mVenus in the nucleus and cytoplasm during the course of meiosis. The region colored in beige represents the cytoplasm, in green the nucleoplasm, and in white the nucleolus. Scale bar: 10 μm.


( B ) Quantitative of signal distribution of versus fraction of CDKA ; 1 : mVenus during prophase I of meiosis as revealed by live imaging Twenty at each time point were used for . Error bars represent mean ± SD two biological replicates were performed .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Quantitative analysis of the signal distribution of the nuclear versus cytoplasmic fraction of CDKA;1:mVenus during prophase I of meiosis as revealed by live cell imaging Twenty cells at each time point were used for the analysis. Error bars represent mean ± SD and two biological replicates were performed.


( C ) Immunolocalization of CDKA ; 1 ( green ) ASY1 ( red ) on spread chromsomes in leptotene zygotene of wild - type expressing a functional PROCDKA ; 1 : CDKA ; 1 : Strep construct . last lane shows a magnification of region marked by red rectangle . Arrowheads indicate synapsed regions of homologous chromosomes where CDKA ; 1 is no longer present . Scale bar : 5 μm .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O

(C) Immunolocalization of CDKA;1 (green) and ASY1 (red) on spread chromsomes in leptotene and zygotene of wild-type plants expressing a functional PROCDKA;1:CDKA;1:Strep construct. The last lane shows a magnification of the region marked by the red rectangle. Arrowheads indicate synapsed regions of homologous chromosomes where CDKA;1 is no longer present. Scale bar: 5 μm.


( D ) Chromosome spread of wildtype hypomorphic cdka ; 1 mutant CDKA ; 1T161D . ( a , h ) zygotene or zygotene - like stages ; ( b , i ) pachytene or pachytene - like stages ; ( c , j , k ) diakinesis or dikinesis - like stages ; ( d ) metaphase I ; ( e , i , m , n ) end of meiosis I with two ( e , m ) or three ( i ) pools of chromosomes ; ( f ) metaphase II ; ( g ) tetrad . Red arrowheads indicate initiated formation of a phragmoplast . arrowheads depict mitochondria . Scale bars : 10 μm .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Chromosome spread analysis of the wildtype and the hypomorphic cdka;1 mutant CDKA;1T161D. (a, h) zygotene or zygotene-like stages; (b, i) pachytene or pachytene-like stages; (c, j, k) diakinesis or dikinesis-like stages; (d) metaphase I; (e, i, m, n) end of meiosis I with two (e, m) or three (i) pools of chromosomes; (f) metaphase II; (g) tetrad. Red arrowheads indicate the initiated formation of a phragmoplast. White arrowheads depict mitochondria. Scale bars: 10 μm.


( A ) ASY1 : GFP ASY1T142D : GFP localization in late G2 leptotene of meiocytes of wildtype CDKA ; 1T161D mutants . ASY3 : RFP , highlighting chromsomes , was used as a marker for staging of meiosis . Scale bar : 5 μm .

O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(A) ASY1:GFP and ASY1T142D:GFP localization in late G2 and leptotene of male meiocytes of the wildtype and CDKA;1T161D mutants. ASY3:RFP, highlighting chromsomes, was used as a marker for the staging of meiosis. Scale bar: 5 μm.


( B ) Kinase assays of CDKA ; 1 - SDS , - TAM , - CYCA3 ; 1 complexes using ASY1 puried from baculovirus - infected insect as a substrate . upper panel shows autoradiograph . control reaction without CDKA ; 1 - cyclin indicates a background activity co - purified from insect . lower panel indicates loading by coomassie briliant blue ( CBB ) . Arrowheads indicate ASY1 asterisks depict relevant cyclin used which also gets phosphorylated in . Numbers indicate relative intensities of ASY1 bands .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

(B) Kinase assays of CDKA;1-SDS, -TAM, and -CYCA3;1 complexes using ASY1 puried from baculovirus-infected insect cells as a substrate. The upper panel shows the autoradiograph. The control reaction without CDKA;1-cyclin complex indicates a background activity co-purified from insect cells. The lower panel indicates protein loading by coomassie briliant blue (CBB) staining. Arrowheads indicate ASY1 proteins and asterisks depict the relevant cyclin used which also gets phosphorylated in the assay. Numbers indicate the relative intensities of ASY1 bands.


( C ) upper panel shows a phos - tag gel of ASY11 - 300 ASY11 - 300 / T142V ; T184V with without CDKA ; 1 - SDS kinase complexes using an anti - MBP antibody . lower panel denotes loading of CDKA ; 1 using an anti - Strep antibody . Arrowheads represent as indicated .

