GFP - ORAI1 expressing HEK293 were transfected with mCherry - STIM1 ( WT or L374P ) left unstimulated or stimulated with 1 μM TG in Ca2 + - free Ringer ' s solution . ( D , E ) Representative TIRF microscopy from one of two repeat experiments . Scale bars , 10 μm .

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GFP-ORAI1 expressing HEK293 cells were transfected with mCherry-STIM1 (WT or L374P) and left unstimulated or stimulated with 1 μM TG in Ca2+-free Ringer's solution. (D,E) Representative TIRF microscopy images from one of two repeat experiments. Scale bars, 10 μm.


GFP - ORAI1 expressing HEK293 were transfected with mCherry - STIM1 ( WT or L374P ) left unstimulated or stimulated with 1 μM TG in Ca2 + - free Ringer ' s solution . ( F ) Percentages of WT or mutant mCherry - STIM1 present at plasma ( i . e . visible in TIRFM evanescent field ) before TG stimulation . Data are mean ± calculated from 550 ( WT ) 830 ( L374P ) from two independent experiments . ( G ) Change of mCherry - STIM1 ( WT or L374P mutant ) [ F ( t ) / F ( 0 ) ] in TIRFM evanescence field . normalized to values at time of TG addition in Ca2 + - free Ringer ' s buffer at 0 sec . Traces show mean ± of 40 - 50 from two independent experiments . ( H ) Colocalization of mCherry - STIM1 ( WT or L374P mutant ) GFP - ORAI1 from time of TG addition at 0 sec . Colocalization measured using Pearson ' s coefficient for GFP mCherry .

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GFP-ORAI1 expressing HEK293 cells were transfected with mCherry-STIM1 (WT or L374P) and left unstimulated or stimulated with 1 μM TG in Ca2+-free Ringer's solution. (F) Percentages of WT or mutant mCherry-STIM1 present at the plasma membrane (i.e. visible in the TIRFM evanescent field) before TG stimulation. Data are the mean ± SEM calculated from 550 (WT) and 830 (L374P) cells from two independent experiments. (G) Change of mCherry-STIM1 (WT or L374P mutant) fluorescence [F(t)/F(0)] in the TIRFM evanescence field. Fluorescence normalized to values at the time of TG addition in Ca2+-free Ringer's buffer at 0 sec. Traces show mean ± SEM of 40-50 cells from two independent experiments. (H) Colocalization of mCherry-STIM1 (WT or L374P mutant) and GFP-ORAI1 from the time of TG addition at 0 sec. Colocalization measured using the Pearson's coefficient for GFP and mCherry.


( A ) of CD4 + T from P1 ( red ) , P2 ( blue ) , mother ( grey ) HD ( black ) stimulated with anti - CD3 ( 5 μg / ml ) anti - CD28 ( 10 μg / ml ) in presence or absence of 1 μM FK506 for 24 hours . ( A ) Representative histograms of FSC ( left panel ) percentages of T blasts ( defined as to right of dotted vertical ) analyzed by flow cytometry ( right panel ) .

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Cell size (A) of CD4+ T cells from P1 (red), P2 (blue), the mother (grey) and HD (black) stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 hours. (A) Representative histograms of FSC (left panel) and percentages of T cell blasts (defined as cells to the right of the dotted vertical line) analyzed by flow cytometry (right panel).


proliferation ( B ) of CD4 + T from P1 ( red ) , P2 ( blue ) , mother ( grey ) HD ( black ) stimulated with anti - CD3 ( 5 μg / ml ) anti - CD28 ( 10 μg / ml ) in presence or absence of 1 μM FK506 for 24 hours . ( B ) Representative histograms of CFSE dilution ( left panel ) percentages of proliferating ( defined as to left of dotted vertical ) ( right panel ) . Bar graphs in A B are mean ± from two independent experiments .

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proliferation (B) of CD4+ T cells from P1 (red), P2 (blue), the mother (grey) and HD (black) stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 hours. (B) Representative histograms of CFSE dilution (left panel) and percentages of proliferating cells (defined as cells to the left of the dotted vertical line) (right panel). Bar graphs in A and B are the mean ± SEM from two independent experiments.


( C , D ) Cytokine production by PBMC from P1 , P2 , mother an unrelated HD after stimulation with PMA ( 40 ng / ml ) ionomycin ( 500 ng / ml ) for 4 hours . Cytokines were analyzed by flow cytometry following surface with antibodies against CD3 , CD4 CD45RO , permeabilization intracellular cytokine for GM - CSF , IL - 22 IL - 17A . Representative flow cytometry plots ( C ) of Th17 ( GM - CSF , IL - 22 , IL - 17A ) , Th1 ( TNF - α , IFN - γ ) Th2 ( IL - 4 ) cytokines ( D ) . Data represent mean ± from two independent experiments .

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(C,D) Cytokine production by PBMC from P1, P2, the mother and an unrelated HD after stimulation with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 hours. Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3, CD4 and CD45RO, permeabilization and intracellular cytokine staining for GM-CSF, IL-22 and IL-17A. Representative flow cytometry plots (C) and quantification of Th17 (GM-CSF, IL-22, IL-17A), Th1 (TNF-α, IFN-γ) and Th2 (IL-4) cytokines (D). Data represent the mean ± SEM from two independent experiments.


A ) Severe onychomycosis in P2 ( STIM1 p . L374P ) .

A) Severe onychomycosis in P2 (STIM1 p.L374P).


B ) PBMC of a healthy donor ( HD1 ) cultured for 6 days in presence of 6 . 5 mg / ml C . albicans . At day 6 , PBMC were restimulated with PMA ionomycin in presence of 10 μM GSK - 7975A or DMSO for 6 h CD4 + CD8 + T were analyzed for cytokine production by flow cytometry . Bar graphs represent mean ± from two independent experiments .

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B) PBMC of a healthy donor (HD1) cultured for 6 days in the presence of 6.5 mg/ml C. albicans protein. At day 6, PBMC were restimulated with PMA and ionomycin in the presence of 10 μM GSK-7975A or DMSO for 6 h and CD4+ and CD8+ T cells were analyzed for cytokine production by flow cytometry. Bar graphs represent the mean ± SEM from two independent experiments.


Human T from two healthy donors , P1 his mother were expanded cultured in presence of C . albicans IL - 2 for 2 - 4 . ( C ) T were restimulated with PMA ionomycin for 6 h in presence or absence of 10 μM GSK - 7975A analyzed for cytokine production by flow cytometry . Bar graphs represent mean ± from two independent experiments .

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Human T cells from two healthy donors, P1 and his mother were expanded and cultured in the presence of C. albicans and IL-2 for 2-4 weeks. (C) T cells were restimulated with PMA and ionomycin for 6 h in the presence or absence of 10 μM GSK-7975A and analyzed for cytokine production by flow cytometry. Bar graphs represent the mean ± SEM from two independent experiments.


Human T from two healthy donors , P1 his mother were expanded cultured in presence of C . albicans IL - 2 for 2 - 4 . ( D ) Fura - 2 loaded T were stimulated with thapsigargin ( TG ) in Ca2 + - free buffer followed by readdition of 2 mM Ca2 + to induce SOCE . Bar graphs represent mean ± from two independent experiments .

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Human T cells from two healthy donors, P1 and his mother were expanded and cultured in the presence of C. albicans and IL-2 for 2-4 weeks. (D) Fura-2 loaded T cells were stimulated with thapsigargin (TG) in Ca2+-free buffer followed by readdition of 2 mM Ca2+ to induce SOCE. Bar graphs represent the mean ± SEM from two independent experiments.


Sublingual infection of WT , Stim1fl / flVav - iCre Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( A ) ( BW ) measured for 7 days normalized to BW at day 0 of infection . Data are mean ± measured from 4 repeat experiments 13 WT , 7 Stim1fl / flVav - iCre , 8 Stim1fl / flStim2fl / flVav - iCre mice .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (A) Body weight (BW) measured for 7 days and normalized to BW at day 0 of infection. Data are mean ± SEM measured from 4 repeat experiments and 13 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.


Sublingual infection of WT , Stim1fl / flVav - iCre Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( B ) of CFU of C . albicans per gram tongue tissue of indicated mice at day 7 p . i . Data are mean ± from 4 repeat experiments 12 WT , 7 Stim1fl / flVav - iCre , 8 Stim1fl / flStim2fl / flVav - iCre mice .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (B) Quantification of CFU of C. albicans per gram tongue tissue of the indicated mice at day 7 p.i. Data are mean ± SEM from 4 repeat experiments and 12 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.


Sublingual infection of WT , Stim1fl / flVav - iCre Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( C ) Macroscopic of C . albicans - infected tongues of mice at day 7 p . i . Arrows dashes lines indicate C . albicans infiltrated areas .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (C) Macroscopic images of the C. albicans-infected tongues of mice at day 7 p.i. Arrows and dashes lines indicate C. albicans infiltrated areas.


Sublingual infection of WT , Stim1fl / flVav - iCre Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( D ) Histological of H & E ( left ) PAS ( right ) stained tongue tissue at day 7 p . i . Clusters of polymorphonuclear ( PMN ) ( neutrophils , arrows ) Candida ( black arrows ) are indicated .

Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (D) Histological images of H&E (left) and PAS (right) stained tongue tissue at day 7 p.i. Clusters of polymorphonuclear (PMN) cells (neutrophils, white arrows) and Candida (black arrows) are indicated.


