( A ) Comparison of IFNγ production ( y - axis ) after incubation with plate - coated antibodies or ( 10 µg / ml each ) ( x - axis ) . Box plots showing IFNγ 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 . ( A , Samples were acquired in duplicate mean was calculated for each donor condition . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

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 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

(A) Comparison of IFNγ production (y-axis) after incubation with plate-coated antibodies or proteins (10 µg/ml each) (x-axis). Box plots showing IFNγ 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. (A, Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


( B ) Comparison of IFNγ in supernatant after co - culture of NK with 721 . 221 target in presence or absence of either 10 µg / ml αTRAIL or isotype control ( Iso ) using ELISA ( n = 8 different donors per condition ) . Effector : target was 1 : 1 . Left panel : Box plots showing IFNγ ( y - axis ) for described culture conditions ( x - axis ) . Right panel : Box plots display inhibition of IFNγ production by NK ( y - axis ) in presence of either isotype or αTRAIL as relative reduction compared to no antibody . Data information : Wilcoxon signed - rank . B , Samples were acquired in duplicate mean was calculated for each donor condition . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

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 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) Comparison of IFNγ levels in the supernatant after co-culture of NK cells with 721.221 target cells in the presence or absence of either 10 µg/ml αTRAIL or isotype control (Iso) using ELISA (n = 8 different donors per condition). Effector:target ratio was 1:1. Left panel: Box plots showing IFNγ levels (y-axis) for the described culture conditions (x-axis). Right panel: Box plots display inhibition of IFNγ production by NK cells (y-axis) in presence of either isotype or αTRAIL as relative reduction compared to no antibody. Data information: Wilcoxon signed-rank test. B, Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


( C ) Representative contour plots showing conjugate formation between NK 721 . 221 target as determined by flow cytometry . Plots showing three distinct populations at beginning of co - culture ( 0 min ) after 50 min : single 721 . 221 ( CT Orange - positive ) , single NK ( CT Violet - positive ) conjugates ( double - positive ) . Relative frequency of NK in conjugates is calculated on total number of gated NK . ( D ) Comparison of conjugate formation after co - culture between NK 722 . 221 after 0 min after 50 min in presence or absence of 10 µg / ml αTRAIL or isotype control ( n = 8 different donors ) . Left panel : Box plots showing relative frequency of NK in conjugates ( y - axis ) for different time points conditions ( x - axis ) . Right panel : Box plots display inhibition of conjugate formation ( y - axis ) in presence of either isotype or αTRAIL as relative reduction compared to no antibody . Data information : Wilcoxon signed - rank . D ) Samples were acquired in duplicate mean was calculated for each donor condition . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative contour plots showing conjugate formation between NK cells and 721.221 target cells as determined by flow cytometry. Plots showing three distinct populations at the beginning of the co-culture (0 min) and after 50 min: single 721.221 cells (CT Orange-positive), single NK cells (CT Violet-positive) and conjugates (double-positive). Relative frequency of NK cells in conjugates is calculated based on the total number of gated NK cells. (D) Comparison of conjugate formation after co-culture between NK cells and 722.221 cells after 0 min and after 50 min in the presence or absence of 10 µg/ml αTRAIL or isotype control (n = 8 different donors). Left panel: Box plots showing relative frequency of NK cells in conjugates (y-axis) for different time points and conditions (x-axis). Right panel: Box plots display inhibition of conjugate formation (y-axis) in the presence of either isotype or αTRAIL as relative reduction compared to no antibody. Data information: Wilcoxon signed-rank test. D) Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


( E ) Expression of phosphorylated signaling . Upper panel : Concatenated contour plots of one donor depicting expression of phosphorylated signaling Syk , p38 MAPK , Akt PLC - γ2 for following culture conditions controls ( x - axis : left to right ) : FMO , PBS , isotype , αTRAIL , αNKp46 , αNKG2D , αCD16 . Lower panel : Bar graphs showing of p - Syk + , p - p38 MAPK + , p - Akt + p - PLC - γ2 + NK after 30 min of stimulation ( n = 3 different donors ) . Bar graphs represent median , whiskers display 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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-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 B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR 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

(E) Expression levels of phosphorylated signaling proteins. Upper panel: Concatenated contour plots of one donor depicting the expression of phosphorylated signaling proteins Syk, p38 MAPK, Akt and PLC-γ2 for the following culture conditions and controls (x-axis: left to right): FMO, PBS, isotype, αTRAIL, αNKp46, αNKG2D, αCD16. Lower panel: Bar graphs showing the percentage of p-Syk+, p-p38 MAPK+, p-Akt+ and p-PLC-γ2+ NK cells after 30 min of stimulation (n = 3 different donors). Bar graphs represent the median, whiskers display minimum and maximum data points.


Lysis of different target in co - culture with NK was quantified in various cytotoxicity assays . ( A ) Left panel : Representative contour plots showing depletion of 721 . 221 target in presence of NK . Middle panel : of target remaining ( y - axis ) after co - culture with NK in presence of either αTRAIL or isotype control , in reference to target kept alone . Right panel : Box plots displaying difference of target remaining ( y - axis ) between αTRAIL isotype conditions displayed as p . p . ( n = 12 different donors ) . Each data point represents mean of at least 2 technical replicates . Data information : Wilcoxon signed - rank . Experiments were performed in four batches with three different donors each . " No NK " control samples served as a reference for donors in each batch . Lines connect each data value of NK condition with their designated " No NK " control . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays. (A) Left panel: Representative contour plots showing depletion of 721.221 target cells in the presence of NK cells. Middle panel: Percentage of target cells remaining (y-axis) after co-culture with NK cells in the presence of either αTRAIL or isotype control, in reference to target cells kept alone. Right panel: Box plots displaying difference of target cells remaining (y-axis) between αTRAIL and isotype conditions displayed as p.p. (n = 12 different donors). Each data point represents the mean of at least 2 technical replicates. Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


Lysis of different target in co - culture with NK was quantified in various cytotoxicity assays . ( B ) Left panel : Representative contour plots showing of . 221 - DR4 / 5KO ( control ) . 221 - Cas9 ( target ) in presence or absence of NK . Middle panel : between . 221 - DR4 / 5KO . 221 - Cas9 ( y - axis ) in presence or absence of NK . Right panel : Specific lysis of . 221 - Cas9 displayed as % ( n = 12 different donors ) . Each data point represents mean of at least 3 technical replicates . Data information : Wilcoxon signed - rank . Experiments were performed in four batches with three different donors each . " No NK " control samples served as a reference for donors in each batch . Lines connect each data value of NK condition with their designated " No NK " control . 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays. (B) Left panel: Representative contour plots showing the percentage of .221-DR4/5KO (control) and .221-Cas9 cells (target) in the presence or absence of NK cells. Middle panel: Ratio between .221-DR4/5KO and .221-Cas9 cells (y-axis) in the presence or absence of NK cells. Right panel: Specific lysis of .221-Cas9 cells displayed as % (n = 12 different donors). Each data point represents the mean of at least 3 technical replicates. Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


Lysis of different target in co - culture with NK was quantified in various cytotoxicity assays . ( C ) Left panel : Representative contour plots showing of Raji - pSIP ( control ) Raji - DR5 + + ( target ) in presence or absence of NK . Middle panel : between Raji - pSIP Raji - DR5 + + ( y - axis ) in presence or absence of NK . Right panel : Specific lysis of Raji - DR5 + + displayed as % ( n = 12 different donors ) . Each data point represents mean of at least three technical replicates . Data information : Wilcoxon signed - rank . Experiments were performed in four batches with three different donors each . " No NK " control samples served as a reference for donors in each batch . Lines connect each data value of NK condition with their designated " No NK " control . 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 B-MEASURED_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 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

Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays. (C) Left panel: Representative contour plots showing the percentage of Raji-pSIP (control) and Raji-DR5++ (target) in the presence or absence of NK cells. Middle panel: Ratio between Raji-pSIP and Raji-DR5++ (y-axis) in the presence or absence of NK cells. Right panel: Specific lysis of Raji-DR5++ cells displayed as % (n = 12 different donors). Each data point represents the mean of at least three technical replicates. Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


A . Immunoblot of of indicated in lysates from non - infected ( ni ) infected wild - type iBMDMs for 60 or 120 min .

O O 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. Immunoblot analysis of the levels of the indicated proteins in lysates from non-infected (ni) and infected wild-type iBMDMs for 60 or 120 min.


C . Immunoblot of Klf4 tubulin in lysates from non - infected ( ni ) infected wild - type iBMDMs for 60 or 120 min .

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

C. Immunoblot analysis of Klf4 and tubulin levels in lysates from non-infected (ni) and infected wild-type iBMDMs for 60 or 120 min.


H . Immunoblot of Arg1 tubulin in lysates from non - infected ( ni ) infected wild - type stat6 - / - iBMDMs for 60 or 120 min .

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H. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type and stat6-/- iBMDMs for 60 or 120 min.


D . Immunofluorescence confocal microscopy of colocalization of Kp52145 harbouring pFPV25 . 1Cm , cresyl violet dye in wild - type ( WT ) stat6 - / - macrophages . were taken 90 min post infection . are representative of duplicate coverslips of three independent experiments . E . of Kp52145 harbouring pFPV25 . 1Cm co - localization with cresyl violet over a time course . Values are given as mean of Kp52145 co - localizing with marker ± . number of infected counted per time in three independent experiments are indicated in figure .