O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

(C) The upper panel shows a phos-tag gel analysis of ASY11-300 and ASY11-300/T142V;T184V with and without CDKA;1-SDS kinase complexes using an anti-MBP antibody. The lower panel denotes loading of CDKA;1 using an anti-Strep antibody. Arrowheads represent the proteins as indicated.


( B ) Localization patterns of different ASY1 : GFP variants together with ASY3 : RFP ( for staging of zygotene pachytene ) in a asy1 mutant background during prophase I . Bar : 5

O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

(B) Localization patterns of different ASY1:GFP variants together with ASY3:RFP (for staging of zygotene and pachytene) in a asy1 mutant background during prophase I. Bar: 5 μ


( C ) Signal distribution profiles of ASY1 : GFP ASY1T142V : GFP at leptotene many small peaks with low amplitude in ASY1T142V : GFP indicate diffused localization as opposed to clear peaks seen in wildtype .

O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(C) Signal distribution profiles of ASY1:GFP and ASY1T142V:GFP at leptotene The many small peaks with low amplitude in ASY1T142V:GFP indicate diffused localization as opposed to the clear peaks seen in the wildtype.


( D , E ) Immunolocalization of ASY1 ( D ) ZYP1 ( E ) in ASY1 : GFP ( asy1 ) ASY1T142V : GFP ( asy1 ) using anti - GFP anti - ZYP1 antibodies , respectively . was stained with DAPI ( blue ) . Scale bars : 5 μm .

O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(D, E) Immunolocalization of ASY1 (D) and ZYP1 (E) in ASY1:GFP (asy1) and ASY1T142V:GFP (asy1) plants using anti-GFP and anti-ZYP1 antibodies, respectively. DNA was stained with DAPI (blue). Scale bars: 5 μm.


( A ) Yeast two - hybrid interaction assays of ASY3 with different ASY1 variants . Monomeric GFP ( mGFP ) fused with AD ( activating domain ) BD ( binding domain ) were used as controls . Yeast harboring both AD BD plasmids were grown on synthetic medium supplied with glucose in absence of Leu Trp ( - L / T , left panel ) , on Synthetic Drop - out ( SD ) medium in absence of Leu , Trp , His ( - L / T / H , middle panel ) , on SD medium in absence of Leu , Trp , His Ade ( - L / T / H / A , right panel ) . Yeast were incubated until OD600 = 1 then diluted 10 - , 100 - 1000 - fold for assays .

O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Yeast two-hybrid interaction assays of ASY3 with different ASY1 variants. Monomeric GFP (mGFP) fused with AD (activating domain) and BD (binding domain) were used as controls. Yeast cells harboring both the AD and BD plasmids were grown on synthetic medium supplied with glucose in the absence of Leu and Trp (-L/T, left panel), on Synthetic Drop-out (SD) medium in the absence of Leu, Trp, and His (-L/T/H, middle panel), and on SD medium in the absence of Leu,Trp, His and Ade (-L/T/H/A, right panel). Yeast cells were incubated until OD600 = 1 and then diluted 10-, 100- and 1000-fold for the assays.


( B ) GST pull down of ASY3 with different ASY1 variants . numbers above bands show relative of bands . input pull down fractions were analyzed by immuno - blotting with anti - GST ( ASY3 ) anti - MBP ( ASY1 ) antibodies . ( C ) of pull down fractions of ASY1 as shown in ( B ) . band in pull down of ASY11 - 300 / T142V ; T184V at a Triton X - 100 of 0 . 5 % was defined as 1 . relative amount of ASY1 in pull down fractions was normalized by band of pulled - down ASY3 fraction . average band of ASY1 at different concentrations of Triton X - 100 used was plotted . Asteriks indicate significant difference ( two - t - , p < 0 . 05 ) . Error bars represent mean ± SD two biological replicates were performed . .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) GST pull down of ASY3 with different ASY1 variants. The numbers above the bands show the relative intensity of the bands. The input and pull down fractions were analyzed by immuno-blotting with the anti-GST (ASY3) and anti-MBP (ASY1) antibodies. (C) Quantification of the pull down fractions of ASY1 as shown in (B). The band intensity in the pull down of ASY11-300/T142V;T184V at a Triton X-100 concentration of 0.5% was defined as 1. The relative amount of ASY1 in the pull down fractions was normalized by the band intensity of the pulled-down ASY3 fraction. The average band intensity of ASY1 at different concentrations of Triton X-100 used was plotted. Asteriks indicate significant difference (two-tailed t-test, p < 0.05). Error bars represent mean ± SD and two biological replicates were performed..