Sublingual infection of WT , Stim1fl / flVav - iCre Stim1fl / flStim2fl / flVav - iCre mice with 2x107 CFU of C . albicans . ( E ) Frequencies of Cd11b + Gr1 + neutrophils in tongues from indicated mice at day 7 p . i . Bar graphs represent mean ± from 3 experiments 9 WT , 7 Stim1fl / flVav - iCre , 8 Stim1fl / flStim2fl / flVav - iCre mice .

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Sublingual infection of WT, Stim1fl/flVav-iCre and Stim1fl/flStim2fl/flVav-iCre mice with 2x107 CFU of C. albicans. (E) Frequencies of Cd11b+Gr1+ neutrophils in the tongues from the indicated mice at day 7 p.i. Bar graphs represent the mean ± SEM from 3 experiments and 9 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.


( F , G ) Cytokine production by CD4 + T isolated from submandibular lymph nodes of WT , Stim1fl / flVav - iCre , Stim1fl / flStim2fl / flVav - iCre Stim1fl / flCd4Cre mice 7 days after sublingual infection with 2x107 CFU C . albicans . Representative contour plots of cytokine production ( F ) percentages of CD4 + T producing TNF - α , IFN - γ , IL - 17A , IL - 17F GM - CSF ( G ) . Data are mean ± from two independent experiments 9 WT , 7 Stim1fl / flVav - iCre , 8 Stim1fl / flStim2fl / flVav - iCre mice .

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(F,G) Cytokine production by CD4+ T cells isolated from submandibular lymph nodes of WT, Stim1fl/flVav-iCre, Stim1fl/flStim2fl/flVav-iCre and Stim1fl/flCd4Cre mice 7 days after sublingual infection with 2x107 CFU C. albicans. Representative contour plots of cytokine production (F) and percentages of CD4+ T cells producing TNF-α, IFN-γ, IL-17A, IL-17F and GM-CSF (G). Data are the mean ± SEM from two independent experiments and 9 WT, 7 Stim1fl/flVav-iCre, 8 Stim1fl/flStim2fl/flVav-iCre mice.


Candicidal function of neutrophils depend on SOCE . ( H ) Coculture of Cd11b + Gr - 1 + neutrophils from WT , Stim1fl / flMx1Cre Stim1fl / flStim2fl / flMx1Cre mice with C . albicans in vitro for 0 , 30 , 60 90 min ( MOI 0 . 5 ) . Percentages of live C . albicans isolated from neutrophils that were lysed at indicated time points . Data are mean ± from 6 mice per genotype 3 independent repeat experiments .

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Candicidal function of neutrophils depend on SOCE. (H) Coculture of Cd11b+Gr-1+ neutrophils from WT, Stim1fl/flMx1Cre and Stim1fl/flStim2fl/flMx1Cre mice with C.albicans in vitro for 0, 30, 60 and 90 min (MOI 0.5). Percentages of live C.albicans isolated from neutrophils that were lysed at the indicated time points. Data are the mean ± SEM from 6 mice per genotype and 3 independent repeat experiments.


ROS production of neutrophils depend on SOCE . ( I ) ROS production by Cd11b + Gr - 1 + neutrophils from WT , Stim1fl / flMx1Cre Stim1fl / flStim2fl / flMx1Cre mice was measured after loading of with dihydrorhodamine 123 stimulation with 10 nM PMA for 30 minutes . Representative histogram plots bar graphs indicating percentages of ROS + neutrophils normalized to WT neutrophils stimulated with PMA .

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ROS production of neutrophils depend on SOCE. (I) ROS production by Cd11b+Gr-1+ neutrophils from WT, Stim1fl/flMx1Cre and Stim1fl/flStim2fl/flMx1Cre mice was measured after loading of cells with dihydrorhodamine 123 and stimulation with 10 nM PMA for 30 minutes. Representative histogram plots and bar graphs indicating the percentages of ROS+ neutrophils normalized to WT neutrophils stimulated with PMA.


Sublingual C . albicans infection of WT Stim1fl / flCd4Cre mice as described in Figure 5 . ( A ) ( BW ) change measured for 7 days p . i . relative to BW at day of infection . Data are mean ± measured from 4 repeat experiments 13 WT 7 Stim1fl / flCd4Cre mice .

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Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (A) Body weight (BW) change measured for 7 days p.i. relative to BW at the day of infection. Data are mean ± SEM measured from 4 repeat experiments and 13 WT and 7 Stim1fl/flCd4Cre mice.


Sublingual C . albicans infection of WT Stim1fl / flCd4Cre mice as described in Figure 5 . ( B ) of CFU of C . albicans isolated from tongues of mice at day 7 post - infection ( p . i . ) . Data are mean ± from 4 repeat experiments 7 - 13 mice per genotype .

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Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (B) Quantification of CFU of C. albicans isolated from the tongues of mice at day 7 post-infection (p.i.). Data are mean ± SEM from 4 repeat experiments and 7-13 mice per genotype.


Sublingual C . albicans infection of WT Stim1fl / flCd4Cre mice as described in Figure 5 . ( C ) Macroscopic of C . albicans - infected tongues of mice at day 7 p . i .

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Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (C) Macroscopic images of the C. albicans-infected tongues of mice at day 7 p.i.


Sublingual C . albicans infection of WT Stim1fl / flCd4Cre mice as described in Figure 5 . ( D ) Histological of H & E ( left ) PAS - stained ( right ) tongue tissue at day 7 p . i . Scale bars 100 μm . Arrows point to clusters of polymorphonuclear ( PMN ) ( neutrophil ) infiltrates .

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Sublingual C. albicans infection of WT and Stim1fl/flCd4Cre mice as described in Figure 5. (D) Histological images of H&E (left) and PAS-stained (right) tongue tissue at day 7 p.i. Scale bars 100 μm. Arrows point to clusters of polymorphonuclear (PMN) cell (neutrophil) infiltrates.


Systemic C . albicans infection . WT Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( E ) Kaplan - Meier survival plot showing survival of mice . Data from 5 Stim1fl / fl Cd4Cre mice 12 WT mice is shown .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (E) Kaplan-Meier survival plot showing survival of mice. Data from 5 Stim1fl/fl Cd4Cre mice and 12 WT mice is shown.


Systemic C . albicans infection . WT Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( F ) ( BW ) of mice normalized to BW at day of infection . Data are mean ± from 4 independent experiments 8 WT 7 Stim1fl / flCd4Cre mice .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (F) Body weight (BW) of mice normalized to BW at the day of infection. Data are the mean ± SEM from 4 independent experiments and 8 WT and 7 Stim1fl/flCd4Cre mice.


Systemic C . albicans infection . WT Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( G ) Representative macroscopic of kidneys at day 6 p . i .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (G) Representative macroscopic images of kidneys at day 6 p.i.


Systemic C . albicans infection . WT Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( H ) CFUs of C . albicans per gram tissue isolated from liver , right kidney lung of mice at day 6 p . i . Each dot represents one mouse .

Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (H) CFUs of C. albicans per gram tissue isolated from the liver, the right kidney and the lung of mice at day 6 p.i. Each dot represents one mouse.


Systemic C . albicans infection . WT Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( I ) Representative histological of H & E PAS stained kidneys of mice at day 6 p . i . Magnification 6 . 4x ( left ) 40x ( right ) . arrows define areas infiltrated with leukocytes ( H & E ) C . albicans ( PAS ) .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (I) Representative histological images of H&E and PAS stained kidneys of mice at day 6 p.i. Magnification 6.4x (left) and 40x (right). White arrows define areas infiltrated with leukocytes (H&E) and C. albicans (PAS).


Systemic C . albicans infection . WT Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( J ) Frequencies of CD4 + T isolated from spleens of mice at day 6 p . i . producing indicated cytokines after restimulation with 20 nM PMA 1 μM ionomycin for 6 hours analyzed by flow cytometry .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (J) Frequencies of CD4+ T cells isolated from the spleens of mice at day 6 p.i. producing the indicated cytokines after restimulation with 20 nM PMA and 1 μM ionomycin for 6 hours and analyzed by flow cytometry.


Systemic C . albicans infection . WT Stim1fl / fl Cd4Cre mice were injected i . v . with 2x105 CFU C . albicans or PBS as control . ( K ) Frequencies CD45 + Cd11b + Gr - 1 + neutrophils in blood of mice at day 6 post - infection analyzed by flow cytometry . Data in J are mean ± from 2 independent experiments 6 WT 4 Stim1fl / flCd4Cre mice ; data in K are mean ± from one experiment 2 WT 3 Stim1fl / flCd4Cre mice .

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Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (K) Frequencies CD45+Cd11b+Gr-1+ neutrophils in the blood of mice at day 6 post-infection analyzed by flow cytometry. Data in J are the mean ± SEM from 2 independent experiments and 6 WT and 4 Stim1fl/flCd4Cre mice; data in K are the mean ± SEM from one experiment and 2 WT and 3 Stim1fl/flCd4Cre mice.


- seq of CD4 + T isolated from 3 WT 3 Stim1fl / flCd4Cre mice that were differentiated into pathogenic Th17 ( pTh17 : IL - 1β , IL - 6 , IL - 23 ) non - pathogenic Th17 ( npTh17 : IL - 6 , TGFβ1 ) . ( B ) Volcano plots of DEGs in npTh17 ( gray dots ) . Highlighted in red ( upregulated in Stim1fl / flCd4Cre ) blue ( downregulated in Stim1fl / flCd4Cre ) are DEGs that belong to a gene expression signature of npTh17 pTh17 defined previously ( Gaublomme et al . , 2015 , Lee et al . , 2012 ) .