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D. Immunofluorescence confocal microscopy of the colocalization of Kp52145 harbouring pFPV25.1Cm, and cresyl violet dye in wild-type (WT) and stat6-/- macrophages. The images were taken 90 min post infection. Images are representative of duplicate coverslips of three independent experiments. E. Percentage of Kp52145 harbouring pFPV25.1Cm co-localization with cresyl violet over a time course. Values are given as mean percentage of Kp52145 co-localizing with the marker ± SEM. The number of infected cells counted per time in three independent experiments are indicated in the figure.


A . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , tlr2 - / - , tlr4 - / - tlr2 / 4 - / - iBMDMs for 60 or 120 min . B . Immunoblot of Arg1and tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , tlr2 - / - , tlr4 - / - tlr2 / 4 - / - iBMDMs for 60 or 120 min .

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

A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs for 60 or 120 min. B. Immunoblot analysis of Arg1and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs for 60 or 120 min.


F . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , myd88 - / - , tram / trif - / - for 60 or 120 min . G . Immunoblot of Arg1 tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , myd88 - / - , tram / trif - / - for 60 or 120 min .

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

F. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), myd88-/-, tram/trif-/- for 60 or 120 min. G. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), myd88-/-, tram/trif-/- for 60 or 120 min.


A . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or ifnar1 - / - for 60 or 120 min . B . Immunoblot of Arg1 tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or ifnar1 - / - for 60 or 120 min .

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A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or ifnar1-/- for 60 or 120 min. B. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or ifnar1-/- for 60 or 120 min.


K . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or irf3 - / - for 60 or 120 min . L Immunoblot of Arg1 tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or irf31 - / - for 60 or 120 min .

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K. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or irf3-/- for 60 or 120 min. L Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or irf31-/- for 60 or 120 min.


A . Immunoblot of phospho - CREB ( pCREB ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or tlr4 - / - for 60 or 120 min . B . Immunoblot of phospho - CREB ( pCREB ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or myd88 - / - for 60 or 120 min .

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

A. Immunoblot analysis of phospho-CREB (pCREB) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or tlr4-/- for 60 or 120 min. B. Immunoblot analysis of phospho-CREB (pCREB) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or myd88-/- for 60 or 120 min.


D . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or il10 - / - for 60 or 120 min .

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

D. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or il10-/- for 60 or 120 min.


F . Immunoblot of Arg1 tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or il10 - / - for 60 or 120 min .

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-CONTROLLED_VAR O O O O O O O O O

F. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or il10-/- for 60 or 120 min.


L . Immunofluorescence confocal microscopy of colocalization of Kp52145 harbouring pFPV25 . 1Cm , cresyl violet dye in wild - type ( WT ) il10 - / - macrophages . were taken 90 min post infection . are representative of duplicate coverslips of three independent experiments . M . of Kp52145 harbouring pFPV25 . 1Cm co - localization with cresyl violet over a time course in wild - type ( WT ) il10 - / - macrophages . Values are given as mean of Kp52145 co - localizing with marker ± . number of infected counted per time in three independent experiments are indicated in figure .

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 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

L. Immunofluorescence confocal microscopy of the colocalization of Kp52145 harbouring pFPV25.1Cm, and cresyl violet dye in wild-type (WT) and il10-/- macrophages. The images were taken 90 min post infection. Images are representative of duplicate coverslips of three independent experiments. M. Percentage of Kp52145 harbouring pFPV25.1Cm co-localization with cresyl violet over a time course in wild-type (WT) and il10-/- macrophages. Values are given as mean percentage of Kp52145 co-localizing with the marker ± SEM. The number of infected cells counted per time in three independent experiments are indicated in the figure.


A . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from wild - type macrophages non - infected ( ni ) , or infected with Kp52145 or LPS O - polysaccharide mutant , 52145 - Δglf , for 60 or 120 min .

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

A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from wild-type macrophages non-infected (ni), or infected with Kp52145 or the LPS O-polysaccharide mutant, strain 52145-Δglf, for 60 or 120 min.


C . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from wild - type macrophages non - infected ( ni ) , or infected with Kp52145 or CPS mutant , 52145 - ΔwcaK2 , for 60 or 120 min .

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

C. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from wild-type macrophages non-infected (ni), or infected with Kp52145 or the CPS mutant, strain 52145-ΔwcaK2, for 60 or 120 min.


A . arg1 mRNA were assessed by qPCR in hM - CSF - treated PBMCs from 6 donors non - infected ( ni ) or infected Kp52145 for 1 , 3 or 5 h . D . ido mRNA were assessed by qPCR in hM - CSF - treated PBMCs from 6 donors non - infected ( ni ) or infected Kp52145 for 1 , 3 or 5 h .

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A. arg1 mRNA levels were assessed by qPCR in hM-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h. D. ido mRNA levels were assessed by qPCR in hM-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h.


B . il10 mRNA were assessed by qPCR in M - CSF - treated PBMCs from 6 donors non - infected ( ni ) or infected Kp52145 for 1 , 3 or 5 h . C . chi3l mRNA were assessed by qPCR in hM - CSF - treated PBMCs from 6 donors non - infected ( ni ) or infected Kp52145 for 1 , 3 or 5 h .

B. il10 mRNA levels were assessed by qPCR in M-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h. C. chi3l mRNA levels were assessed by qPCR in hM-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h.


E . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from PMA - treated THP - 1 macrophages non - infected ( ni ) , or infected with Kp52145 for 60 or 120

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

E. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from PMA-treated THP-1 macrophages non-infected (ni), or infected with Kp52145 for 60 or 120 min


O . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from PMA - treated THP - 1 macrophages non - infected ( ni ) , or infected with Kp52145 or CPS mutant , 52145 - ΔwcaK2 , for 60 or 120 min .

O. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from PMA-treated THP-1 macrophages non-infected (ni), or infected with Kp52145 or the CPS mutant, strain 52145-ΔwcaK2, for 60 or 120 min.


SARS - CoV - 2 . For weigh loss record , hACE2 mice ( n = 7 ) WT mice ( n = 3 ) were experimentally challenged intranasally with SARS - CoV - 2 , ACE2 - Mock mice ( n = 3 ) were used as control . then loss was recorded over 14 days ( a , ANOVA , * * * p < 0 . 001 ) .

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SARS-CoV-2.For weigh loss record, hACE2 mice (n=7) and WT mice (n=3) were experimentally challenged intranasally with SARS-CoV-2, and the ACE2-Mock mice (n=3) were used as control. And then the weight loss was recorded over 14 days (a, ANOVA, ***p<0.001).


To screen replication , 12 mice were infected in each group , 3 mice per group were sacrificed their major organs harvested for viral load titer at 1 dpi , 3 dpi , 5 dpi 7 dpi respectively . distribution of SARS - CoV - 2 in organs of ACE2 - HB - 01 mice was detected by qRT - PCR ( b ) .

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To screen virus replication, 12 mice were infected in each group, and 3 mice per group were sacrificed and their major organs harvested for viral load and virus titer at 1 dpi, 3 dpi, 5 dpi and 7 dpi respectively. The distribution of SARS-CoV-2 in the primary organs of ACE2-HB-01 mice was detected by qRT-PCR (b).


titers of lungs were determined on Vero E6 ( c , unpaired t - , * * * p < 0 . 001 )

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Virus titers of lungs were determined on Vero E6 cells (c, unpaired t-test, ***p<0.001)


isolated from lungs of ACE2 - HB - 01 mice at 3 dpi was observed by electron microscope ( d ) .

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the virus isolated from lungs of ACE2-HB-01 mice at 3 dpi was observed by electron microscope (d).


specific IgG against SARS - CoV - 2 was detected at 21 dpi by ELISA ( e , unpaired t - , * * * p < 0 . 001 ) .

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The specific IgG against SARS-CoV-2 was detected at 21 dpi by ELISA (e, unpaired t-test, ***p<0.001).


mice . a . Gross pathology histopathology of lungs from WT - HB - 01 mice ( 3 dpi ) , ACE2 - Mock mice ( 3 dpi ) ACE2 - HB - 01 mice ( 3 dpi 5 dpi ) . Postmortem examinations showed focal red lesions ( red arrow ) throughout dorsal of right middle lobe of lung at 3dpi . lesions progressed into multifocally scattered - reddish purple areas palpable nodules ( red arrow ) throughout right lobe of lung at 5 dpi . Histopathological observation indicated that moderate interstitial pneumonia with thickened alveolar septa ( black arrows ) infiltration of lymphocytes ( red arrows ) . swollen degenerative alveolar macrophages ( green arrows ) are scattered within alveolar cavities at 3dpi 5 dpi .

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mice.a. Gross pathology and histopathology of lungs from WT-HB-01 mice (3 dpi), ACE2-Mock mice (3 dpi) and ACE2-HB-01 mice (3 dpi and 5 dpi). Postmortem examinations showed focal dark red lesions (red arrow) throughout the dorsal of the right middle lobe of the lung at 3dpi. The lesions progressed into multifocally scattered-dark reddish purple areas and palpable nodules (red arrow) throughout the right lobe of the lung at 5 dpi. Histopathological observation indicated that moderate interstitial pneumonia with thickened alveolar septa (black arrows) and infiltration of lymphocytes (red arrows). The swollen and degenerative alveolar macrophages (green arrows) are scattered within the alveolar cavities at 3dpi and 5 dpi.


b . Immunohistochemical examination of lungs of each group . sequential sections were stained by HE , respectively . viral antigens were observed in alveolar macrophage ( green arrows ) , alveolar epithelia ( blue arrows ) , degenerative being desquamated bronchial epithelial ( black arrows ) . Black bar = 100 µm , red bar = 50 µm .