( A ) Localization patterns of ASY1T142V ; T184V : GFP together with ASY3 - RFP in wildtype pch2 mutants . Please note that of ASY1T142V ; T184V : GFP in pch2 mutants were taken with increased sensitivity for a better visibility . Scale bar : 5 μm .

O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(A) Localization patterns of ASY1T142V;T184V:GFP together with ASY3-RFP in the wildtype and pch2 mutants. Please note that images of ASY1T142V;T184V:GFP in pch2 mutants were taken with increased sensitivity for a better visibility. Scale bar: 5 μm.


( B ) Seed sets ( mean ± SD , n = 5 ) of WT , asy1 , pch2 , asy1 pch2 , ASY1T142V ; T184V : GFP ( asy1 ) ASY1T142V ; T184V : GFP ( asy1 pch2 ) . Asteriks indicate significant differences ( two - t - , P < 0 . 01 ) ns depicts no significant difference . Scale bar : 2 mm .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Seed sets (mean ± SD, n = 5) of WT, asy1, pch2, asy1 pch2, ASY1T142V;T184V:GFP (asy1) and ASY1T142V;T184V:GFP (asy1 pch2) plants. Asteriks indicate significant differences (two-tailed t-test, P < 0.01) and ns depicts no significant difference. Scale bar: 2 mm.


( C ) Localization patterns of ASY1 : GFP , ASY1T142V ; T184V : GFP , ASY11 - 570 : GFP in wildtype ( WT ) / or in pch2 mutant at early prophase I . Scale bars : 5 μm .

O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(C) Localization patterns of ASY1:GFP, ASY1T142V;T184V:GFP, and ASY11-570:GFP in the wildtype (WT) and/or in pch2 mutant plants at early prophase I. Scale bars: 5 μm.


( D ) Localization pattern of PCH2 : GFP together with ASY1 : RFP in meiocytes of wildtype . Arrowheads indicate chromosomal regions where ASY1 removal was concomitant with localization of PCH2 . Scale bar : 10 μm .

O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O

(D) Localization pattern of PCH2:GFP together with ASY1:RFP in the male meiocytes of wildtype. Arrowheads indicate the chromosomal regions where the ASY1 removal was concomitant with the localization of PCH2. Scale bar: 10 μm.


( B ) Interaction assays of different ASY1 fragments ( with without closure motif ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O

(B) Interaction assays of different ASY1 fragments (with and without the closure motif).


( C ) Interaction of ASY1 closure motif ( ASY1571 - 596 ) with different ASY1 HORMA domain variants .

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O

(C) Interaction analysis of the ASY1 closure motif (ASY1571-596) with different ASY1 HORMA domain variants.


( A ) GalNAc - T expression in human liver ( map data for human liver was kindly provided by " Gene Expression Applications research group at Illumina " , http : / / www . ebi . ac . uk / arrayexpress / experiments / E - MTAB - 513 / ) HepG2 wild type quantified by RNAseq ( n = 2 , bars representing mean with SD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) GalNAc-T expression levels in human liver (The body map data for human liver was kindly provided by the "Gene Expression Applications research group at Illumina", http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-513/) and HepG2 wild type cells quantified by RNAseq (n=2, bars representing mean with SD).


( B ) VVA lectin immunoblot of secretomes from HepG2 SC , SCΔT1 , SCΔT2 SCΔT3 .

(B) VVA lectin immunoblot of secretomes from HepG2 SC, SCΔT1, SCΔT2 and SCΔT3.


Histograms showing distribution of M / L quantitation ratios of monoglycosylated peptides identified in ( A ) HepG2SCΔT1 , ( B ) ΔT2 ( C ) + T3 . Glycopeptides with M / L < - 1 are colored red glycopeptides with a M / L > + 1 are colored green . ( D ) Venn diagram showing distribution of candidates for isoform - specific sites among HepG2SCΔT1 , T2 + T3 applying a log10 ( + / - 1 ) cut - off ( excluding sites identified in both TCL SEC for each isoform ) , ( E ) TCL alone ( F ) SEC alone .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Histograms showing the distribution of M/L quantitation ratios of monoglycosylated peptides identified in (A) HepG2SCΔT1, (B) ΔT2 and (C) +T3. Glycopeptides with M/L ratio < -1 are colored red and glycopeptides with a M/L ratio > +1 are colored green. (D) Venn diagram showing the distribution of candidates for isoform-specific sites among HepG2SCΔT1, T2 and +T3 applying a log10 (+/-1) cut-off (excluding sites identified in both TCL and SEC for each isoform), and (E) TCL alone and (F) SEC alone.