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RNA-seq analysis of CD4+ T cells isolated from 3 WT and 3 Stim1fl/flCd4Cre mice that were differentiated into pathogenic Th17 cells (pTh17: IL-1β, IL-6, IL-23) and non-pathogenic Th17 cells (npTh17: IL-6, TGFβ1). (B) Volcano plots of DEGs in npTh17 cells (gray dots). Highlighted in red (upregulated in Stim1fl/flCd4Cre) and blue (downregulated in Stim1fl/flCd4Cre) are DEGs that belong to a gene expression signature of npTh17 cells and pTh17 cells defined previously (Gaublomme et al., 2015, Lee et al., 2012).


( C ) Normalized expression of Th17 - associated cytokines receptors in npTh17 derived from WT Stim1fl / flCd4Cre mice . Bar graphs represent mean ± from 3 mice per group . Statistical by unpaired Student ' s t - with following significance : * p < 0 . 05 , * * * p < 0 . 001 .

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(C) Normalized expression of Th17 cell-associated cytokines and receptors in npTh17 cells derived from WT and Stim1fl/flCd4Cre mice. Bar graphs represent the mean ± SEM from 3 mice per group. Statistical analysis by unpaired Student's t-test with the following significance levels: *p<0.05, ***p<0.001.


( F ) Heat map of DEGs ( p ( adj . ) < 0 . 1 ) within KEGG pathways " Foxo signaling " " HIF - 1 signaling " that are specifically deregulated in npTh17 . Numerical values are relative gene expression .

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(F) Heat map of DEGs (p (adj.) < 0.1) within the KEGG pathways "Foxo signaling" and "HIF-1 signaling" that are specifically deregulated in npTh17 cells. Numerical values are relative gene expression.


( G ) Gene set enrichment ( GSEA ) of DEGs in WT STIM1 - deficient npTh17 . Normalized enrichment score ( NES ) p - values are as indicated for following genesets : Myc targets : " Menssen _ myc _ targets " ; Glycolysis : " Humancyc _ mm _ glycolysis _ i " ; Mitochondrial function : " Wong _ mitochondria _ gene _ module " ; TCA cycle : " Kegg _ mm _ citrate _ cycle " .

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(G) Gene set enrichment analysis (GSEA) of DEGs in WT and STIM1-deficient npTh17 cells. Normalized enrichment score (NES) and p-values are as indicated for the following genesets: Myc targets: "Menssen_myc_targets"; Glycolysis: "Humancyc_mm_glycolysis_i"; Mitochondrial function: "Wong_mitochondria_gene_module"; TCA cycle: "Kegg_mm_citrate_cycle".


( A ) Heat map of metabolism - associated DEGs ( p ( adj . ) < 0 . 10 ) in npTh17 of WT Stim1fl / flCd4Cre mice determined by - Seq as described in Figure 7 . shows relative minimum maximum values per gene ( row min / max ) . Numerical values are relative gene expression . TCA , tricarboxylic acid .

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(A) Heat map of metabolism-associated DEGs (p (adj.) < 0.10) in npTh17 cells of WT and Stim1fl/flCd4Cre mice determined by RNA-Seq as described in Figure 7. Heatmap shows relative minimum and maximum values per gene (row min/max). Numerical values are relative gene expression. TCA, tricarboxylic acid.


Glucose mitochondrial metabolism in npTh17 from WT ( grey or black ) Stim1fl / flCd4Cre ( blue ) mice differentiated for 3 days in vitro . ( B ) Glucose uptake by npTh17 were loaded with 2 - NBDG ( + ) or not ( - ) analyzed by flow cytometry 90 min later . Bar graphs show delta MFI of 2 - NBDG normalized to unlabeled . Data represent mean ± from 3 independent experiments .

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Glucose and mitochondrial metabolism in npTh17 cells from WT (grey or black) and Stim1fl/flCd4Cre (blue)mice differentiated for 3 days in vitro. (B) Glucose uptake by npTh17 cells were loaded with 2-NBDG (+) or not (-) and analyzed by flow cytometry 90 min later. Bar graphs show the delta MFI of 2-NBDG fluorescence normalized to unlabeled cells. Data represent the mean ± SEM from 3 independent experiments.


( C ) Extracellular acidification rate ( ECAR ) of npTh17 before after addition of 25 mM glucose , 5 μM oligomycin 100 mM 2 - deoxy - D - glucose ( 2 - DG ) . Seahorse graphs bar graphs depicting glycolysis , glycolytic capacity reserve mean ± from of 2 - 3 mice per genotype from one representative out of three independent experiments .

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(C) Extracellular acidification rate (ECAR) of npTh17 cells before and after addition of 25 mM glucose, 5 μM oligomycin and 100 mM 2-deoxy-D-glucose (2-DG). Seahorse graphs and bar graphs depicting glycolysis, glycolytic capacity and reserve the mean ± SEM from of 2-3 mice per genotype from one representative out of three independent experiments.


Glucose mitochondrial metabolism in npTh17 from WT ( grey or black ) Stim1fl / flCd4Cre ( blue ) mice differentiated for 3 days in vitro . ( D ) Oxygen consumption rate ( OCR ) of npTh17 before after addition of 1 μM oligomycin , 1 . 5 μM FCCP 100 nM rotenone / 1 μM antimycin . Seahorse graphs bar graphs depicting basal maximal respiration , coupling efficiency ATP production represent mean ± from of 2 - 3 mice per genotype from one representative out of three independent experiments .

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Glucose and mitochondrial metabolism in npTh17 cells from WT (grey or black) and Stim1fl/flCd4Cre (blue)mice differentiated for 3 days in vitro. (D) Oxygen consumption rate (OCR) of npTh17 cells before and after addition of 1 μM oligomycin, 1.5 μM FCCP and 100 nM rotenone / 1 μM antimycin. Seahorse graphs and bar graphs depicting basal and maximal respiration, coupling efficiency and ATP production represent the mean ± SEM from of 2-3 mice per genotype from one representative out of three independent experiments.


Glucose mitochondrial metabolism in human CD4 + T isolated from PBMC of a HD P1 stimulated with 5 μg / ml anti - CD3 10 μg / ml anti - CD28 for 24 hours before . Some HD T were stimulated in presence of 1μM FK506 . ( E , F ) ECAR before after addition of 25 mM glucose , 5 μM oligomycin 100 mM 2 - DG .

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Glucose and mitochondrial metabolism in human CD4+ T cells isolated from PBMC of a HD and P1 and stimulated with 5 μg/ml anti-CD3 and 10 μg/ml anti-CD28 for 24 hours before analysis. Some HD T cells were stimulated in the presence of 1μM FK506. (E,F) ECAR before and after addition of 25 mM glucose, 5 μM oligomycin and 100 mM 2-DG.


Glucose mitochondrial metabolism in human CD4 + T isolated from PBMC of a HD P1 stimulated with 5 μg / ml anti - CD3 10 μg / ml anti - CD28 for 24 hours before . Some HD T were stimulated in presence of 1μM FK506 . ( G , H ) OCR before after addition of 1 μM oligomycin , 1 . 5 μM FCCP 100 nM rotenone / 1 μM antimycin . Seahorse graphs ( G ) bar graphs ( H ) depicting basal maximal respiration , SRC ATP production ( right ) represent mean ± of 2 technical replicates from isolated T from one experiment .

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Glucose and mitochondrial metabolism in human CD4+ T cells isolated from PBMC of a HD and P1 and stimulated with 5 μg/ml anti-CD3 and 10 μg/ml anti-CD28 for 24 hours before analysis. Some HD T cells were stimulated in the presence of 1μM FK506. (G,H) OCR before and after addition of 1 μM oligomycin, 1.5 μM FCCP and 100 nM rotenone / 1 μM antimycin. Seahorse graphs (G) and bar graphs (H) depicting basal and maximal respiration, SRC and ATP production (right) represent the mean ± SEM of 2 technical replicates from isolated T cells from one experiment.


A . Immunoblotting of c - MYC ATF4 in melanoma treated with NT - siRNA ( - ) or siASNS ( # 1 or # 2 ) for 72 hr .

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A. Immunoblotting of c-MYC and ATF4 in melanoma cells treated with NT-siRNA (-) or siASNS (#1 or #2) for 72 hr.


Reverse transcription with quantitative PCR ( RT - qPCR ) of indicated transcripts in melanoma 36 hr after treatment with siASNS ( # 1 or # 2 ) ( B )

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Reverse transcription with quantitative PCR (RT-qPCR) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS (#1 or #2) (B)


C . Reverse transcription with quantitative PCR ( RT - qPCR ) of indicated transcripts in melanoma 36 hr after treatment with siASNS , siATF4 , or both ( C ) .

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C. Reverse transcription with quantitative PCR (RT-qPCR) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS, siATF4, or both (C).


D . Immunoblotting of ATF4 ASNS in melanoma 72 hr after treatment with siASNS , siATF4 , or both .

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D. Immunoblotting of ATF4 and ASNS in melanoma cells 72 hr after treatment with siASNS, siATF4, or both.


E . Proliferation of melanoma over indicated times after treatment with siASNS , siATF4 , or both .

E. Proliferation of melanoma cells over indicated times after treatment with siASNS, siATF4, or both.


F . RT - qPCR of indicated transcripts in melanoma 36 hr after treatment with siASNS ( # 1 or # 2 ) .

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F. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS (#1 or #2).


Immunoblotting of c - MYC in melanoma treated for 72 hr with siASNS , L - Asn , or both ( G )

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Immunoblotting of c-MYC in melanoma cells treated for 72 hr with siASNS, L-Asn, or both (G)


H . Immunoblotting of c - MYC in A375 treated as indicated for 72 hr ( H ) .

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H. Immunoblotting of c-MYC in A375 cells treated as indicated for 72 hr (H).