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b. Immunohistochemical examination of lungs of each group. The sequential sections were stained by HE and IHC, respectively. The viral antigens were observed in the alveolar macrophage (green arrows), the alveolar epithelia (blue arrows), and the degenerative and being desquamated bronchial epithelial cells (black arrows). Black bar = 100 µm, red bar = 50 µm.


Immunofluorescence of viral antigens in lungs of SARS - CoV - 2 - infected hACE2 mice . Co - localization of SARS - CoV - 2 S hACE2 receptor in hACE2 mouse lungs , sections were incubated with anti - SARS - CoV - 2 S antibody , anti - human ACE2 antibody , DAPI . lung sections of ACE2 - Mock mice ( a - d ) . lung sections of ACE2 - HB - 01 mice ( e - h ) . arrows showed viral S ( f ) hACE2 ( g ) , respectively , yellow arrow showed merge of viral S hACE2 ( h ) . bar = 25 µm .

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Immunofluorescence analysis of viral antigens in lungs of SARS-CoV-2-infected hACE2 mice.Co-localization of SARS-CoV-2 S protein and hACE2 receptor in hACE2 mouse lungs, the sections were incubated with anti-SARS-CoV-2 S protein antibody, anti-human ACE2 antibody, and DAPI. The lung sections of ACE2-Mock mice (a-d). The lung sections of ACE2-HB-01 mice (e-h). The white arrows showed the viral S protein (f) and hACE2 (g), respectively, the yellow arrow showed the merge of viral S protein and hACE2 (h). White bar=25 µm.


( C ) Clonal lines expressing p53 - Venus were engineered to monitor p53 expression dynamics ( " input " ) two canonically regulated p53 target promoters expressing mCherry ( " output " ) in response to Nutlin - 3 treatment .

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(C) Clonal cell lines expressing p53-Venus were engineered to monitor p53 expression dynamics ("input") and two canonically regulated p53 target promoters expressing mCherry ("output") in response to Nutlin-3 treatment.


( D ) Representative phase contrast yellow ( indicating p53 - Venus ) at indicated time points for a exposed to " natural dynamics " Nutlin - 3 dosing regimen .

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(D) Representative phase contrast and yellow fluorescent (indicating p53-Venus levels) images at the indicated time points for a cell exposed to the "natural dynamics" Nutlin-3 dosing regimen.


( E - F ) p53 - Venus expression in response to " natural dynamics " Nutlin - 3 dosing regimen . Single traces ( gray ; E ) mean ( red ; E ) are shown for p53 - Venus expression in response to each Nutlin - 3 regimen . Heat map ( F ) shows an alternative representation of traces as shown in ( E ) . N = 51 .

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(E-F) p53-Venus expression in response to the "natural dynamics" Nutlin-3 dosing regimen. Single cell traces (gray; E) and the mean (red; E) are shown for p53-Venus expression in response to each Nutlin-3 regimen. Heat map (F) shows an alternative representation of all traces as shown in (E). N = 51 cells.


( A ) Representative phase contrast , yellow ( indicating p53 - Venus ) , red ( indicating reporter mCherry ) at indicated time points for MDM2 promoter reporter exposed to " natural dynamics " Nutlin - 3 dosing regimen .

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(A) Representative phase contrast, yellow fluorescent (indicating p53-Venus levels), and red fluorescent (indicating reporter mCherry levels) images at the indicated time points for MDM2 promoter reporter cells exposed to the "natural dynamics" Nutlin-3 dosing regimen.


( B - G ) Single traces of mCherry expression for " responding " ( gray ) or " not responding " ( gray ) MDM2 promoter or CDKN1A promoter exposed to natural dynamics ( B ) , low amplitude ( D ) , high amplitude ( D ) , high frequency ( E ) , low frequency / short duration ( F ) , or long duration ( G ) Nutlin - 3 dosing regimens . average trace for responding not responding is shown in red blue , respectively . Heat maps show alternative representation of single traces below each associated time course plot .

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(B-G) Single cell traces of mCherry expression for the "responding" (light gray) or "not responding" (dark gray) MDM2 promoter or CDKN1A promoter cells exposed to the natural dynamics (B), low amplitude (D), high amplitude (D), high frequency (E), low frequency/short duration (F), or long duration (G) Nutlin-3 dosing regimens. The average trace for responding and not responding cells is shown in red and blue, respectively. Heat maps show alternative representation of all single cell traces below each associated time course plot.


Dose - response curves representing rate of mCherry expression from MDM2 ( pink dots ) CDKN1A ( purple dots ) promoters as a function of total p53 . Values of expression were averaged across in first 15 - h response to long duration Nutlin - 3 dosing regimen . Solid black lines represent best fit to a Hill function model of promoter activation . Dashed lines indicate half - maximal threshold of p53 for promoter activation half maximal promoter activity for each promoter .

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Dose-response curves representing the rate of mCherry expression from the MDM2 (pink dots) and CDKN1A (purple dots) promoters as a function of total p53 levels. Values of expression levels were averaged across all cells in the first 15-h response to the long duration Nutlin-3 dosing regimen. Solid black lines represent the best fit to a Hill function model of promoter activation. Dashed lines indicate the half-maximal threshold of p53 levels for promoter activation and the half maximal promoter activity for each promoter.


( B - G ) Effects of p53 duration modulation on target promoter activation in terms of mean timing ( B , E ) , magnitude ( C , F ) , rate ( D , G ) for MDM2 ( B - D ) CDKN1A ( E - G ) promoters in response to short ( green ) long ( green ) p53 durations . N = at least 45 per condition , error bars = .

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(B-G) Effects of p53 duration modulation on target promoter activation in terms of the mean timing (B, E), magnitude (C, F), and rate (D, G) for the MDM2 (B-D) and CDKN1A (E-G) promoters in response to short (light green) and long (dark green) p53 durations. N = at least 45 cells per condition, error bars = SEM.


( H ) Mean MDM2 - YFP in single at times corresponding to first ( 5 . 5 h ) or second ( 11 h ) of p53 expression in response to natural long duration Nutlin - 3 dosing regimens . N = at least 40 per condition , error bars = .

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(H) Mean nuclear MDM2-YFP levels in single cells at times corresponding to the first pulse (5.5 h) or second pulse (11 h) of p53 expression in response to natural and long duration Nutlin-3 dosing regimens. N = at least 40 cells per condition, error bars = SEM.


( A ) Effects of p53 frequency modulation on target promoter activation in terms of mean timing ( A , D ) , magnitude ( B , E ) , rate ( C , F ) for MDM2 ( A - C ) CDKN1A ( D - F ) promoters in response to low ( blue ) , natural ( medium blue ) , high ( blue ) p53 frequencies . N = at least 45 per condition , error bars = .

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(A) Effects of p53 frequency modulation on target promoter activation in terms of the mean timing (A, D), magnitude (B, E), and rate (C, F) for the MDM2 (A-C) and CDKN1A (D-F) promoters in response to low (light blue), natural (medium blue), high (dark blue) p53 frequencies. N = at least 45 cells per condition, error bars = SEM.


( H ) of responding for MDM2 CDKN1A promoters in response to low , natural , high frequency p53 pulses .

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(H) Percentage of responding cells for the MDM2 and CDKN1A promoters in response to low, natural, and high frequency p53 pulses.


( A - B ) Representative phase contrast , red ( indicating geminin - mCherry ) , yellow ( indicating p53 - Venus ) of in response to a low ( A ) or high ( B ) frequency Nutlin - 3 dosing regimen over 40 h .

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(A-B) Representative phase contrast, red fluorescence (indicating geminin-mCherry levels), and yellow fluorescence (indicating p53-Venus levels) images of cells in response to a low (A) or high (B) frequency Nutlin-3 dosing regimen over 40 h.


( C ) Geminin - mCherry traces in single treated with low , natural , or high frequency Nutlin - 3 dosing regimens . N = at least 50 per condition .