human NK ( n = 19 different donors per condition ) were co - incubated with either no target ( NK ( - ) ) , autologous CD4 T ( Mock ) or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) at an effector : target of 1 : 2 . NK degranulation was quantified by measuring CD107a surface expression using flow cytometry . ( A ) Representative histograms ( overlay ) displaying CD107a expression on NK after culture with described culture conditions ( NK ( - ) , Mock , HIV ) . For HIV - 1 co - culture condition , subsequent analyses of antigen expression were conducted by gating non - responsive ( CD107a - ) responsive ( CD107a + ) NK subsets . ( B ) Relative frequency of CD107a + NK ( y - axis ) after culture in described conditions ( x - axis ) ( n = 19 different donors ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human NK cells (n = 19 different donors per condition) were co-incubated with either no target cells (NK(-)), autologous CD4 T cells (Mock) or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) at an effector:target cell ratio of 1:2. NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry. (A) Representative histograms (overlay) displaying CD107a expression on NK cells after culture with the described culture conditions (NK(-), Mock, HIV). For the HIV-1 co-culture condition, subsequent analyses of antigen expression were conducted by gating non-responsive (CD107a-) and responsive (CD107a+) NK cell subsets. (B) Relative frequency of CD107a+ NK cells (y-axis) after culture in the described conditions (x-axis) (n = 19 different donors). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


human NK ( n = 19 different donors per condition ) were co - incubated with either no target ( NK ( - ) ) , autologous CD4 T ( Mock ) or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) at an effector : target of 1 : 2 . NK degranulation was quantified by measuring CD107a surface expression using flow cytometry . ( C ) HIV - specific response of bulk NK displayed as points ( p . p . ) CD107a + NK after normalization to mock CD4 T ( y - axis ) ( n = 19 different donors ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human NK cells (n = 19 different donors per condition) were co-incubated with either no target cells (NK(-)), autologous CD4 T cells (Mock) or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) at an effector:target cell ratio of 1:2. NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry. (C) HIV-specific response of bulk NK cells displayed as percentage points (p.p.) CD107a+ NK cells after normalization to mock CD4 T cells (y-axis) (n = 19 different donors). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


human NK ( n = 19 different donors per condition ) were co - incubated with HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) ( E ) Representative histograms ( overlay ) showing TRAIL surface expression as on NK after co - incubation with HIV - 1 - infected CD4 T . Histograms show TRAIL expression for CD107a + CD107a - NK subsets ( solid lines ) as well as their corresponding FMO controls ( dotted lines ) . Gate defining TRAIL + is indicated as well . ( F ) Left panel : Relative frequency of TRAIL + ( y - axis ) of CD107a + CD107a - NK subsets ( x - axis ) after co - incubation with HIV - 1 - infected CD4 T . Right panel : Difference in relative frequency of TRAIL + ( y - axis ) between CD107a + CD107a - displayed as p . p . ( n = 19 different donors ) . Data information : Wilcoxon signed - rank . Adjustment for multiplicity : Benjamini Hochberg false discovery rate ( FDR ) F , left panel : ) ; Bonferroni ( F , right panel Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points . Lines connecting CD107a - CD107a + refer to measured values of same donor .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human NK cells (n = 19 different donors per condition) were co-incubated with HIV-1-infected (NL4-3) CD4 T cells (HIV) (E) Representative histograms (overlay) showing TRAIL surface expression as fluorescence intensity on NK cells after co-incubation with HIV-1-infected CD4 T cells. Histograms show TRAIL expression for CD107a+ and CD107a- NK cell subsets (solid lines) as well as their corresponding FMO controls (dotted lines). Gate defining TRAIL+ cells is indicated as well. (F) Left panel: Relative frequency of TRAIL+ cells (y-axis) of CD107a+ and CD107a- NK cell subsets (x-axis) after co-incubation with HIV-1-infected CD4 T cells. Right panel: Difference in relative frequency of TRAIL+ cells (y-axis) between CD107a+ and CD107a- cells displayed as p.p. (n = 19 different donors). Data information: Wilcoxon signed-rank test. Adjustment for multiplicity: Benjamini and Hochberg false discovery rate (FDR) F, left panel:); Bonferroni (F, right panel Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points. Lines connecting CD107a- and CD107a+ cells refer to measured values of the same donor.