A . Immunoblotting of c - MYC in A375 treated with NT - siRNA or siASNS for 24 hr followed by cycloheximide treatment for indicated timepoints ( left ) . Plot showing relative of c - MYC ( y - axis ) as a function of time lapsed ( x - axis ) in left panel ( right ) .

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A. Immunoblotting of c-MYC in A375 cells treated with NT-siRNA or siASNS for 24 hr followed by cycloheximide treatment for indicated timepoints (left). Plot showing relative protein levels of c-MYC (y-axis) as a function of time lapsed (x-axis) in the left panel (right).


Immunoblotting of indicated in melanoma 72 hr after treatment with NT - siRNA ( - ) or siASNS ( # 1 or # 2 ) ( B C )

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Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with NT-siRNA (-) or siASNS (#1 or #2) (B and C)


Immunoblotting of indicated in melanoma 72 hr after treatment with siASNS , PLX - 4032 , or both ( D ) .

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Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS, PLX-4032, or both (D).


E . Immunoblotting of indicated in A375 cultured in L - Asn treated with L - A ' ase , PLX - 4032 , or both for 72 hr .

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E. Immunoblotting of indicated proteins in A375 cells cultured in L-Asn and treated with L-A'ase, PLX-4032, or both for 72 hr.


F . A375 were treated with siASNS , SCH772984 , or both for 72 hr followed by immunoblotting of phosphorylated total GSK3 - β .

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F. A375 cells were treated with siASNS, SCH772984, or both for 72 hr followed by immunoblotting of phosphorylated and total GSK3-β.


G . A375 were treated with siASNS , HA - GSK3 - β ( S9A ) , or both for 72 hr followed by immunoblotting of indicated .

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G. A375 cells were treated with siASNS, HA-GSK3-β (S9A), or both for 72 hr followed by immunoblotting of indicated proteins.


H . RT - qPCR of indicated transcripts in melanoma 36 hr after treatment with siASNS , HA - GSK3 - β ( S9A ) , or both .

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H. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS, HA-GSK3-β (S9A), or both.


I . A375 were treated as indicated for 72 hr followed by immunoblotting of c - MYC .

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I. A375 cells were treated as indicated for 72 hr followed by immunoblotting of c-MYC.


J . Immunoblotting of c - MYC HA in A375 treated as indicated for 72 hr .

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J. Immunoblotting of c-MYC and HA proteins in A375 cells treated as indicated for 72 hr.


A . Immunoblotting of indicated in melanoma treated with siASNS , sicMYC ( # 1 or # 2 ) , or a combination of siASNS sicMYC ( # 1 or # 2 ) for 72 hr .

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A. Immunoblotting of indicated proteins in melanoma cells treated with siASNS, sicMYC (#1 or #2), or a combination of siASNS and sicMYC (#1 or #2) for 72 hr.


B . RT - qPCR of indicated transcripts in melanoma 36 hr after treatment with siASNS , sicMYC , or both .

B. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS, sicMYC, or both.


C . Immunoblotting of indicated in melanoma 72 hr after treatment with siASNS , sicMYC , PLX - 4032 , or a combination of siASNS sicMYC , or siASNS PLX - 4032 .

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C. Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS, sicMYC, PLX-4032, or a combination of siASNS and sicMYC, or siASNS and PLX-4032.


D . Immunoblotting of indicated in A375 72 hr after treatment with siASNS , HA - GSK3 - β ( S9A ) , or both .

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D. Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS, HA-GSK3-β (S9A), or both.


E . RT - qPCR of indicated transcripts in melanoma after 36 hr treatment with siASNS , HA - GSK3 - β ( S9A ) , or both .

E. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, HA-GSK3-β (S9A), or both.


F . A375 were treated as indicated for 72 hr followed by immunoblotting of indicated .

F. A375 cells were treated as indicated for 72 hr followed by immunoblotting of indicated proteins.


Proliferation of melanoma over indicated times after treatment with siASNS , sicMYC , or both ( G ) ,

Proliferation of melanoma cells over indicated times after treatment with siASNS, sicMYC, or both (G),


Proliferation of melanoma over indicated times after treatment with siASNS , HA - GSK3 - β ( S9A ) , or both ( H ) .

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Proliferation of melanoma cells over indicated times after treatment with siASNS, HA-GSK3-β (S9A), or both (H).


A . Immunoblotting of indicated in A375 72 hr after treatment with siASNS , siSLC1A5 , siSLC7A5 , or a combination of siASNS siSLC1A5 , or siASNS siSLC7A5 .

A. Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS, siSLC1A5, siSLC7A5, or a combination of siASNS and siSLC1A5, or siASNS and siSLC7A5.


B . RT - qPCR of indicated transcripts in melanoma after 36 hr treatment with siASNS , siSLC7A5 , or both .

B. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, siSLC7A5, or both.


C . Proliferation of melanoma over indicated times after treatment with siASNS , siSLC7A5 , or both .

C. Proliferation of melanoma cells over indicated times after treatment with siASNS, siSLC7A5, or both.


D . GC - MS - estimation of intracellular acid in A375 treated with siASNS , siATF4 , or both ( top left ) ; siASNS , sicMYC , or both ( top right ) ; siASNS , siSLC1A5 , or both ( bottom left ) ; or siASNS , siSLC7A5 , or both ( bottom right ) . fold change is relative to NT - siRNA - treated A375 .

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D. GC-MS-based estimation of intracellular amino acid levels in A375 cells treated with siASNS, siATF4, or both (top left); siASNS, sicMYC, or both (top right); siASNS, siSLC1A5, or both (bottom left); or siASNS, siSLC7A5, or both (bottom right). The fold change is relative to NT-siRNA-treated A375 cells.


Immunoblotting of indicated in A375 72 hr after indicated treatment ( E ) ,

Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment (E),


Immunoblotting of indicated in A375 72 hr after , treatment with siASNS , sicMYC , or

Immunoblotting of indicated proteins in A375 cells 72 hr after , treatment with siASNS, sicMYC, or both


Immunoblotting of indicated in A375 72 hr after indicated treatment with , siASNS , siSLC7A5 , or both ( G )

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Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS, siSLC7A5, or both (G)


Immunoblotting of indicated in A375 72 hr after indicated treatment with siASNS , BCH , or both ( H )

Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, BCH, or both (H)


Immunoblotting of indicated in A375 72 hr after indicated treatment with siASNS , HA - GSK3 - β ( S9A ) , or both ( I )

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Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, HA-GSK3-β (S9A), or both (I)


Immunoblotting of indicated in A375 72 hr after indicated treatment with , siASNS or indicated combinations ( J )

Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS or indicated combinations (J)


Immunoblotting of indicated in A375 72 hr after indicated treatment with siASNS , siSLC3A2 , or both ( K ) .

Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, siSLC3A2, or both (K).


L . RT - qPCR of indicated transcripts in melanoma after 36 hr treatment with siASNS , siSLC3A2 , or both .

L. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, siSLC3A2, or both.


M . Proliferation of melanoma over indicated times after treatment with siASNS , siSLC3A2 , or both .

M. Proliferation of melanoma cells over indicated times after treatment with siASNS, siSLC3A2, or both.


( A ) CRISPR - guided AID ∆ C could support CSR in Aicda - / - CH12F3 . Left , CRISPR - guided AID is schematically illustrated with each component listed . Right , CRISPR - guided AID initiated CSR to IgA . Blue arrows represent multiple CRISPR recognition sites . number in parentheses indicates CRISPR recognition sites in each S region . Three biological replicates are plotted by dots mean with standard deviation ( SD ) . Data information : For panels A one - way ANOVA followed by Dunnett ' s multiple comparisons was used for significance assessment . For panels , * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 ; ns , p > 0 . 05 .

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(A) CRISPR-guided AID∆C protein could support CSR in Aicda-/- CH12F3 cells. Left, CRISPR-guided AID is schematically illustrated with each component listed. Right, CRISPR-guided AID initiated CSR levels to IgA. Blue arrows represent multiple CRISPR recognition sites. The number in the parentheses indicates CRISPR recognition sites in each S region. Three biological replicates are plotted by dots and mean with standard deviation (SD). Data information: For panels A one-way ANOVA followed by Dunnett's multiple comparisons test was used for significance assessment. For all panels, ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, p>0.05.


( B ) CRISPR - guided AID generated breaks at S region in Aicda - / - CH12F3 were quantitatively detected by PEM - seq . Top , schematic illustration shows bait SaCas9 site at Iγ3 region CRISPR - guided AID variants targeting sites at S regions . Bottom , percentages of translocation junctions between Iγ3 Sμ or Sα regions are plotted as mean with SD of three biological replicates in bar plot . Data information : For panels B , one - way ANOVA followed by Dunnett ' s multiple comparisons was used for significance assessment . For panels , * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 ; ns , p > 0 . 05 .

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(B) CRISPR-guided AID generated breaks at the S region in Aicda-/- CH12F3 cells were quantitatively detected by PEM-seq. Top, schematic illustration shows the bait SaCas9 site at Iγ3 region and CRISPR-guided AID variants targeting sites at S regions. Bottom, percentages of translocation junctions between Iγ3 and Sμ or Sα regions are plotted as mean with SD of three biological replicates in the bar plot. Data information: For panels B, one-way ANOVA followed by Dunnett's multiple comparisons test was used for significance assessment. For all panels, ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, p>0.05.


( C ) AID - AIDE58Q dimer were expressed in AID - deficient CSR - activated B , AID dimer is schematically illustrated on top CSR to IgG1 are shown . Three biological replicates are plotted by dots mean with SD is shown . Data information : For panels C , one - way ANOVA followed by Dunnett ' s multiple comparisons was used for significance assessment . For panels , * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 ; ns , p > 0 . 05 .