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(C) Geminin-mCherry traces in single cells treated with the low, natural, or high frequency Nutlin-3 dosing regimens. N = at least 50 cells per condition.


of undergoing division ( D ) within 40 h imaging in response to low , natural , high frequency Nutlin - 3 dosing

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Percentage of cells undergoing cell division (D) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens


of positive for geminin - mCherry expression ( E ) within 40 h imaging in response to low , natural , high frequency Nutlin - 3 dosing

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Percentage of cells positive for geminin-mCherry expression (E) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens


A Representative immunofluorescence for PH3 ( green ) , combined with DAPI ( ) , of E14 . 5 wildtype ( WT ) 11B mouse dorsolateral neocortex rostrally . B Representative immunofluorescence for pVim ( yellow ) , combined with DAPI ( ) , of E14 . 5 WT 11B mouse dorsolateral neocortex rostrally . Boxes ( 50 µm x 65 µm ) in ( B ) indicate areas that are shown at higher magnification in ( B ' ) . Arrow , pVim + BP with basal process ( mitotic bRG ) ; notched arrowheads , basal process ; arrowhead , pVim + BP without basal process ( mitotic bIP ) . C - E of PH3 + APs ( C ) , PH3 + BPs ( D ) pVim + bRG ( E left ) bIPs ( E right ) in a 200 µm - wide field of E14 . 5 WT ( ) 11B ( black ) mouse dorsolateral neocortex rostrally . Note difference in ordinate scales in ( E ) . F Data information : are single optical sections Scale bars , 50 µm 10 µm Data are mean of 6 - 19 ( WT ) 6 - 26 ( 11B ) littermate , which were derived from 3 - 9 separate litters . Error bars indicate SD , two - unpaired Student ' s t - , * P < 0 . 05 ; * * P < 0 . 01 P = 0 . 0269 ( D ) ; P = 0 . 0302 ( E , left ) ; P = 0 . 0139 ( E , right )

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A Representative immunofluorescence for PH3 (green), combined with DAPI staining (white), of E14.5 wildtype (WT) and 11B mouse dorsolateral neocortex rostrally. B Representative immunofluorescence for pVim (yellow), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (50 µm x 65 µm) in (B) indicate areas that are shown at higher magnification in (B'). Arrow, pVim+ BP with basal process (mitotic bRG); notched arrowheads, basal process; arrowhead, pVim+ BP without basal process (mitotic bIP). C-E Quantification of PH3+ APs (C), PH3+ BPs (D) and pVim+ bRG (E left) and bIPs (E right) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. Note the difference in ordinate scales in (E). F Data information: Images are single optical sections Scale bars, 50 µm 10 µm Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0269 (D); P = 0.0302 (E, left); P = 0.0139 (E, right)


F Representative immunofluorescence for Pax6 ( cyan ) Tbr2 ( magenta ) , combined with DAPI ( ) , of E14 . 5 WT 11B mouse dorsolateral neocortex rostrally . Boxes ( 25 µm x 25 µm ) in ( F ) indicate areas that are shown at higher magnification in ( F ' ) . Outlined by dashed lines , Pax6 + & Tbr2 + BP ; outlined by solid lines , Pax6 + & Tbr2 - BP . G - K of Pax6 + & Tbr2 - in VZ ( G ) , Pax6 + & Tbr2 + in VZ ( H ) , Pax6 + & Tbr2 - in SVZ IZ ( I ) , Pax6 + & Tbr2 + in SVZ IZ ( J ) Pax6 - & Tbr2 + in SVZ IZ ( K ) in a 200 µm - wide field of E14 . 5 WT ( ) 11B ( black ) mouse dorsolateral neocortex rostrally . L Data information : are Z projections of 4 optical sections Scale bars , 50 µm 10 µm Data are mean of 6 - 19 ( WT ) 6 - 26 ( 11B ) littermate , which were derived from 3 - 9 separate litters . Error bars indicate SD , two - unpaired Student ' s t - , * P < 0 . 05 ; * * P < 0 . 01 P = 0 . 0281 ( I ) ; P = 0 . 0088 ( J ) ; P = 0 . 0014 ( K )

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F Representative immunofluorescence for Pax6 (cyan) and Tbr2 (magenta), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (25 µm x 25 µm) in (F) indicate areas that are shown at higher magnification in (F'). Outlined by dashed white lines, Pax6+ & Tbr2+ BP; outlined by solid white lines, Pax6+ & Tbr2- BP. G-K Quantification of Pax6+ & Tbr2- cells in VZ (G), Pax6+ & Tbr2+ cells in VZ (H), Pax6+ & Tbr2- cells in SVZ and IZ (I), Pax6+ & Tbr2+ cells in SVZ and IZ (J) and Pax6- & Tbr2+ cells in SVZ and IZ (K) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. L Data information: Images are Z projections of 4 optical sections Scale bars, 50 µm 10 µm Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0281 (I); P = 0.0088 (J); P = 0.0014 (K)


L - N , Sequential BrdU - EdU labelling . BrdU was injected into pregnant mice at E13 . 5 , i . e . 24 hr before sacrifice at E14 . 5 , EdU was injected into same pregnant mice 0 . 5 hr before sacrifice at E14 . 5 . ( L ) Representative immunofluorescence for BrdU ( magenta ) , combined with EdU ( cyan ) DAPI ( ) , of E14 . 5 WT 11B mouse dorsolateral neocortex rostrally . ( M , N ) of BrdU + & EdU + ( M ) of BrdU + that are BrdU + & EdU + ( N ) in VZ SVZ in a 200 µm - wide field of E14 . 5 WT ( ) 11B ( black ) mouse dorsolateral neocortex rostrally . Data information : Data are mean of 6 - 19 ( WT ) 6 - 26 ( 11B ) littermate , which were derived from 3 - 9 separate litters . Error bars indicate SD , two - unpaired Student ' s t - , * P < 0 . 05 ; * * P < 0 . 01 P = 0 . 0049 ( M , SVZ ) ; P = 0 . 0323 ( N , SVZ ) .

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L-N, Sequential BrdU-EdU labelling. BrdU was injected into pregnant mice at E13.5, i.e. 24 hr before sacrifice at E14.5, and EdU was injected into the same pregnant mice 0.5 hr before sacrifice at E14.5. (L) Representative immunofluorescence for BrdU (magenta), combined with EdU staining (cyan) and DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. (M, N) Quantification of BrdU+ & EdU+ cells (M) and the percentage of BrdU+ cells that are BrdU+ & EdU+ (N) in VZ and SVZ in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. Data information: Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0049 (M, SVZ); P = 0.0323 (N, SVZ).


H Representative immunofluorescence for Tbr1 ( cyan ) , Ctip2 ( magenta ) Satb2 ( green ) , combined with DAPI ( ) , of E18 . 5 WT 11B mouse neocortex at position where cortical thickness was measured ( A , red lines ) . I of zone thickness of E18 . 5 WT ( ) 11B ( black ) mouse neocortex at position where cortical thickness was measured ( A , red lines ) . J Data information : are single optical sections Scale bar , 20 µm Data are mean of 7 ( WT ) 8 - 12 ( 11B ) littermate , which were derived from 4 separate litters . Error bars indicate SD . Two - way ANOVA , followed by Bonferroni ' s multiple comparisons ( B - D ) ; two - unpaired Student ' s t - P = 0 . 0287 ( I , IZ ) ; P = 0 .

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H Representative immunofluorescence for Tbr1 (cyan), Ctip2 (magenta) and Satb2 (green), combined with DAPI staining (white), of E18.5 WT and 11B mouse neocortex at the position where cortical thickness was measured (A, red lines). I Quantification of zone thickness of E18.5 WT (white) and 11B (black) mouse neocortex at the position where cortical thickness was measured (A, red lines). J Data information: Images are single optical sections Scale bar, 20 µm Data are the mean of 7 (WT) and 8-12 (11B) littermate embryos, which were derived from 4 separate litters. Error bars indicate SD. Two-way ANOVA, followed by Bonferroni's multiple comparisons test (B-D); two-tailed unpaired Student's t-test P = 0.0287 (I, IZ); P = 0.0039


A Dorsal view of adult wildtype ( WT ) 11B mouse brain at P56 . Orange lines illustrate measurements of neocortex width ; red lines illustrate measurements of neocortex . B , C of neocortex width , . D Data information : Scale bar , 1 mm Data are mean of 8 ( WT ) 8 ( 11B ) adult littermate mice . Error bars indicate SD , two - unpaired Student ' s t - , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 . P < 0 . 0001 ( B , neocortex width ) ; P < 0 . 0001 ( C )

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A Dorsal view of adult wildtype (WT) and 11B mouse brain at P56. Orange lines illustrate the measurements of neocortex width and length; red lines illustrate the measurements of neocortex area. B, C Quantification of neocortex width, length and area. D Data information: Scale bar, 1 mm Data are the mean of 8 (WT) and 8 (11B) adult littermate mice. Error bars indicate SD, two-tailed unpaired Student's t-test , * P < 0.05, ** P < 0.01, **** P < 0.0001. P < 0.0001 (B, neocortex width); P < 0.0001 (C)


G , H Representative immunofluorescence for Ctip2 ( magenta ) , Satb2 ( green ) Brn2 ( yellow ) , combined with DAPI ( ) , of P56 adult WT 11B mouse neocortex at position where cortical thickness was measured ( D , red lines ) . Data information : are single optical sections Scale bar , 20

O O O O O O B-MEASURED_VAR 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 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

G, H Representative immunofluorescence for Ctip2 (magenta), Satb2 (green) and Brn2 (yellow), combined with DAPI staining (white), of P56 adult WT and 11B mouse neocortex at the position where cortical thickness was measured (D, red lines). Data information: Images are single optical sections Scale bar, 20 µm


IntelliCage was used to evaluate memory flexibility of adult 11B mice . Four separate sessions were carried out : ( i ) 5 WT together with 5 11B ; ( ii ) 5 other WT together with 5 other 11B ; ( iii ) 5 WT together with 7 11B ; ( iv ) 4 other WT together with 8 other 11B . Data are mean of total 10 ( WT ) , 10 ( 11B ) , 9 ( WT ) 15 ( 11B ) adult littermate mice . Data points were obtained at consecutive days , error bars indicate SD . ( A , B ) of discrimination error rate in first 100 visits during drinking phase of acquisition stage of adult wildtype ( WT ) 11B mice ( , A ; , B ) in IntelliCage . Overall place learning performance of WT 11B mice was analysed using two - way ANOVA followed by Fisher ' s LSD ; data points during acquisition phase were included . For ( A ) , WT vs . 11B , P > 0 . 05 ; data over time , * * * * P < 0 . 0001 ; interaction , P > 0 . 05 . For ( B ) , WT vs . 11B , P > 0 . 05 ; data over time , * * * * P < 0 . 0001 ; interaction , P > 0 . 05 . Note that ( i ) two sessions two sessions yielded essentially same results , ( ii ) not shown in panels two separate sessions yielded essentially same results , two separate sessions yielded essentially same results .