human NK ( n = 19 different donors per condition ) were co - incubated with HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) ( G ) Left panel : TRAIL surface expression ( y - axis ) of CD107a + CD107a - NK subsets ( x - axis ) after co - incubation with HIV - 1 - infected CD4 T displayed as median . Right panel : Differences in TRAIL surface expression ( y - axis ) between CD107a + CD107a - displayed as fold - difference ( n = 19 different donors ) . Data information : Wilcoxon signed - rank . Adjustment for multiplicity : ; Bonferroni G ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points . Lines connecting CD107a - CD107a + refer to measured values of same donor .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human NK cells (n = 19 different donors per condition) were co-incubated with HIV-1-infected (NL4-3) CD4 T cells (HIV) (G) Left panel: TRAIL surface expression (y-axis) of CD107a+ and CD107a- NK cell subsets (x-axis) after co-incubation with HIV-1-infected CD4 T cells displayed as median fluorescence intensity. Right panel: Differences in TRAIL surface expression (y-axis) between CD107a+ and CD107a- cells displayed as fold-difference (n = 19 different donors). Data information: Wilcoxon signed-rank test. Adjustment for multiplicity: ; Bonferroni G). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points. Lines connecting CD107a- and CD107a+ cells refer to measured values of the same donor.


Comparison of CD107a expression between various NK subsets after co - incubation with either no target present ( - ) , autologous CD4 T ( Mock ) , or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) ( n = 13 different donors per condition ) . Predefined NK subsets comprise CD56Dim , CD56Bright , KIR - educated , defined as expressing at least one self - inhibitory KIR ( 2DL1 / L2 / L3 , 3DL1 ) negative for NKG2A , NKG2A - educated , expressing NKG2A but lacking self - inhibitory KIR , uneducated , lacking self - inhibitory KIR NKG2A altogether . ( A ) Relative frequency of CD107a + ( y - axis ) of predefined NK subsets ( x - axis ) . Left panel displays data for bulk NK , middle panel for CD56Dim CD56Bright NK , right panel for uneducated , KIR - educated NKG2A - educated ( n = 13 different donors ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of CD107a expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether. (A) Relative frequency of CD107a+ cells (y-axis) of predefined NK cell subsets (x-axis). Left panel displays data for bulk NK cells, middle panel for CD56Dim and CD56Bright NK cells, right panel for uneducated, KIR-educated and NKG2A-educated cells (n = 13 different donors). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Comparison of CD107a expression between various NK subsets after co - incubation with either no target present ( - ) , autologous CD4 T ( Mock ) , or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) ( n = 13 different donors per condition ) . Predefined NK subsets comprise CD56Dim , CD56Bright , KIR - educated , defined as expressing at least one self - inhibitory KIR ( 2DL1 / L2 / L3 , 3DL1 ) negative for NKG2A , NKG2A - educated , expressing NKG2A but lacking self - inhibitory KIR , uneducated , lacking self - inhibitory KIR NKG2A altogether . ( B ) HIV - specific responses of predefined NK sub - populations ( x - axis ) displayed as points ( p . p . ) CD107a + NK after normalization to mock CD4 T ( y - axis ) ( n = 13 different donors ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of CD107a expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether. (B) HIV-specific responses of predefined NK cell sub-populations (x-axis) displayed as percentage points (p.p.) CD107a+ NK cells after normalization to mock CD4 T cells (y-axis) (n = 13 different donors). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Comparison of TRAIL expression between various NK subsets after co - incubation with either no target present ( - ) , autologous CD4 T ( Mock ) , or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) ( n = 13 different donors per condition ) . Predefined NK subsets comprise CD56Dim , CD56Bright , KIR - educated , defined as expressing at least one self - inhibitory KIR ( 2DL1 / L2 / L3 , 3DL1 ) negative for NKG2A , NKG2A - educated , expressing NKG2A but lacking self - inhibitory KIR , uneducated , lacking self - inhibitory KIR NKG2A altogether . ( C ) TRAIL expression ( y - axis ) of predefined NK subsets ( x - axis ) . Left panel displays data for bulk NK , middle panel for CD56Dim CD56Bright NK , right panel for uneducated , KIR - educated NKG2A - educated . Expression is displayed as median ( MdFI ) ( n = 13 different donors ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of TRAIL expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether. (C) TRAIL expression levels (y-axis) of predefined NK cell subsets (x-axis). Left panel displays data for bulk NK cells, middle panel for CD56Dim and CD56Bright NK cells, right panel for uneducated, KIR-educated and NKG2A-educated cells. Expression is displayed as median fluorescence intensity (MdFI) (n = 13 different donors). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Comparison of TRAIL expression between various NK subsets after co - incubation with either no target present ( - ) , autologous CD4 T ( Mock ) , or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) ( n = 13 different donors per condition ) . Predefined NK subsets comprise CD56Dim , CD56Bright , KIR - educated , defined as expressing at least one self - inhibitory KIR ( 2DL1 / L2 / L3 , 3DL1 ) negative for NKG2A , NKG2A - educated , expressing NKG2A but lacking self - inhibitory KIR , uneducated , lacking self - inhibitory KIR NKG2A altogether . ( D ) Comparison of TRAIL expression ( y - axis ) between CD107a - CD107a + of predefined NK subsets . Left panel displays data for bulk NK , middle panel for CD56Dim CD56Bright NK , right panel for uneducated , KIR - educated NKG2A - educated . Expression is displayed as MdFI . ( n = 13 different donors ) . ( E ) Fold - differences in TRAIL surface expression ( y - axis ) between CD107a + CD107a - of pre - defined NK sub - populations ( x - axis ) . ( n = 13 different donors ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points . Lines connecting CD107a - CD107a + refer to measured values of same donor .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of TRAIL expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether. (D) Comparison of TRAIL expression levels (y-axis) between CD107a- and CD107a+ cells of predefined NK cell subsets. Left panel displays data for bulk NK cells, middle panel for CD56Dim and CD56Bright NK cells, right panel for uneducated, KIR-educated and NKG2A-educated cells. Expression is displayed as MdFI. (n = 13 different donors). (E) Fold-differences in TRAIL surface expression (y-axis) between CD107a+ and CD107a- cells of pre-defined NK cell sub-populations (x-axis). (n = 13 different donors). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points. Lines connecting CD107a- and CD107a+ cells refer to measured values of the same donor.