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

(C) AID-AIDE58Q dimer proteins were expressed in AID-deficient CSR-activated B cells, AID dimer is schematically illustrated on top and CSR levels to IgG1 are shown. Three biological replicates are plotted by dots and mean with SD is shown. Data information: For panels C, one-way ANOVA followed by Dunnett's multiple comparisons test was used for significance assessment. For all panels, ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, p>0.05.


( D ) CSR to IgA in Aicda - / - CH12F3 - miniS with indicated AID variants or CRISPR - guided AID variants . of CSR to IgA are plotted mean with SD of three to six biological replicates . Data information : Unpaired two - Student ' s t - was performed for panel D . For panels , * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 ; ns , p > 0 . 05 .

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(D) CSR levels to IgA in Aicda-/- CH12F3-miniS cells with indicated AID variants or CRISPR-guided AID variants. Levels of CSR to IgA are plotted and mean with SD of three to six biological replicates. Data information: Unpaired two-tailed Student's t-test was performed for panel D. For all panels, ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, p>0.05.


( E ) Mutation profiles of miniS region in Ung - / - Msh2 - / - Aicda - / - miniS lines complemented with indicated AID . Mutation frequency at each nucleotide along 756 - bp miniS region is plotted as a bar graph with green error bars , representing mean with standard error of mean ( ) of three biological replicates . Positions of AID - preferred AGCT other RGYW motifs are marked with orange yellow respectively under each plot .

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(E) Mutation profiles of miniS region in Ung-/-Msh2-/-Aicda-/- miniS cell lines complemented with indicated AID. Mutation frequency at each nucleotide along the 756-bp miniS region is plotted as a bar graph with green error bars, representing mean with standard error of the mean (SEM) of three biological replicates. Positions of AID-preferred AGCT and other RGYW motifs are marked with orange and yellow respectively under each plot.


( F ) Mutation profiles of miniS region in Ung - / - Msh2 - / - Aicda - / - miniS lines expressing CRISPR - guided AID variants . Mutation frequency at each nucleotide is plotted as mean with of three biological replicates in bar graph with green error bar . Plots are labeled as in ( B ) . sgRNA targeting position is indicated with blue lines at bottom .

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(F) Mutation profiles of miniS region in Ung-/-Msh2-/-Aicda-/- miniS cell lines expressing CRISPR-guided AID variants. Mutation frequency at each nucleotide is plotted as mean with SEM of three biological replicates in the bar graph with green error bar. Plots are labeled as in (B). The sgRNA targeting position is indicated with blue lines at the bottom.


( A ) Representative showing localization of AID ( green ) , AIDR190X ( green ) , endogenous IgH loci ( red ) in nucleofected Aicda - / - CH12F3 . IgH loci are visualized using Sμ - gRNA indicated by arrows . An illustration of CRISPR - Sirius IgH locus visualization is shown on top . Scale bar , 5 µm .

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

(A) Representative images showing the localization of AID (green), AIDR190X (green), and endogenous IgH loci (red) in nucleofected Aicda-/- CH12F3 cells. The IgH loci are visualized using Sμ-gRNA and indicated by white arrows. An illustration of CRISPR-Sirius IgH locus visualization is shown on top. Scale bar, 5 µm.


( B ) OptoDroplet formation in CH12F3 . Schematic illustration of - induced optoDroplet system is depicted on top . AID variants were tagged to N terminus of optoDroplet construct blue was employed to promote formation of three times followed by imaging each time . Representative after third blue activation in Aicda - / - CH12F3 with indicated AID variants are shown at bottom . dashed green depict shapes of nucleus nucleolus on Hoechst or GFP - FBL , respectively . Scale bar , 5 µm .

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 B-MEASURED_VAR 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) OptoDroplet formation in CH12F3 cells. Schematic illustration of light-induced optoDroplet system is depicted on top. AID variants were tagged to the N terminus of the optoDroplet construct and blue light was employed to promote the formation of puncta three times followed by imaging each time. Representative images after the third blue light activation in Aicda-/- CH12F3 cells with the indicated AID variants are shown at the bottom. White dashed line and green line depict the shapes of nucleus and nucleolus based on Hoechst staining or GFP-FBL, respectively. Scale bar, 5 µm.


( C ) Representative showing optoDroplet formation of indicated AID variants in HEK293T . CRM1 inhibitor ( CRM1i ) was used for selective trapping of AID in nuclei . Scale bar , 5 µm .

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(C) Representative images showing optoDroplet formation of indicated AID variants in HEK293T cells. CRM1 inhibitor (CRM1i) was used for selective trapping of AID in nuclei. Scale bar, 5 µm.


( D ) of indicated AID variants in each HEK293T nucleus was measured normalized relative to of whole . Data are shown as box plots . Values between lower quartile upper quartile are represented by box ranges , a horizontal within box represents median , whisker extends from minimum value to maximum value . " + " indicates mean level . Each dot indicates one , more than 20 represent two biological replicate samples were counted . One - way ANOVA followed by Dunnett ' s multiple comparisons was performed . * * * * , p < 0 . 0001 ; * , p < 0 . 05 ; ns , p > 0 . 05 .

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(D) The fluorescence intensity of indicated AID variants in each HEK293T cell nucleus was measured and normalized relative to the fluorescence intensity of the whole cell. Data are shown as box plots. Values between lower quartile and upper quartile are represented by box ranges, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. "+" indicates the mean level. Each dot indicates one cell, and more than 20 cells represent two biological replicate samples were counted. One-way ANOVA followed by Dunnett's multiple comparisons test was performed. ****, p<0.0001; *, p<0.05; ns, p>0.05.


( E ) recovery lines for optoAID ∆ C droplets in FRAP experiment in HEK293T . Bleaching occurs at t = 0 s . background - subtracted intensities are normalized by pre - bleach values . mean with SD of 10 pairs of optoAIDR190X 10 pairs of optoAIDcry is shown , respectively .

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(E) Intensity recovery lines for optoAID∆C droplets in FRAP experiment in HEK293T cells. Bleaching occurs at t = 0 s. The background-subtracted fluorescence intensities are normalized by pre-bleach values. The mean with SD of 10 pairs of optoAIDR190X and 10 pairs of optoAIDcry is shown, respectively.


( F ) Formation of in B complemented with AID - FUS - GFP or AID - FUSm - GFP . FUSm contains 13 mutations on either Y or R residue . Left : Representative showing localization of AID - FUS - GFP AID - FUSm - GFP with without CRM1 inhibitor ( CRM1i ) treatment in Aicda - / - B . Scale bar , 5 µm . Right : of number of with condensed ( 3 biological replicates ) . Unpaired two - Student ' s t - was performed . * * * * , p < 0 . 0001 .

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(F) Formation of puncta in primary B cells complemented with AID-FUS-GFP or AID-FUSm-GFP. FUSm contains 13 mutations on either Y or R residue. Left: Representative images showing cellular localization of AID-FUS-GFP and AID-FUSm-GFP with and without CRM1 inhibitor (CRM1i) treatment in Aicda-/- primary B cells. Scale bar, 5 µm. Right: Quantification of the number of cells with condensed puncta (3 biological replicates). Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.


( G ) CSR to IgG1 in activated Aicda - / - B complemented with indicated . Left , representative flow cytometry plots measuring CSR to IgG1 . Co - expressed GFP was used to indicate infected fractions . Right , mean with SD of three biological replicates is shown in bar plot . Unpaired two - Student ' s t - was performed . * * * * , p < 0 . 0001 .

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(G) CSR levels to IgG1 in activated Aicda-/- primary B cells complemented with the indicated protein. Left, representative flow cytometry plots measuring CSR to IgG1. Co-expressed GFP was used to indicate the infected fractions. Right, the mean with SD of three biological replicates is shown in the bar plot. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.


( A ) Representative immunofluorescence of endogenous AID in presence of ectopically - expressed EV , AID , AIDR190X , or AIDcry in cytokine - stimulated CH12F3 . EV , empty vector control . An antibody against AID C terminus ( 185 - 198 aa ) detected both endogenous ectopically - expressed full - AID . nucleolus is indicated by co - nucleofected mCherry - FBL . dashed depicts shape of nucleus on Hoechst . Arrows indicate location of formed by endogenous AID . were taken by N - SIM microscope processed via N - SIM software . A middle slice of was shown . Scale bar , 5 µm .

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

(A) Representative immunofluorescence images of endogenous AID in the presence of ectopically-expressed EV, AID, AIDR190X, or AIDcry in cytokine-stimulated CH12F3 cells. EV, empty vector control. An antibody against AID C terminus (185-198 aa) detected both endogenous and ectopically-expressed full-length AID. The nucleolus is indicated by co-nucleofected mCherry-FBL. The white dashed line depicts the shape of the nucleus based on Hoechst staining. Arrows indicate the location of puncta formed by endogenous AID. The images were taken by the N-SIM microscope and processed via N-SIM software. A middle slice of the images was shown. Scale bar, 5 µm.


( B ) Co - condensation of AID AIDR190X in nucleus of CH12F3 in early G1 phase . Top : experimental layout . GFP - tagged wild - type AID ( green ) mCherry - tagged AID variants ( red ) were co - nucleofected in Aicda - / - CH12F3 . resulting were arrested with nocodazole for 12 hours after transfection . Six hours later , nocodazole was removed from system for 1 . 5 hours before imaging . Bottom left : representative showing subcellular localization of indicated AID variants . dashed depicts shape of nucleus on Hoechst . arrow indicates location of co - condensed . Scale bar , 5 µm . Bottom right : plot profiles of . Cyto : cytoplasm , Nuc : nucleus .