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 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 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 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-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

IntelliCage was used to evaluate the memory flexibility of adult 11B mice. Four separate sessions were carried out: (i) 5 WT males together with 5 11B males; (ii) 5 other WT males together with 5 other 11B males; (iii) 5 WT females together with 7 11B females; (iv) 4 other WT females together with 8 other 11B females. Data are the mean of the total 10 males (WT), 10 males (11B), 9 females (WT) and 15 females (11B) adult littermate mice. Data points were obtained at consecutive days, error bars indicate SD. (A, B) Quantification of the discrimination error rate in the first 100 visits during the drinking phase of the acquisition stage of adult wildtype (WT) and 11B mice (male, A; female, B) in the IntelliCage. Overall place learning performance of WT and 11B mice was analysed using two-way ANOVA followed by Fisher's LSD test; all data points during the acquisition phase were included. For males (A), WT vs. 11B, P > 0.05; data over time, **** P < 0.0001; interaction, P > 0.05. For females (B), WT vs. 11B, P > 0.05; data over time, **** P < 0.0001; interaction, P > 0.05. Note that (i) the two male sessions and the two female sessions yielded essentially the same results, and (ii) not shown in panels the two separate male sessions yielded essentially the same results, and the two separate female sessions yielded essentially the same results.


of NANOG - positive iPSC colonies obtained after 14 days of OSKM induction in MEFs treated with either non - target or Kdm3b siRNA in FBS media . Values represent fold change over non - target control providing relative quantitation . of NANOG - positive iPSC colonies obtained after 12 or 14 days of OSKM induction in MEFs treated with either non - targeting or Kdm3b siRNA in KSR media . Values represent fold change over non - target control providing relative quantitation .

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-CONTROLLED_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 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

Quantification of NANOG-positive iPSC colonies obtained after 14 days of OSKM induction in MEFs treated with either non-target or Kdm3b siRNA in FBS media. Values represent fold change over non-target control providing relative quantitation. Quantification of NANOG-positive iPSC colonies obtained after 12 or 14 days of OSKM induction in MEFs treated with either non-targeting or Kdm3b siRNA in KSR media. Values represent fold change over non-target control providing relative quantitation.


of NANOG positive colonies after retroviral expression of OSKM ( Left ) or OSK ( Right ) in MEF treated with non - targeting or siRNA targeting Kdm3 . Nanog were performed on Day 16 for OSKM or Day 21 for OSK reprogramming . Values represent fold change over non - target control providing relative quantitation .

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-CONTROLLED_VAR 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

Quantification of NANOG positive colonies after retroviral expression of OSKM (Left) or OSK (Right) in MEF treated with non-targeting or siRNA targeting Kdm3. Nanog counts were performed on Day 16 for OSKM or Day 21 for OSK reprogramming. Values represent fold change over non-target control providing relative quantitation.


Immunoblot of H3K9me1 / 2 histone demethylase in WT or Kdm3b CRISPR targeted pre - iPSC clones . These pre - iPSCs carry a Nanog - GFP reporter . Parent ( WT ) , two Kdm3b - KO clones using gRNA targeting either exon 1 ( KO # 1 ) or exon 2 ( KO # 2 ) are shown . Polymerase II are loading control .

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Immunoblot of H3K9me1/2 histone demethylase in WT or Kdm3b CRISPR targeted pre-iPSC clones. These pre-iPSCs carry a Nanog-GFP reporter. Parent line (WT), and two Kdm3b-KO clones using gRNA targeting either exon 1 (KO#1) or exon 2 (KO#2) are shown. RNA Polymerase II levels are the loading control.


of Nanog - GFP positive after 10 days of AA + 2i treatment of WT or Kdm3b - deleted pre - iPSCs .

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Quantification of Nanog-GFP positive cells after 10 days of AA+2i treatment of WT or Kdm3b-deleted pre-iPSCs.


Immunoblot of H3K9 methylation upon addition of H3K9 methyltransferase inhibitor UNC0638 in WT or Kdm3b - KO pre - iPSCs .

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

Immunoblot of H3K9 methylation levels upon addition of H3K9 methyltransferase inhibitor UNC0638 in WT or Kdm3b-KO pre-iPSCs.


of Nanog - GFP positive after 10 days of AA + 2i or AA + 2i with 5µM UNC0638 in WT or Kdm3b deleted pre - iPSCs .

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Quantification of percent Nanog-GFP positive cells after 10 days of AA+2i or AA+2i with 5µM UNC0638 in WT or Kdm3b deleted pre-iPSCs.


Pearson correlation of transcripts per million ( TPM ) of WT Kdm3b - KO pre - iPSC upon DMSO or AA + 2i treatment for 2 or 10 days mESC , reprogramming populations from Chronis et . al ( 2017 ) , ( labelled as CC ) .

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Pearson correlation of transcripts per million (TPM) of WT and Kdm3b-KO pre-iPSC upon DMSO or AA+2i treatment for 2 or 10 days and mESC, reprogramming populations from Chronis et. al (2017), (labelled as CC).


k - means clustering of union of DEG ( FDR > 0 . 99 ) between day 2 DMSO in WT Kdm3b - KO pre - iPSCs . Values represent a log2 - fold change of day 2 over DMSO . Key pluripotency genes are shown on right . k - means clustering of union of DEG ( FDR > 0 . 99 ) between day 10 DMSO in WT Kdm3b - KO pre - iPSCs . Values represent a log2 - fold change of day 10 over DMSO . Key pluripotency genes or epigenetic regulators are shown on right .

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 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

k-means clustering of the union of DEG (FDR > 0.99) between day 2 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 2 over DMSO. Key pluripotency genes are shown on the right. k-means clustering of the union of DEG (FDR > 0.99) between day 10 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 10 over DMSO. Key pluripotency genes or epigenetic regulators are shown on the right.


Expression of Tet enzymes Tdg during AA + 2i treatment in WT or Kdm3b - KO pre - iPSCs . Error bars represent standard deviation of two biological replicates from - Seq sample .

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Expression of Tet enzymes and Tdg during AA+2i treatment in WT or Kdm3b-KO pre-iPSCs. Error bars represent the standard deviation of two biological replicates from the RNA-Seq sample.


of Nanog - GFP of WT , Kdm3b - KO Kdm3b - KO pre - iPSC clones expressing Tet1 catalytic domain ( Tet1CD ) , Nanog , or both .

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

Quantification of percent Nanog-GFP cells of WT, Kdm3b-KO and Kdm3b-KO pre-iPSC clones expressing Tet1 catalytic domain (Tet1CD), Nanog, or both.


Representative 5hmC profiles during AA + 2i conversion of WT pre - iPSCs . Illustration of 5hmC pattern is shown in bottom panel .

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Representative 5hmC profiles during AA+2i conversion of WT pre-iPSCs. Illustration of the 5hmC pattern is shown in the bottom panel.


of k - means clustered 5hmC patterns with histone modifications in pre - iPSCs mESCs from Chronis et al ( 2017 ) .

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Heatmap of k-means clustered 5hmC patterns with histone modifications in pre-iPSCs and mESCs from Chronis et al (2017).


Representative profiles of regions that undergo demethylation through a 5hmC intermediate . Top - 5hmC profile during AA + 2i treatment . Middle - methylation via bisulfite sequencing in MEFs iPSCs , data from Rais et al 2013 . Bottom - 5hmC via Tet - assisted bisulfite sequencing of MEFs expressing OSKM for 0 or 5 days , data from Hu et al 2014 .

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Representative profiles of regions that undergo demethylation through a 5hmC intermediate. Top - 5hmC profile during AA+2i treatment. Middle - Percent methylation via bisulfite sequencing in MEFs and iPSCs, data from Rais et al 2013. Bottom - Percent 5hmC via Tet-assisted bisulfite sequencing of MEFs expressing OSKM for 0 or 5 days, data from Hu et al 2014.


Left - of Nanog - GFP positive colonies . Right - Total Nanog - GFP positive colonies obtained after ten days of AA + 2i treatment in Tdg - depleted pre - iPSC ( Tdg ) or control ( Empty ) .

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Left - Quantification of percent Nanog-GFP positive colonies. Right - Total Nanog-GFP positive colonies obtained after ten days of AA+2i treatment in Tdg-depleted pre-iPSC (Tdg) or control (Empty).


of Nanog - GFP positive of WT or Kdm3b - KO pre - IPSC clones expressing Nanog , Tet1CD , or Nanog + Tet1CD after 15 days of AA + 2i or AA + 2i with 4 or 5µM of UNC0638 .