human CD4 T from eight different donors ( n = 8 ) were separately infected with lab - adapted HIV - 1 strains . Combined expression of DR4 DR5 was assessed by flow cytometry four days after infection . Uninfected CD4 T were determined as CD4 - positive HIV - 1 p24 - negative , infected as CD4 - negative p24 - positive . ( A ) Histograms ( overlay ) of one representative donor displaying combined DR4 / 5 surface expression on CD4 T previously incubated with either NL4 - 3 , JR - CSF , WITO , CH198 , or CH236 . Expression is displayed as ( x - axis ) . ( B ) Comparison of combined surface expression of DR4 / 5 between mock ( circles ) , uninfected ( grey circles ) infected CD4 T ( orange circles ) , from left to right : Mock : n = 8 ; NL4 - 3 : n = 8 ; JR - CSF : n = 8 ; WITO : n = 7 ; CH198 : n = 7 ; CH236 : n = 8 ( 7 - 8 different donors per condition ) . Expression is displayed as relative ( RFI ) after normalization to respective secondary - AB - only control ( y - axis ) . Data information Wilcoxon signed - rank . Values for non - infected infected of same donor are connected with lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human CD4 T cells from eight different donors (n = 8) were separately infected with lab-adapted and primary HIV-1 strains. Combined expression of DR4 and DR5 was assessed by flow cytometry four days after infection. Uninfected CD4 T cells were determined as CD4-positive and HIV-1 p24-negative, infected cells as CD4-negative and p24-positive. (A) Histograms (overlay) of one representative donor displaying combined DR4/5 surface expression on CD4 T cells previously incubated with either NL4-3, JR-CSF, WITO, CH198, or CH236. Expression is displayed as fluorescence intensity (x-axis). (B) Comparison of the combined surface expression of DR4/5 between mock (white circles), uninfected (grey circles) and infected CD4 T cells (orange circles), from left to right: Mock: n = 8; NL4-3: n = 8; JR-CSF: n = 8; WITO: n = 7; CH198: n = 7; CH236: n = 8 (7-8 different donors per condition). Expression is displayed as relative fluorescence intensity (RFI) after normalization to the respective secondary-AB-only control (y-axis). Data information Wilcoxon signed-rank test. Values for non-infected and infected cells of the same donor are connected with lines.