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 B-CONTROLLED_VAR 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 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) Co-condensation of AID and AIDR190X in the nucleus of CH12F3 cells in the early G1 phase. Top: experimental layout. GFP-tagged wild-type AID (green) and mCherry-tagged AID variants (red) were co-nucleofected in Aicda-/- CH12F3 cells. The resulting cells were arrested with nocodazole for 12 hours after transfection. Six hours later, nocodazole was removed from the system for 1.5 hours before imaging. Bottom left: representative images showing subcellular localization of indicated AID variants. The white dashed line depicts the shape of the nucleus based on Hoechst staining. The white arrow indicates the location of the co-condensed puncta. Scale bar, 5 µm. Bottom right: plot profiles of the images. Cyto: cytoplasm, Nuc: nucleus.


( C ) of relative of AID - GFP in diluted phase of shown in ( B ) . of AID - GFP in diluted phase was normalized by total from whole . Data are shown as box plots . Values between lower quartile upper quartile are represented by box ranges , a horizontal within box represents median , whisker extends from minimum value to maximum value . " + " indicates mean level . Each dot indicates one ( n = 25 ) , representative result from three biological replicates were shown . Unpaired two - Student ' s t - was performed . * * * * , p < 0 . 0001 .

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(C) Quantification of the relative fluorescence intensity of AID-GFP in the diluted phase of cells shown in (B). The fluorescence intensity of AID-GFP in the diluted nuclear phase was normalized by the total intensity from the whole cell. Data are shown as box plots. Values between lower quartile and upper quartile are represented by box ranges, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. "+" indicates the mean level. Each dot indicates one cell (n = 25), and representative result from three biological replicates were shown. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.


( D ) Dynamics of trapping of wild - type AID in optoAIDR190X condensates . Top , representative at different time points . Dashed circles indicate co - condensed formation of AID - GFP . Scale bar , 5 µm . Bottom , a summary of 12 observed is shown as mean ± SD in a bar graph . Percentages of optoAIDR190X with trapped wild - type AID are plotted for second third blue - activations , colored dot indicates one .

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(D) Dynamics of trapping of wild-type AID in optoAIDR190X condensates. Top, representative fluorescence images at different time points. Dashed circles indicate the co-condensed puncta formation of AID-GFP. Scale bar, 5 µm. Bottom, a summary of 12 observed cells is shown as mean ±SD in a bar graph. Percentages of optoAIDR190X puncta with trapped wild-type AID are plotted for the second and third blue-light activations, and the colored dot indicates one cell.


( B ) optoDroplet formation of indicated AID mutants along with stimulation . Top , procedure of optoDroplet is illustrated with indicated stimulation ( blue arrow ) image acquisition ( black arrow ) time points . Bottom , plots show of with optoDroplet formation as mean ± SD at each acquisition time point . total acquired numbers of each genotype are indicated in parentheses . Significance assessment between AIDR190X AIDR190X - 4R was shown at each time point . Unpaired two - Student ' s t - was performed . * * * * , p < 0 . 0001 .

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(B) The optoDroplet formation of indicated AID mutants along with light stimulation. Top, the procedure of optoDroplet assay is illustrated with indicated light stimulation (blue arrow) and image acquisition (black arrow) time points. Bottom, line plots show quantification of cells with optoDroplet formation as mean ± SD at each acquisition time point. The total acquired cell numbers of each genotype are indicated in parentheses. Significance assessment between AIDR190X and AIDR190X-4R was shown at each time point. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.


( C ) CSR of ectopically - expressed AID variants in AID - proficient CH12F3 . Left , representative flow cytometry plots of CSR to IgA . Co - expressed GFP indicates infected fractions . Right , CSR mean with SD of three biological replicates is shown in bar plot . ( D ) CSR of ectopically - expressed AID variants in CSR - activated B . Left , representative flow cytometry plots of CSR to IgG1 . Right , bar plot shows CSR mean with SD of four biological replicates . Data information : One - way ANOVA followed by Dunnett ' s multiple comparisons was performed for ( C ) ( D ) . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 .

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(C) CSR levels of ectopically-expressed AID variants in AID-proficient CH12F3 cells. Left, representative flow cytometry plots of CSR to IgA. Co-expressed GFP indicates the infected cell fractions. Right, the CSR mean with SD of three biological replicates is shown in the bar plot. (D) CSR levels of ectopically-expressed AID variants in CSR-activated B cells. Left, representative flow cytometry plots of CSR to IgG1. Right, the bar plot shows the CSR mean with SD of four biological replicates. Data information: One-way ANOVA followed by Dunnett's multiple comparisons test was performed for (C) and (D). ***, p<0.001; **, p<0.01; *, p<0.05.


( A ) Left , representative of MBP - GFP - AID in presence of 10 % PEG8000 . Scale bar , 10 µm 2 µm , respectively . Right , schematic sequences .

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(A) Left, representative images of MBP-GFP-AID in the presence of 10% PEG8000. Scale bar, 10 µm and 2 µm, respectively. Right, the schematic protein sequences.


( B ) Left , representative of MBP - GFP - AIDcry + CTT or MBP - GFP - AIDcry in presence 10 % PEG8000 at indicated concentrations . Scale bar , 10 µm . Right , sizes of MBP - GFP - AIDcry + CTT or MBP - GFP - AIDcry condensates are plotted as box plots with three technical replicates . box represents values between lower quartile upper quartile , a horizontal within box represents median , whisker extends from minimum value to maximum value . points are listed mean is indicated by " + " . Unpaired two - Student ' s t - was performed . * * * * , p < 0 . 0001 .

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(B) Left, representative images of MBP-GFP-AIDcry+CTT or MBP-GFP-AIDcry in the presence 10% PEG8000 at the indicated protein concentrations. Scale bar, 10 µm. Right, sizes of MBP-GFP-AIDcry+CTT or MBP-GFP-AIDcry protein condensates are plotted as box plots with three technical replicates. The box represents values between the lower quartile and the upper quartile, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. All points are listed and mean is indicated by "+". Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.


( A ) AID deamination was performed in vitro . Left , a schematic diagram of deamination activity . Right , deamination reaction with indicated combinations was processed visualized . " + " : 0 . 25 μM of indicated ; " + + " : 0 . 5 μM of indicated ; " - " : not added .

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(A) AID deamination assay was performed in vitro. Left, a schematic diagram of deamination activity assay. Right, the deamination reaction with indicated protein combinations was processed and visualized. "+": 0.25 μM of indicated protein; "++": 0.5 μM of indicated protein; "-": not added.


( B ) CSR to IgA induced by AID variants in CH12F3 . Left , representative flow cytometry plots . Right , CSR mean with SD of three biological replicates is shown in bar plot . One - way ANOVA followed by Dunnett ' s multiple comparisons was performed . * * * , p < 0 . 001 ; * * , p < 0 . 01 .

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(B) CSR to IgA induced by AID variants in CH12F3 cells. Left, representative flow cytometry plots. Right, the CSR mean with SD of three biological replicates is shown in the bar plot. One-way ANOVA followed by Dunnett's multiple comparisons test was performed. ***, p<0.001; **, p<0.01.


( C ) Representative of co - condensation of MBP - mCherry - AIDcry MBP - GFP - AID . concentrations molecular ratios are indicated . Scale bar , 10 µm .

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(C) Representative images of the co-condensation of MBP-mCherry-AIDcry and MBP-GFP-AID. The protein concentrations and molecular ratios are indicated. Scale bar, 10 µm.


( C ) Immunocytochemistry showing post - synaptic marker PSD - 95 ( red ) , pre - synaptic marker Synapsin - I ( green ) , dendritic marker Map2 ( blue ) co - localization of PSD - 95 Synapsin - I from dissociated Nedd4 - 2 WT or Nedd4 - 2 cKO hippocampal neurons at DIV 16 . Representative secondary dendrites ( left ) of colocalized synaptic number ( right ) ( n = 36 for Nedd4 - 2 WT Nedd4 - 2 cKO neurons ) are shown . Scale bar : 5 µm .

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

(C) Immunocytochemistry showing post-synaptic marker PSD-95 (red), pre-synaptic marker Synapsin-I (green), dendritic marker Map2 (blue) and co-localization of PSD-95 and Synapsin-I from dissociated Nedd4-2 WT or Nedd4-2 cKO hippocampal neurons at DIV 16. Representative secondary dendrites (left) and quantification of colocalized synaptic puncta number and area (right) (n = 36 for Nedd4-2 WT and Nedd4-2 cKO neurons) are shown. Scale bar: 5 µm.


( F ) A train of high frequency stimulation ( HFS ; 100 Hz for 1s ) induced early - phase LTP ( E - LTP ) at Schaffer collateral synapses in Nedd4 - 2 WT ( n = 7 slices from 6 mice ) Nedd4 - 2 cKO ( n = 6 slices from 5 mice ) mice . Representative fEPSP traces were recorded before 60 min after LTP induction ( Scale bars : 0 . 5 mV , 5 ms ) . Summary bar graphs showing fEPSP slopes measured 50 - 60 min after a HFS at Schaffer collateral synapses was on right . ( G ) Late - phase LTP ( L - LTP ) induced by four trains of HFS in Nedd4 - 2 WT slices ( n = 7 slices from 7 mice ) Nedd4 - 2 cKO slices ( n = 9 slices from 8 mice ) . Representative fEPSP traces were recorded before 180 min after LTP induction ( Scale bars : 0 . 5 mV , 5 ms ) . Summary bar graphs showing fEPSP slopes measured 160 - 180 min after L - LTP induction at Schaffer collateral synapses was on right .