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

Quantification of percent Nanog-GFP positive cells of WT or Kdm3b-KO pre-IPSC clones expressing Nanog, Tet1CD, or Nanog+Tet1CD after 15 days of AA+2i or AA+2i with 4 or 5µM of UNC0638.


Left - of K - mean clustered 5hmC patterns in WT pre - iPSC with 5hmC profile of Kdm3b - KO pre - iPSC treated with AA + 2i for 0 , 2 or 10 days . Middle - Metaplot of 5hmC signal on day 10 between WT ( orange ) Kdm3b - KO ( black ) for each cluster . Y - axis scale is in normalized coverage data ( 1x106 / Total ) . Right - Illustration of 5hmC pattern in WT Kdm3b - KO during AA + 2i treatment .

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 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 B-CONTROLLED_VAR O O O O O O O O

Left - Heatmap of K-mean clustered 5hmC patterns in WT pre-iPSC with 5hmC profile of Kdm3b-KO pre-iPSC treated with AA+2i for 0, 2 or 10 days. Middle - Metaplot of 5hmC signal on day 10 between WT (orange) and Kdm3b-KO (black) for each cluster. Y-axis scale is in normalized coverage data (1x106/Total Count). Right - Illustration of 5hmC pattern in WT and Kdm3b-KO during AA+2i treatment.


Representative 5hmC profiles during AA + 2i conversion of WT Kdm3b - KO pre - iPSC .

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Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC.


Scheme for identifying genes with both misregulated 5hmC expression . of two - fold misregulated 5hmC two - fold differential expression between WT Kdm3b - KO after 10 days of AA + 2i treatment ( 607 genes ) . Peaks are sorted by 5hmC signal in WT . seq values represent a log2 - fold change of day 10 over DMSO for either WT or Kdm3b KO pre - iPSCs .

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Scheme for identifying genes with both misregulated 5hmC and expression. Heatmap of two-fold misregulated 5hmC and two-fold differential expression between WT and Kdm3b-KO after 10 days of AA+2i treatment (607 genes). Peaks are sorted by 5hmC signal in WT. RNA seq values represent a log2-fold change of day 10 over DMSO for either WT or Kdm3b KO pre-iPSCs.


Box plot of expression for genes that are two - fold misregulated 5hmC expression between WT Kdm3b - KO on day 10 ( 607 genes ) .

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Box plot of expression for genes that are two-fold misregulated 5hmC and expression between WT and Kdm3b-KO on day 10 (607 genes).


of POU5F1 bound peaks in both mESC pre - IPSC ( Cluster 1 ) mESC alone ( Cluster 2 ) with 5hmC during Day 0 , 2 10 of AA + 2i treatment .

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Heatmap of POU5F1 bound peaks in both mESC and pre-IPSC (Cluster 1) and mESC alone (Cluster 2) with 5hmC levels during Day 0, 2 and 10 of AA+2i treatment.


Metaplots of POU5F1 binding for Cluster 1 ( Left ) Cluster 2 ( Right ) regions in pre - iPSCs mESCs .

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Metaplots of POU5F1 binding for Cluster 1 (Left) and Cluster 2 (Right) regions in pre-iPSCs and mESCs.


Metaplots of 5hmC Enrichment for Cluster 1 ( Left ) Cluster 2 ( Right ) regions for Day 0 , 2 10 of AA + 2i treatment .

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Metaplots of 5hmC Enrichment for Cluster 1 (Left) and Cluster 2 (Right) regions for Day 0, 2 and 10 of AA+2i treatment.


Left - Scheme for identifying sites that lose 5hmC their association with POU5F1 binding . Right - of 5hmC profile during AA + 2i treatment of WT pre - iPSC POU5F1 binding in pre - iPSCs or mESCs at loci identified in left panel .

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Left - Scheme for identifying sites that lose 5hmC and their association with POU5F1 binding. Right - Heatmap of 5hmC profile during AA+2i treatment of WT pre-iPSC and POU5F1 binding in pre-iPSCs or mESCs at the loci identified in the left panel.


Representative 5hmC profiles during AA + 2i conversion of WT Kdm3b - KO pre - iPSC with POU5F1 binding in pre - iPSCs mESCs . Loci were assigned to nearest gene .

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Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC with POU5F1 binding in pre-iPSCs and mESCs. Loci were assigned to the nearest gene.


ChIP - PCR for POU5F1 during AA + 2i reprogramming of WT Kdm3b - KO pre - iPSCs . Values are fold change of enrichment between WT Kdm3b KO ( WT / Kdm3bKO ) .

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ChIP-PCR for POU5F1 during AA+2i reprogramming of WT and Kdm3b-KO pre-iPSCs. Values are fold change of enrichment between WT and Kdm3b KO (WT/Kdm3bKO).


tSNE of Tet1 , Tet2 , Nanog , Cdh1 expression in single sequencing of MEF undergoing OSKM reprogramming from Tran et al 2019b . MEF , Reprogramming ( REPROG ) , mESCs are highlighted in red dotted lines . Specific reprogramming timepoints are highlighted within reprogramming cluster in arrows . Color corresponds to log10 of transcript for each .

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tSNE of Tet1, Tet2, Nanog, and Cdh1 expression in single cell RNA sequencing of MEF undergoing OSKM reprogramming from Tran et al 2019b. MEF, Reprogramming (REPROG), and mESCs cells are highlighted in red dotted lines. Specific reprogramming timepoints are highlighted within the reprogramming cluster in arrows. Color corresponds to log10 of transcript counts for each individual cell.


Bar graphs indicating number of expressing Tet1 , Tet2 , Cdh1 or Nanog from reprograming population ( Day 3 , 6 , 9 12 ) . Number of per reprogramming population is highlighted within legend .

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

Bar graphs indicating number of cells expressing Tet1, Tet2, Cdh1 or Nanog from the reprograming population (Day 3, 6, 9 and 12). Number of cells per reprogramming population is highlighted within legend.


Scheme for deriving Tet1 Tet2 double knockout pre - iPSC lines . Tet1KO / KO Tet2fl / fl or Tet1WT / WT Tet2fl / fl MEFs were treated with retroviruses containing OSKM . Tet1KO / KO Tet2fl / fl or Tet1WT / WT Tet2fl / fl pre - iPSC clones were isolated treated with either Adenoviral expressed Cre recombinase or Empty vector to generate a population of pre - iPSCs with Tet1KO / KO Tet2fl / fl , Tet1KO / KO Tet2KO / KO , Tet1WT / WT Tet2fl / fl , Tet1WT / WT , Tet2KO / KO . Scale bar , 50 μm .

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

Scheme for deriving Tet1 and Tet2 double knockout pre-iPSC lines. Tet1KO/KO Tet2fl/fl or Tet1WT/WT Tet2fl/fl MEFs were treated with retroviruses containing OSKM. Tet1KO/KO Tet2fl/fl or Tet1WT/WT Tet2fl/fl pre-iPSC clones were isolated and treated with either Adenoviral expressed Cre recombinase or Empty vector to generate a population of pre-iPSCs with Tet1KO/KO Tet2fl/fl, Tet1KO/KO Tet2KO/KO, Tet1WT/WT Tet2fl/fl, and Tet1WT/WT, and Tet2KO/KO. Scale bar, 50 μm.


of Nanog positive by intracellular flow cytometry in each genotype as indicated of Tet1WT / WT Tet2fl / fl , Tet1WT / WT Tet2KO / KO , Tet1KO / KO Tet2fl / fl , Tet1KO / KO Tet2KO / KO after 10 days of AA + 2i treatment .

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

Quantification of percent Nanog positive cells by intracellular flow cytometry in each genotype as indicated of Tet1WT/WT Tet2fl/fl, Tet1WT/WT Tet2KO/KO, Tet1KO/KO Tet2fl/fl, and Tet1KO/KO Tet2KO/KO after 10 days of AA+2i treatment.


RT - PCR of SPPL2c expression in different murine tissues . Total from indicated tissues was either transcribed into cDNA prior to PCR amplification ( + RT ) or , as negative control , used directly as template ( - RT ) . A fragment of actin ORF was amplified as control .

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 B-CONTROLLED_VAR 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

RT-PCR analysis of SPPL2c expression in different murine tissues. Total RNA from the indicated tissues was either transcribed into cDNA prior to PCR amplification (+RT) or, as negative control, used directly as template (-RT). A fragment of the actin ORF was amplified as control.


Western Blot of total lysates from testis isolated from wild type or SPPL2c - / - using antibodies against C - ( D ) or N - terminal ( E ) epitopes of SPPL2c . Lysates from HeLa transiently expressing SPPL2c isoforms were analysed in parallel .

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Western Blot analysis of total lysates from testis isolated from wild type or SPPL2c-/- using antibodies against C- (D) or N-terminal (E) epitopes of SPPL2c. Lysates from HeLa cells transiently expressing the SPPL2c isoforms were analysed in parallel.


SPPL2c was visualised by indirect immunofluorescence in HeLa transiently expressing C - terminally Myc - tagged murine SPPL2c isoform A . For detection of SPPL2c , either anti - Myc or SPPL2c antiserum ( C - terminal epitope ) were employed as indicated together with anti - KDEL , anti - GM130 or anti - ERGIC53 as indicated in order to label ER , cis Golgi apparatus or ER - Golgi intermediate compartment ( ERGIC ) , respectively .