Degranulation of human NK after co - culture with various target in presence or absence of αTRAIL ( A ) Comparison of CD107a expression after co - culture with 721 . 221 target with either 10 µg / ml αTRAIL or isotype control ( Iso ) using flow cytometry ( n = 10 different donors per condition ) . Each data point represents mean of 2 technical replicates . Effector : target was 1 : 1 . Left panel : Concatenated density plot depicting CD107a expression as for one representative donor . Right panel : Box plots showing relative frequency of CD107a + NK ( y - axis ) . ( B ) Box plots display inhibition of degranulation ( y - axis ) after co - culture with 721 . 221 target in presence of either isotype or αTRAIL as relative reduction compared to no antibody ( n = 10 different donors per condition ) . ( C ) Comparison of CD107a expression after co - culture with autologous HIV - I - infected CD4 T with either 10 µg / ml αTRAIL or isotype control ( Iso ) using flow cytometry ( n = 9 different donors per condition ) Each data point represents mean of 2 technical replicates . Left panel : Concatenated density plot depicting CD107a expression as for one representative donor . Right panel : Box plots showing relative frequency of CD107a + NK ( y - axis ) . ( D ) Box plots display inhibition of degranulation ( y - axis ) after co - culture with autologous HIV - I - infected CD4 T in presence of either isotype or αTRAIL as relative reduction compared to no antibody ( n = 9 different donors per condition ) . Data information : Wilcoxon signed - rank . Adjustment for multiple comparisons was performed using Bonferroni . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points . Bar graphs represent mean associated whiskers display SD .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after co-culture with various target cells in the presence or absence of αTRAIL (A) Comparison of CD107a expression after co-culture with 721.221 target cells with either 10 µg/ml αTRAIL or isotype control (Iso) using flow cytometry (n = 10 different donors per condition). Each data point represents the mean of 2 technical replicates. Effector:target ratio was 1:1. Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis). (B) Box plots display inhibition of degranulation (y-axis) after co-culture with 721.221 target cells in presence of either isotype or αTRAIL as relative reduction compared to no antibody (n = 10 different donors per condition). (C) Comparison of CD107a expression after co-culture with autologous HIV-I-infected CD4 T cells with either 10 µg/ml αTRAIL or isotype control (Iso) using flow cytometry (n = 9 different donors per condition) Each data point represents the mean of 2 technical replicates. Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis). (D) Box plots display inhibition of degranulation (y-axis) after co-culture with autologous HIV-I-infected CD4 T cells in the presence of either isotype or αTRAIL as relative reduction compared to no antibody (n = 9 different donors per condition). Data information: Wilcoxon signed-rank test. Adjustment for multiple comparisons was performed using Bonferroni. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Bar graphs represent the mean and the associated whiskers display the SD.


Degranulation of human NK after co - culture with various target in presence or absence of αDR4 / 5 . ( E ) Representative histograms ( overlay , left panel ) bar graphs ( n = 3 independent experiments , right panel ) showing combined surface expression of DR4 DR5 on 721 . 221 . Each data point represents mean of 3 technical replicates . Data information : Wilcoxon signed - rank . Adjustment for multiple comparisons was performed using Bonferroni . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points . Bar graphs represent mean associated whiskers display SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after co-culture with various target cells in the presence or absence of αDR4/5. (E) Representative histograms (overlay, left panel) and bar graphs (n = 3 independent experiments, right panel) showing the individual and combined surface expression of DR4 and DR5 on 721.221 cells. Each data point represents the mean of 3 technical replicates. Data information: Wilcoxon signed-rank test. Adjustment for multiple comparisons was performed using Bonferroni. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Bar graphs represent the mean and the associated whiskers display the SD.


Degranulation of human NK after co - culture with various target in presence or absence of αDR4 / 5 . ( F ) Comparison of CD107a expression after co - culture with 721 . 221 target in presence of either αDR4 / 5 ( 10 µg / ml each ) , or 20 µg / ml isotype control ( Iso ) using flow cytometry ( n = 9 different donors per condition ) . Effector : target was 1 : 1 . Box plots showing relative frequency of CD107a + NK ( y - axis ) . ( G ) Box plots display inhibition of degranulation after co - culture with 721 . 221 target in presence of either isotype or αDR4 / 5 as relative reduction compared to no antibody ( n = 9 different donors per condition ) . Data information : Wilcoxon signed - rank . Adjustment for multiple comparisons was performed using Bonferroni . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points . Bar graphs represent mean associated whiskers display SD .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after co-culture with various target cells in the presence or absence of αDR4/5. (F) Comparison of CD107a expression after co-culture with 721.221 target cells in the presence of either αDR4/5 (10 µg/ml each), or 20 µg/ml isotype control (Iso) using flow cytometry (n = 9 different donors per condition). Effector:target ratio was 1:1. Box plots showing relative frequency of CD107a+ NK cells (y-axis). (G) Box plots display inhibition of degranulation after co-culture with 721.221 target cells in presence of either isotype or αDR4/5 as relative reduction compared to no antibody (n = 9 different donors per condition). Data information: Wilcoxon signed-rank test. Adjustment for multiple comparisons was performed using Bonferroni. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Bar graphs represent the mean and the associated whiskers display the SD.