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

(F) A train of high frequency stimulation (HFS; 100 Hz for 1s) induced early-phase LTP (E-LTP) at Schaffer collateral synapses in Nedd4-2 WT (n = 7 slices from 6 mice) and Nedd4-2 cKO (n = 6 slices from 5 mice) mice. Representative fEPSP traces were recorded before and 60 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showing the fEPSP slopes measured 50-60 min after a HFS at Schaffer collateral synapses was on the right. (G) Late-phase LTP (L-LTP) induced by four trains of HFS in Nedd4-2 WT slices (n = 7 slices from 7 mice) and Nedd4-2 cKO slices (n = 9 slices from 8 mice). Representative fEPSP traces were recorded before and 180 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showing the fEPSP slopes measured 160-180 min after L-LTP induction at Schaffer collateral synapses was on the right.


( B ) Barnes maze from Nedd4 - 2 WT ( n = 8 ) or Nedd4 - 2 cKO ( n = 8 ) mice ( 6 - 8 old ) . Schematic representation of Barnes maze ( top left ) . Mice were trained for 4 days with three training trials on each day to locate escape box ( black circle ) . Learning curve during 4 training days was shown by escape latency number of errors before escaping ( top right ) . Schematic representation of Barnes maze on probe trials ( bottom left ) . Quadrant occupancy on probe trial at day5 at day12 ( bottom right ) were assessed by time spent in target ( green quadrant ) .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Barnes maze test from Nedd4-2 WT (n = 8) or Nedd4-2 cKO (n = 8) mice (6-8 weeks old). Schematic representation of Barnes maze (top left). Mice were trained for 4 days with three training trials on each day to locate the escape box (black circle). Learning curve during 4 training days was shown by the escape latency and the number of errors before escaping (top right). Schematic representation of Barnes maze on probe trials (bottom left). Quadrant occupancy on probe trial at day5 and at day12 (bottom right) were assessed by the time spent in the target area (green quadrant).


( B ) Western blots of F - actin to G - actin with or without cLTP induction from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured neurons . Schematic representation of experimental design for cLTP inductions ( top left ) . Representative western blots ( bottom left ) of F - actin to G - actin from Nedd4 - 2 WT or Nedd4 - 2 cKO ( right ) cultures after cLTP induction recovery in normal aCSF for 10 or 30 min before lysis ( n = 11 8 for Nedd4 - 2 WT Nedd4 - 2 cKO cultures , respectively ) . For , data from cLTP induction were normalized to data from control ( CTL ) condition .

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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 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

(B) Western blots of F-actin to G-actin ratio with or without cLTP induction from Nedd4-2 WT or Nedd4-2 cKO cultured neurons. Schematic representation of the experimental design for cLTP inductions (top left). Representative western blots (bottom left) and quantification of F-actin to G-actin from Nedd4-2 WT or Nedd4-2 cKO (right) cultures after cLTP induction and recovery in normal aCSF for 10 or 30 min before lysis (n = 11 and 8 for Nedd4-2 WT and Nedd4-2 cKO cultures, respectively). For quantification, data from cLTP induction were normalized to data from control (CTL) condition.


( A ) Western blots of Nedd4 - 2 , total cofilin , phospho ( p ) - cofilin GAPDH from Nedd4 - 2 WT or Nedd4 - 2 cKO forebrains at postnatal day 14 . Representative western blots of total cofilin relative to GAPDH p - cofilin relative to total cofilin ( n = 6 for Nedd4 - 2 WT Nedd4 - 2 cKO brains ) were shown .

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

(A) Western blots of Nedd4-2, total cofilin, phospho (p)-cofilin and GAPDH from Nedd4-2 WT or Nedd4-2 cKO forebrains at postnatal day 14. Representative western blots and quantification of total cofilin relative to GAPDH and p-cofilin relative to total cofilin (n = 6 for Nedd4-2 WT and Nedd4-2 cKO brains) were shown.


( B ) Immunocytochemistry of cofilin , p - cofilin dendritic marker MAP2 from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured hippocampal neurons at DIV 16 . Representative dendrites of Nedd4 - 2 WT Nedd4 - 2 cKO neurons of total cofilin relative to MAP2 p - cofilin relative to total cofilin are shown ( n = 25 26 for Nedd4 - 2 WT Nedd4 - 2 cKO neurons , respectively ) . Scale bar : 5 µm .

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

(B) Immunocytochemistry of cofilin, p-cofilin and dendritic marker MAP2 from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 16. Representative dendrites of Nedd4-2 WT and Nedd4-2 cKO neurons and quantification of total cofilin relative to MAP2 and p-cofilin relative to total cofilin are shown (n = 25 and 26 for Nedd4-2 WT and Nedd4-2 cKO neurons, respectively). Scale bar: 5 µm.


( C ) Immunocytochemistry showing cofilin , p - cofilin , dendritic marker MAP2 from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured hippocampal neurons induced with or without cLTP at DIV 16 . Representative dendrites from Nedd4 - 2 WT or Nedd4 - 2 cKO cultures are shown ( n = 36 - 39 neurons ) . Scale bar : 5 µm . For , data from cLTP induction were normalized to data from control ( CTL ) condition .

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

(C) Immunocytochemistry showing cofilin, p-cofilin, and dendritic marker MAP2 from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons induced with or without cLTP at DIV 16. Representative dendrites and quantification from Nedd4-2 WT or Nedd4-2 cKO cultures are shown (n = 36-39 neurons). Scale bar: 5 µm. For quantification, data from cLTP induction were normalized to data from control (CTL) condition.


( A ) Immunocytochemistry showing post - synaptic marker PSD - 95 ( red ) , pre - synaptic marker Synapsin - I ( green ) , dendritic marker Map2 ( blue ) co - localization of PSD - 95 Synapsin - I from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured hippocampal neurons at DIV 16 treated with TAT , TAT - S3 , or TAT - pS3 peptide for 12 hrs ( 10 μM ) . Representative secondary dendrites of Nedd4 - 2 WT ( top left ) Nedd4 - 2 cKO ( bottom left ) neurons are shown . of colocalized synaptic number synaptic for Nedd4 - 2 WT ( top right ) Nedd4 - 2 cKO ( bottom right ) neurons is on right ( n = 32 - 36 neurons ) . Scale bar : 5 µm .

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

(A) Immunocytochemistry showing post-synaptic marker PSD-95 (red), pre-synaptic marker Synapsin-I (green), dendritic marker Map2 (blue) and co-localization of PSD-95 and Synapsin-I from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 16 treated with TAT, TAT-S3, or TAT-pS3 peptide for 12 hrs (10 μM). Representative secondary dendrites of Nedd4-2 WT (top left) and Nedd4-2 cKO (bottom left) neurons are shown. Quantification of colocalized synaptic puncta number and synaptic puncta area for Nedd4-2 WT (top right) and Nedd4-2 cKO (bottom right) neurons is on the right (n = 32-36 neurons). Scale bar: 5 µm.


( B ) Patch - clamp recordings from Nedd4 - 2 WT or Nedd4 - 2 cKO hippocampal neurons treated with TAT , TAT - S3 , or TAT - pS3 peptide for 12 hrs ( 10 μM ) at DIV 14 - 17 . Representative mEPSC traces from Nedd4 - 2 WT ( top left ) Nedd4 - 2 cKO ( bottom left ) are shown . of mEPSC amplitude frequency from Nedd4 - 2 WT ( top right ) Nedd4 - 2 cKO ( bottom right ) neurons are shown ( n = 22 - 24 neurons ) .

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

(B) Patch-clamp recordings from Nedd4-2 WT or Nedd4-2 cKO hippocampal neurons treated with TAT, TAT-S3, or TAT-pS3 peptide for 12 hrs (10 μM) at DIV 14-17. Representative mEPSC traces from Nedd4-2 WT (top left) and Nedd4-2 cKO (bottom left) are shown. Quantification of mEPSC amplitude and frequency from Nedd4-2 WT (top right) and Nedd4-2 cKO (bottom right) neurons are shown (n = 22-24 neurons).


( C ) Barnes maze after intraperitoneally injecting TAT ( n = 8 ) or TAT - pS3 ( n = 8 ) peptide in Nedd4 - 2 WT mice ( top ) , or TAT ( n = 9 ) or TAT - pS3 ( n = 8 ) peptides in Nedd4 - 2 cKO mice ( bottom ) . Learning curve during 4 training days of Barnes maze task showing escape latency , number of errors before escaping , quadrant occupancy on probe trial at day 5 at day 12 were assessed .

O 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 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-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

(C) Barnes maze test after intraperitoneally injecting TAT (n = 8) or TAT-pS3 (n = 8) peptide in Nedd4-2 WT mice (top), or TAT (n = 9) or TAT-pS3 (n = 8) peptides in Nedd4-2 cKO mice (bottom). Learning curve during 4 training days of Barnes maze task showing the escape latency, number of errors before escaping, and the quadrant occupancy on probe trial at day 5 and at day 12 were assessed.


( B ) Immunoblot of fractions of HEK293 Flp - In stably overexpressing both mouse TREM2 mouse DAP12 upon treatment with 4D9 antibody reveals increased of - bound TREM2 similar to what can be achieved by ADAM protease inhibition using GM6001 inhibitor . An isotype antibody was used as a negative control . Calnexin served as a loading control . of - bound TREM2 were quantified by MSD ELISA . Data represent mean ± ( n = 3 ) . One - way ANOVA , Tukey ' s post hoc ; p ( DMSO vs GM ) = 0 . 0011 ; p ( DMSO vs isotype ) = 0 . 992 ; p ( isotype vs 4D9 ) = 0 . 0005 ; n . s . , not significant .