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SPPL2c was visualised by indirect immunofluorescence in HeLa cells transiently expressing C-terminally Myc-tagged murine SPPL2c isoform A. For detection of SPPL2c, either anti-Myc or the SPPL2c antiserum (C-terminal epitope) were employed as indicated together with anti-KDEL, anti-GM130 or anti-ERGIC53 as indicated in order to label the ER, the cis Golgi apparatus or the ER-Golgi intermediate compartment (ERGIC), respectively.


Lysates from HEK293 transiently transfected with murine SPPL2c - myc ( isoform A ) were treated with glycosidases PNGase F or Endo H as indicated prior to Western blot with anti - SPPL2c ( C - term ) . Deglycosylation of endogenously expressed LIMP2 was analysed as control .

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Lysates from HEK293 cells transiently transfected with murine SPPL2c-myc (isoform A) were treated with the glycosidases PNGase F or Endo H as indicated prior to Western blot analysis with anti-SPPL2c (C-term). Deglycosylation of endogenously expressed LIMP2 was analysed as control.


N - glycosylation of endogenous SPPL2c is similar to overexpressed . Total lysates from testis of wild type or SPPL2c - / - mice were

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N-glycosylation of endogenous SPPL2c is similar to the overexpressed protein. Total lysates from testis of wild type or SPPL2c-/- mice were treated


Subcellular fractionation of postnuclear supernatant from murine testis using a self - forming Percoll density gradient . Fractions were collected from bottom to top subjected to Western blot for SPPL2c . disulfide isomerase ( PDI ) , Cathepsin D CD44 were detected as organelle marker .

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Subcellular fractionation of postnuclear supernatant from murine testis using a self-forming Percoll density gradient. Fractions were collected from bottom to top and subjected to Western blot analysis for SPPL2c. Protein disulfide isomerase (PDI), Cathepsin D and CD44 were detected as organelle marker proteins.


HEK293 were transiently transfected with murine N - terminally HA - tagged heme oxygenase 1 ( HA - mHO - 1 ) alone or in combination with active or inactive ( D / A ) murine SPP or SPPL2c . Substrate were determined by Western blot with anti - HA . For detection of SPP , an antiserum recognizing both endogenous human ( h ) overexpressed murine ( m ) SPP was employed . Murine SPPL2c was visualised using antiserum generated against C - terminus of . Actin was detected to control for equal loading .

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HEK293 cells were transiently transfected with murine N-terminally HA-tagged heme oxygenase 1 (HA-mHO-1) alone or in combination with active or inactive (D/A) murine SPP or SPPL2c. Substrate levels were determined by Western blot analysis with anti-HA. For detection of SPP, an antiserum recognizing both endogenous human (h) and overexpressed murine (m) SPP was employed. Murine SPPL2c was visualised using the antiserum generated against the C-terminus of the protein. Actin was detected to control for equal protein loading.


Immunofluorescence of HeLa transiently expressing murine HO - 1 ( HA - mHO - 1 ) alone or together with active or inactive ( D / A ) SPPL2c - myc . Substrate proteases were visualised using HA Myc epitopes , respectively .

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Immunofluorescence analysis of HeLa cells transiently expressing murine HO-1 (HA-mHO-1) alone or together with active or inactive (D/A) SPPL2c-myc. Substrate and proteases were visualised using the HA and Myc epitopes, respectively.


TA Ube2J1 is not proteolysed by co - expressed SPP or SPPL2c in HEK293 . Western blot was

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The TA protein Ube2J1 is not proteolysed by co-expressed SPP or SPPL2c in HEK293 cells. Western blot analysis was performed


Differential cleavage of CYB5a , RAMP4 - 2 RAMP4 by SPPL2c SPP . Substrates proteases were co - expressed in HEK293 followed by Western blot as described above . Densitometric from at least n = 3 independent experiments was performed . Substrate were normalised compared to without protease - overexpression .

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Differential cleavage of CYB5a, RAMP4-2 and RAMP4 by SPPL2c and SPP. Substrates and proteases were co-expressed in HEK293 cells followed by Western blot analysis as described above. Densitometric quantification from at least n=3 independent experiments was performed. Substrate levels were normalised and compared to cells without protease-overexpression.


HEK293 expressing HA - RAMP4 - 2 alone or in combination with SPPL2c or SPP were treated with 100 µM ( Z - LL ) 2 - ketone ( ZLL ) , 5 µM inhibitor X ( InX ) , 5 µM DAPT or DMSO as control . RAMP4 - 2 band was quantified from blots of three independent experiments normalised to just over - expressing substrate .

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HEK293 cells expressing HA-RAMP4-2 alone or in combination with SPPL2c or SPP were treated with 100 µM (Z-LL)2-ketone (ZLL), 5 µM inhibitor X (InX), 5 µM DAPT or DMSO as control. RAMP4-2 band intensity was quantified from blots of three independent experiments and normalised to cells just over-expressing the substrate.


Microsomes isolated from testis of wild type SPPL2c - / - mice were solubilised in 1 % digitonin were separated by blue native PAGE . After transfer to PVDF , SPPL2c SPP were detected using polyclonal antisera introduced above .

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Microsomes isolated from testis of wild type and SPPL2c-/- mice were solubilised in 1% digitonin and proteins were separated by blue native PAGE. After transfer to PVDF membrane, SPPL2c and SPP were detected using the polyclonal antisera introduced above.


Histochemical visualization of β - galactosidase ( β - gal ) reporter activity revealed SPPL2c expression within seminiferous tubules . Cryo - sections from PFA - fixed wild type SPPL2c - / - testis were stained with X - Gal as β - gal substrate .

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Histochemical visualization of β-galactosidase (β-gal) reporter activity revealed SPPL2c expression within seminiferous tubules. Cryo-sections from PFA-fixed wild type and SPPL2c-/- testis were stained with X-Gal as β-gal substrate.


SPPL2c were analysed in germ populations from testis of wild type mice sorted for their content which was determined by Hoechst 33342 as depicted in Fig EV4D . While for 4C , 2C 1C fractions 10 µg of were loaded , for testis control samples ( = total lysates ) 20 µg of were subjected to electrophoresis . * , non - specific band .

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SPPL2c protein levels were analysed in germ cell populations from testis of wild type mice sorted for their DNA content which was determined by Hoechst 33342 staining as depicted in Fig EV4D. While for 4C, 2C and 1C fractions 10 µg of protein were loaded, for the testis control samples (=total lysates) 20 µg of protein were subjected to electrophoresis. *, non-specific band.


Immunohistochemical detection of SPPL2c was performed on cryosections of Bouin - fixed wild type SPPL2c - / - testis . SPPL2c antiserum directed against a C - terminal epitope of protease diaminobenzidine as peroxidase substrate were employed . Nuclei were stained with hematoxylin . Scale bar , 100 µm ( upper panel ) or 50 µm ( lower panel ) .

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Immunohistochemical detection of SPPL2c was performed on cryosections of Bouin-fixed wild type and SPPL2c-/- testis. The SPPL2c antiserum directed against a C-terminal epitope of the protease and diaminobenzidine as peroxidase substrate were employed. Nuclei were stained with hematoxylin. Scale bar, 100 µm (upper panel) or 50 µm (lower panel).


SPPL2c were compared in total lysates prepared from testis of wild type mice sacrificed at of 4 , 6 , 8 or 12 by Western blotting . A testis lysate from an adult SPPL2c - / - mouse was used as negative control .

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SPPL2c levels were compared in total lysates prepared from testis of wild type mice sacrificed at the age of 4, 6, 8 or 12 weeks by Western blotting. A testis lysate from an adult SPPL2c-/- mouse was used as negative control.


of both testicles of wild type SPPL2c - / - mice ( n = 12 per genotype ) was determined normalised to .

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The weight of both testicles of wild type and SPPL2c-/- mice (n=12 per genotype) was determined and normalised to the body weight.


Paraffin sections from Bouin - fixed wild type SPPL2c - / - testis were stained with hematoxylin & eosin ( H & E ) . Scale bars , 100 µm .

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Paraffin sections from Bouin-fixed wild type and SPPL2c-/- testis were stained with hematoxylin & eosin (H&E). Scale bars, 100 µm.


In H & E stained paraffin - sections from Bouin - fixed wild type SPPL2c - / - testis , mean number of elongated spermatids per seminiferous tubule was determined . were counted in 20 tubuli in sections from n = 6 mice per genotype .

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In H&E stained paraffin-sections from Bouin-fixed wild type and SPPL2c-/- testis, the mean number of elongated spermatids per seminiferous tubule was determined. Cells were counted in 20 tubuli in sections from n=6 mice per genotype.


SPPL2c is not present in mature spermatozoa . Western blot of total lysates of wild type ( + / + ) SPPL2c - / - testis , epididymis spermatozoa recovered from cauda epididymidis / vas deferens for SPPL2c .

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SPPL2c is not present in mature spermatozoa. Western blot analysis of total lysates of wild type (+/+) and SPPL2c-/- testis, epididymis and spermatozoa recovered from the cauda epididymidis/vas deferens for SPPL2c.


Mature sperm were isolated from epididymis of wild type or SPPL2c - deficient mice analysed by phase - contrast microscopy . Representative pictures of morphologically normal spermatozoa are depicted . Scale bars , 10 µm .

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Mature sperm were isolated from the epididymis of wild type or SPPL2c-deficient mice and analysed by phase-contrast microscopy. Representative pictures of morphologically normal spermatozoa are depicted. Scale bars, 10 µm.