Degranulation of human NK after incubation with plate - coated antibodies ( A ) Comparison of CD107a expression after incubation in either uncoated wells ( PBS ) or wells coated with αTRAIL , αNKG2D , αNKp46 , or isotype using flow cytometry ( n = 12 different donors per condition ) . Left panel : Concatenated density plot depicting CD107a expression as ( y - axis ) for one representative donor 10 µg / ml antibody . Right panel : Box plots showing relative frequency of CD107a + NK ( y - axis ) after incubation with plate - coated antibodies of different concentrations ( x - axis ) . ( B ) Comparison of CD107a expression after incubation with plate - coated DR4 , DR5 or human IgG using flow cytometry ( n = 11 different donors per condition ) . Left panel : Concatenated density plot depicting CD107a expression as ( y - axis ) for one representative donor 10 µg / ml . Right panel : Box plots showing relative frequency of CD107a + NK ( y - axis ) after incubation with plate - coated of different concentrations ( x - axis ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Spearman rank . ( A , B , Each data point represents mean of 2 technical replicates . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after incubation with plate-coated antibodies (A) Comparison of CD107a expression after incubation in either uncoated wells (PBS) or wells coated with αTRAIL, αNKG2D, αNKp46, or isotype using flow cytometry (n = 12 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity (y-axis) for one representative donor and 10 µg/ml antibody concentration. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis) after incubation with plate-coated antibodies of different concentrations (x-axis). (B) Comparison of CD107a expression after incubation with plate-coated DR4 protein, DR5 protein or human IgG using flow cytometry (n = 11 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity (y-axis) for one representative donor and 10 µg/ml protein concentration. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis) after incubation with plate-coated proteins of different concentrations (x-axis). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. (A, B, Each data point represents the mean of 2 technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


Degranulation of human NK after incubation with plate - coated antibodies ( C ) Comparison of granzyme B release after incubation with various stimuli ( 10 µg / ml each ) . Box plots showing Granzyme B in supernatant as determined by ELISA ( left panel : n = 8 different donors per condition , right panel : n = 9 different donors per condition ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Spearman rank . C , Each data point represents mean of 2 technical replicates . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after incubation with plate-coated antibodies (C) Comparison of granzyme B release after incubation with various stimuli (10 µg/ml each). Box plots showing Granzyme B concentration in the supernatant as determined by ELISA (left panel: n = 8 different donors per condition, right panel: n = 9 different donors per condition). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. C, Each data point represents the mean of 2 technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


Degranulation of human NK after incubation with plate - coated antibodies ( D ) Correlation between relative frequency of CD107a + NK granzyme B ( n = 53 , data points obtained from A , B , C , 11 different donors ) .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after incubation with plate-coated antibodies (D) Correlation analysis between relative frequency of CD107a+ NK cells and granzyme B concentration (n = 53, data points obtained from A, B, and C, 11 different donors).


Degranulation of human NK after incubation with plate - coated whole . ( E ) Comparison of CD107a expression after incubation with plate - coated DcR1 , osteoprotegerin ( OPG ) or human IgG using flow cytometry ( n = 9 different donors ) . Box plots showing relative frequency of CD107a + NK ( y - axis ) after incubation with plate - coated of different concentrations ( x - axis ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Spearman rank . E ) Each data point represents mean of 2 technical replicates . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after incubation with plate-coated whole proteins. (E) Comparison of CD107a expression after incubation with plate-coated DcR1 protein, osteoprotegerin (OPG) or human IgG using flow cytometry (n = 9 different donors). Box plots showing relative frequency of CD107a+ NK cells (y-axis) after incubation with plate-coated proteins of different concentrations (x-axis). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. E) Each data point represents the mean of 2 technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.