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

(B) Immunoblot analysis of membrane fractions of HEK293 Flp-In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals increased levels of membrane-bound TREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. Calnexin served as a loading control. Levels of membrane-bound TREM2 were quantified by MSD ELISA. Data represent the mean ± SEM (n=3). One-way ANOVA, Tukey's post hoc test; p (DMSO vs GM) = 0.0011; p (DMSO vs isotype) = 0.992; p (isotype vs 4D9) = 0.0005; n.s., not significant.


( C ) Immunoblot of conditioned media from HEK293 Flp - In stably overexpressing both mouse TREM2 mouse DAP12 upon treatment with 4D9 antibody reveals decreased of sTREM2 similar to what can be achieved by ADAM protease inhibition using GM6001 inhibitor . An isotype antibody was used as a negative control . sAPPα served as a loading control . Note that heavy chains of antibodies used for treatment are also detected annotated . of sTREM2 were quantified by MSD ELISA . Data represent mean ± ( n = 3 ) . One - way ANOVA , Tukey ' s post hoc ; p ( DMSO vs GM ) < 0 . 0001 ; p ( DMSO vs isotype ) = 0 . 6372 ; p ( isotype vs 4D9 ) < 0 . 0001 ; n . s . , not significant .

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

(C) Immunoblot analysis of conditioned media from HEK293 Flp-In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals decreased levels of sTREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. sAPPα served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated. Levels of sTREM2 were quantified by MSD ELISA. Data represent the mean ± SEM (n=3). One-way ANOVA, Tukey's post hoc test; p (DMSO vs GM) < 0.0001; p (DMSO vs isotype) = 0.6372; p (isotype vs 4D9) < 0.0001; n.s., not significant.


( D ) 4D9 antibody selectively detects TREM2 on surface of HEK293 Flp - In stably overexpressing mouse TREM2 mouse DAP12 . An anti - HA antibody was used as a positive control while empty vector transfected HEK293 Flp - In were used as a negative control . Scale bar = 10µm .

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

(D) 4D9 antibody selectively detects TREM2 on the cell surface of HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12. An anti-HA antibody was used as a positive control while empty vector transfected HEK293 Flp-In cells were used as a negative control. Scale bar= 10µm.


( F ) comparison of mouse TREM2 human TREM2 shows substantial conservation around 4D9 epitope ( upper panel ) . Immunoblot demonstrates that antibody 4D9 is highly specific for mouse TREM2 does not detect human TREM2 or mouse TREM1 ( lower panel ) .

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

(F) Sequence comparison of mouse TREM2 and human TREM2 shows substantial sequence conservation around the 4D9 epitope (upper panel). Immunoblot analysis demonstrates that antibody 4D9 is highly specific for mouse TREM2 and does not detect human TREM2 or mouse TREM1 (lower panel).


( G ) 4D9 binding to mouse TREM2 ECD is competed off by a stalk region peptide . A competition ELISA demonstrates that a dose titration of stalk peptide reduces binding of 4D9 to TREM2 ECD with an EC50 of 1 . 3uM . Data represent mean ± ( n = 3 ) .

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

(G) 4D9 binding to mouse TREM2 ECD is competed off by a stalk region peptide. A competition ELISA demonstrates that a dose titration of stalk peptide reduces binding of 4D9 to TREM2 ECD with an EC50 of 1.3uM. Data represent the mean ± SEM (n=3).


( H ) Surface plasmon resonance binding kinetics of increasing concentrations of 4D9 antibody to mouse TREM2 ectodomain evaluated by BIAcore , Kon = 5 . 9 x 105 M - 1s - 1 , Koff = 4 . 0 x 10 - 5 s - 1 , KD = 68 pM . 4D9 binding to human TREM2 or mouse TREM1 was undetectable .

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

(H) Surface plasmon resonance binding kinetics of increasing concentrations of 4D9 antibody to mouse TREM2 ectodomain evaluated by BIAcore, Kon = 5.9 x 105 M-1s-1, Koff = 4.0 x 10-5 s-1, KD = 68 pM. 4D9 binding to human TREM2 or mouse TREM1 was undetectable.


( A ) Flow cytometry dose response curve for binding of 4D9 mAb ( EC50 = 0 . 29nM ) , 4D9 Fab ( EC50 = 0 . 17nM ) , isotype to HEK stably overexpressing mouse TREM2 . Data represent mean ± ( n = 2 ) .

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 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

(A) Flow cytometry dose response curve for cell binding of 4D9 mAb (EC50= 0.29nM), 4D9 Fab (EC50=0.17nM), and isotype to HEK cells stably overexpressing mouse TREM2. Data represent the mean ± SEM (n=2).


( B ) In vitro ADAM17 sheddase activity is blocked by 4D9 - effectorless mAb 4D9 Fab fragment but not an isotype control . polarization of FAM conjugated TREM2 stalk peptide was detected in presence or absence of ADAM17 4D9 mAb , 4D9 Fab isotype control . Data represent mean ± ( n = 6 ) . One - way ANOVA , Tukey ' s post hoc ; p ( 4D9 Fab vs 4D9 mAb ) = 0 . 8855 ; p ( 4D9 Fab vs uncleaved ) < 0 . 0001 ; p ( 4D9 mAb vs uncleaved ) < 0 . 0001 ; n . s . , not significant .

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

(B) In vitro ADAM17 sheddase activity is blocked by 4D9-effectorless mAb and 4D9 Fab fragment but not an isotype control. Fluorescence polarization of FAM conjugated TREM2 stalk peptide was detected in the presence or absence of ADAM17 and 4D9 mAb, 4D9 Fab and isotype control. Data represent the mean ± SEM (n=6). One-way ANOVA, Tukey's post hoc test; p (4D9 Fab vs 4D9 mAb) = 0.8855; p (4D9 Fab vs uncleaved) < 0.0001; p (4D9 mAb vs uncleaved) < 0.0001; n.s., not significant.


( C ) ELISA - mediated of sTREM2 in conditioned media from

(C) ELISA-mediated quantification of sTREM2 in conditioned media from


( D ) AlphaLISA - mediated of pSYK in HEK293 Flp - In stably overexpressing mouse TREM2 mouse DAP12 treated with a dose titration of 4D9 mAb , 4D9 Fab or isotype for 5 minutes . Data represent mean ± ( n = 3 ) .

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

(D) AlphaLISA-mediated quantification of pSYK levels in HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12 treated with a dose titration of 4D9 mAb, 4D9 Fab or isotype for 5 minutes. Data represent the mean ± SEM (n=3).


( E ) AlphaLISA - mediated of pSYK in HEK293 Flp - In stably overexpressing mouse TREM2 mouse DAP12 or empty vector treated with 1mg / ml POPC / POPS liposomes 20µg / ml 4D9 mAb , 4D9 Fab or isotype for 5 minutes . pSYK were also determined for treated with liposomes only . Data represent mean ± ( n = 3 ) . Two - way ANOVA , Tukey ' s post hoc ( effect : F1 , 16 = 365 . 7 , p = < 0 . 0001 ; treatment effect : F3 , 16 = 39 . 35 , p < 0 . 0001 ; x treatment effect : F3 , 16 = 38 . 75 , p < 0 . 0001 ) ; p ( no Ab vs isotype ) = 0 . 6218 ; p ( no Ab vs 4D9 mAb ) < 0 . 0001 ; p ( no Ab vs 4D9 Fab ) = 0 . 7301 ; p ( isotype vs 4D9 mAb ) < 0 . 0001 ; p ( isotype vs 4D9 Fab ) > 0 . 9999 ; p ( 4D9 mAb vs 4D9 Fab ) < 0 . 0001 ; n . s . , not significant .

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

(E) AlphaLISA-mediated quantification of pSYK levels in HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12 or empty vector treated with 1mg/ml POPC/POPS liposomes and 20µg/ml 4D9 mAb, 4D9 Fab or isotype for 5 minutes. pSYK levels were also determined for cells treated with liposomes only. Data represent the mean ± SEM (n=3). Two-way ANOVA, Tukey's post hoc test (cell line effect: F1,16=365.7, p=<0.0001; treatment effect: F3,16=39.35, p<0.0001; cell line x treatment effect: F3,16=38.75, p<0.0001); p (no Ab vs isotype) = 0.6218; p (no Ab vs 4D9 mAb) < 0.0001; p (no Ab vs 4D9 Fab) = 0.7301; p (isotype vs 4D9 mAb) < 0.0001; p (isotype vs 4D9 Fab) > 0.9999; p (4D9 mAb vs 4D9 Fab) < 0.0001; n.s., not significant.


( A ) Flow cytometry detection of surface binding of 4D9 ( blue ) isotype ( green ) to wild type Trem2 - / - mouse bone marrow derived macrophages . Representative histograms are shown as MFI ( mean ) . Graphs represent median signal ± ( n = 3 ) . Student ' s t - ( two - ) ; Trem2 + / + : p = 0 . 0005 ; Trem2 - / - : p = 0 . 4435 ; n . s . , not significant .

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

(A) Flow cytometry detection of cell surface binding of 4D9 (blue) and isotype (green) to wild type and Trem2-/- mouse bone marrow derived macrophages. Representative histograms are shown as MFI (mean fluorescent intensity). Graphs represent the median signal intensity ± SEM (n=3). Student's t-test (two-tailed); Trem2+/+: p = 0.0005; Trem2-/-: p = 0.4435; n.s., not significant.