Reduced motility of SPPL2c - / - sperm . Spermatozoa were recovered from cauda epididymidis of wild type SPPL2c - / - mice ( n = 3 ) subjected to computer - assisted sperm . Average path velocity ( VAP ) , straight velocity ( VSL ) curvilinear velocity ( VCL ) were determined in micrometer per second .

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Reduced motility of SPPL2c-/- sperm cells. Spermatozoa were recovered from the cauda epididymidis of wild type and SPPL2c-/- mice (n=3) and subjected to computer-assisted sperm analysis. Average path velocity (VAP), straight line velocity (VSL) and curvilinear velocity (VCL) were determined in micrometer per second.


Mean litter sizes for different breeding schemes . data depicted are on n = 12 ( + / + X + / + ) , n = 57 ( + / - X + / - ) , n = 11 ( - / - X - / - ) , n = 10 ( - / - X + / + ) n = 11 ( + / + X - / - ) different breedings , respectively .

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Mean litter sizes for different breeding schemes. The data depicted are based on n=12 (+/+X+/+), n=57 (+/-X+/-), n=11 (-/-X-/-), n=10 (-/-X+/+) and n=11 (+/+X-/-) different breedings, respectively.


Carbonate - washed total fractions from wild type SPPL2c - / - testis ( n = 5 ) were subjected to a label - free quantitative proteome . For each quantified , a log2 between SPPL2c - deficient wild type samples was calculated . Negative values indicate a depletion positive values an enrichment in SPPL2c - / - samples . In depicted volcano plot negative log10 of p value ( y axis ) is plotted versus log2 of SPPL2c - / - versus wild type samples ( x axis ) . above significance level of p < 0 . 01 ( unpaired Student ' s t ) are coloured in red . hyperbolic curve indicates threshold for a permutation - FDR correction for multiple hypotheses with p = 0 . 05 s0 = 0 . 1 .

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Carbonate-washed total membrane fractions from wild type and SPPL2c-/- testis (n=5) were subjected to a label-free quantitative proteome analysis. For each quantified protein, a log2 intensity ratio between SPPL2c-deficient and wild type samples was calculated. Negative values indicate a depletion and positive values an enrichment in SPPL2c-/- samples. In the depicted volcano plot the negative log10 of the p value (y axis) is plotted versus the log2 intensity ratio of SPPL2c-/- versus wild type samples (x axis). All proteins above the significance level of p<0.01 (unpaired Student's t test) are coloured in red. The hyperbolic curve indicates the threshold for a permutation-based FDR correction for multiple hypotheses with p = 0.05 and s0 = 0.1.


HeLa were transiently transfected with N - terminally HA - tagged PLN ( HA - PLN ) inactive ( D / A ) SPPL2c - myc expressed were visualised by indirect immunofluorescence via their HA Myc epitopes .

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HeLa cells were transiently transfected with N-terminally HA-tagged PLN (HA-PLN) and inactive (D/A) SPPL2c-myc The expressed proteins were visualised by indirect immunofluorescence via their HA and Myc epitopes.


HEK293 were transiently transfected with HA - PLN alone or in combination with active or inactive ( D / A ) murine SPPL2c Isoform A ( D ) or Isoform B ( E ) . Substrate processing was analysed by Western blotting with anti - HA . SPPL2c expression was confirmed with antiserum against C - terminal epitope . Actin was detected to confirm equal loading .

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HEK293 cells were transiently transfected with HA-PLN alone or in combination with active or inactive (D/A) murine SPPL2c Isoform A (D) or Isoform B (E). Substrate processing was analysed by Western blotting with anti-HA. SPPL2c expression was confirmed with the antiserum against the C-terminal epitope. Actin was detected to confirm equal protein loading.


Subcellular localisation of PLN mutants was analysed by indirect immunofluorescence using anti - HA in combination with anti - disulphide isomerase A6 ( PDIA6 ) to stain ER . 37 - 41A 47 - 51 PLN mutants were not expressed in relevant amounts .

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Subcellular localisation of the PLN mutants was analysed by indirect immunofluorescence using anti-HA in combination with anti-protein disulphide isomerase A6 (PDIA6) to stain the ER. The 37-41A and 47-51 PLN mutants were not expressed in relevant amounts.


HEK293 were transiently transfected with wild type or mutated HA - PLN alone or together with SPPL2c isoform A .

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HEK293 cells were transiently transfected with wild type or mutated HA-PLN alone or together with SPPL2c isoform A.


of n = 5 experiments as shown in ( H ) . For each PLN variant , residual PLN in SPPL2c co - expressing is depicted as % of PLN in absence of protease .

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Quantification of n=5 experiments as shown in (H). For each PLN variant, the residual PLN in SPPL2c co-expressing cells is depicted as % of PLN in absence of the protease.


Accumulation of endogenous PLN in testis of SPPL2c - / - mice . PLN was immuno - precipitated ( IP ) from equal amounts of total lysates of wild type or SPPL2c - / - testis with mouse monoclonal antibody . Western blot of IPs was performed with rabbit monoclonal PLN antibody . SPPL2c actin were detected from total lysates directly .

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Accumulation of endogenous PLN in the testis of SPPL2c-/- mice. PLN was immuno-precipitated (IP) from equal protein amounts of total lysates of wild type or SPPL2c-/- testis with the mouse monoclonal antibody. Western blot analysis of the IPs was performed with the rabbit monoclonal PLN antibody. SPPL2c and actin were detected from total lysates directly.


of normalised PLN / Actin ratios from n = 3 biological replicates per genotype .

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Quantification of normalised PLN/Actin ratios from n=3 biological replicates per genotype.


Expression of PLN in murine testis from wild type ( wt ) SPPL2c - / - ( 2c - / - ) was quantified by qPCR .

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Expression of PLN in murine testis from wild type (wt) SPPL2c-/- (2c-/-) was quantified by qPCR.


Immunohistochemical detection of PLN in paraformaldehyde ( PFA ) - fixed testis from wild type SPPL2c - / - mice . Cryosections were stained using a rabbit monoclonal PLN antibody or normal rabbit IgG as negative control diaminobenzidine as peroxidase substrate .

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Immunohistochemical detection of PLN in paraformaldehyde (PFA)-fixed testis from wild type and SPPL2c-/- mice. Cryosections were stained using a rabbit monoclonal PLN antibody or normal rabbit IgG as negative control and diaminobenzidine as peroxidase substrate.


Testis suspensions were stained with 2 . 5 µM DRAQ5 to determine amount . 1C - co = elongated spermatids , 1C = other haploid including round spermatids , 2C = spermatogonia , secondary spermatocytes , Sertoli , other somatic , 4C = spermatocytes , G2 spermatogonia .

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Testis suspensions were stained with 2.5 µM DRAQ5 to determine cellular DNA amount. 1C-co = elongated spermatids, 1C = other haploid cells including round spermatids, 2C = spermatogonia, secondary spermatocytes, Sertoli cells, other somatic cells, 4C = primary spermatocytes, G2 spermatogonia.


Intracellular Ca2 + concentrations were analysed in testis suspensions from either wild type or SPPL2c - deficient mice using Ca2 + - sensitive probe Fluo4 - AM . germ populations were identified Bars indicate Median Fluo4 - AM ± SD of 8 samples from 3 mice per genotype normalised to those of wild type samples .

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Intracellular Ca2+ concentrations were analysed in testis suspensions from either wild type or SPPL2c-deficient mice using the Ca2+-sensitive probe Fluo4-AM. Individual germ cell populations were identified Bars indicate Median Fluo4-AM fluorescence ± SD of 8 individual samples from 3 mice per genotype normalised to those of wild type samples.


Median forward scatter ( FSC ) was controlled in 1C - co population from datasets Median FSC ± SD of 8 samples from 3 mice per genotype .

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Median forward scatter (FSC) was controlled in the 1C-co population from the datasets Median FSC ± SD of 8 individual samples from 3 mice per genotype.


Expression of SPPL2c in human testis . ( H ) Western blot of lysates from HEK293 expressing human ( h ) SPPL2c , human murine testis using antisera against a C - terminal epitope of murine SPPL2c or human SPPL2c as indicated .

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Expression of SPPL2c in human testis. (H) Western blot analysis of lysates from HEK293 cells expressing human (h) SPPL2c, human and murine testis using antisera against a C-terminal epitope of murine SPPL2c or human SPPL2c as indicated.


Immunohistochemical detection of SPPL2c with monoclonal 1A1 antibody in paraffin - sections from Bouin ' s - fixed human testis . In addition , sections were incubated with control immunoglobulins . Arrows indicate elongated spermatids .

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Immunohistochemical detection of SPPL2c with the monoclonal 1A1 antibody in paraffin-sections from Bouin's-fixed human testis. In addition, sections were incubated with control immunoglobulins. Arrows indicate elongated spermatids.


A intrinsic of NAD ( P ) H co - localizes extensively with mitochondria ( marked by Mitotracker CMXRos )

A The intrinsic fluorescence of NAD(P)H co-localizes extensively with mitochondria (marked by Mitotracker CMXRos)


B 2P - FLIM detects fraction of NAD ( P ) H bound to enzyme partners ( a2 % )

B 2P-FLIM detects the fraction of NAD(P)H bound to enzyme partners (a2%)