Mice were superinfected as described in Fig 3A ( C ) Concentrations of IL - 17 IL - 22 in BAL of mice 12 h after with 2x105 cfu of S . pneumoniae serotype 19F alone ( - Flu ) , or following superinfection ( + Flu ) , assessed by ELISA Data information Dat i ( C ) are representative of two independent experiments with 3 - 4 mice per group . data are represented as mean ± S . E . M . Significanc was determined by unpaired student ' s T - . n . s . = not

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Mice were superinfected as described in Fig 3A (C) Concentrations of IL-17 and IL-22 in BAL of mice 12 h after with 2x105 cfu of S. pneumoniae serotype 19F alone (-Flu), or following superinfection (+Flu), assessed by ELISA Data information Dat i (C) are representative of two independent experiments with 3-4 mice per group. All data are represented as mean ± S.E.M. Significanc was determined by unpaired student's T-test. n.s. = not significant


Mice were superinfected as described in Fig 3A . ( A ) load as determined by enumeration of cfu in BAL following administration of αIL - 17 or αIL - 22 blocking antibody . Control wild - type T - bet - / - groups were administered with IgG isotype control antibody Data information Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) data are represented as mean ± S . E . M . Statistical significance was determined with Kruskal - Wallis followed by Dunn " s multiple comparisons n . s . = not

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Mice were superinfected as described in Fig 3A. (A) Bacterial load as determined by enumeration of cfu in BAL following administration of αIL-17 or αIL-22 blocking antibody. Control wild-type and T-bet-/- groups were administered with IgG isotype control antibody Data information Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined with Kruskal-Wallis test followed by Dunn"s multiple comparisons test n.s. = not significant


Mice were superinfected as described in Fig 3A ( B ) T - bet - / - mice were administered αIL - 17 or IgG isotype control antibody every other day from day - 2 post - secondary infection monitored for survival Data information : Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) data are represented as mean ± S . E . M . Statistical significance was determined wit logrank in (

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Mice were superinfected as described in Fig 3A (B) T-bet-/- mice were administered αIL-17 or IgG isotype control antibody every other day from day -2 post-secondary bacterial infection and monitored for survival Data information: Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined wit logrank test in (B


Mice were superinfected as described in Fig 3A ( C ) Absolute neutrophil in lungs following administration of αIL - 17 or IgG isotype control antibody , as determined by flow cytometry Data information Dat are representative of two independent experiments with 3 - 4 in ( C mice per group data are represented as mean ± S . E . M . Statistical significance was determined wit unpaired - students t - in (

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Mice were superinfected as described in Fig 3A (C) Absolute neutrophil counts in the lungs following administration of αIL-17 or IgG isotype control antibody, as determined by flow cytometry Data information Dat are representative of two independent experiments with 3-4 in (C mice per group All data are represented as mean ± S.E.M. Statistical significance was determined wit unpaired-students t-test in (C


Mice were superinfected as described in Fig 3A ( D ) Pneumococcal cfu in BAL following αLy6G antibody mediated neutrophil depletion . Control T - bet - / - mice were administered with IgG isotype control antibody Data information : Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) data are represented as mean ± S . E . M . Statistical significance was determined wit Mann - Whitney U in (

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Mice were superinfected as described in Fig 3A (D) Pneumococcal cfu in BAL following αLy6G antibody mediated neutrophil depletion. Control T-bet-/- mice were administered with IgG isotype control antibody Data information: Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined wit Mann-Whitney U test in (D


Mice were superinfected as described in Fig 3A ( E ) mRNA expression of antimicrobial peptides in lungs following administration of αIL - 17 , IL - 22 or IgG control antibody , as determined by qPCR Data information Dat are representative of two independent experiments wit 4 - 6 in ( E ) mice per group . data are represented as mean ± S . E . M . Statistical significance was determined wit one - way ANOVA followed by Tukey " s multiple comparisons in ( E ) . n . s . = not

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Mice were superinfected as described in Fig 3A (E) mRNA expression of antimicrobial peptides in lungs following administration of αIL-17, IL-22 or IgG control antibody, as determined by qPCR Data information Dat are representative of two independent experiments wit 4-6 in (E) mice per group. All data are represented as mean ± S.E.M. Statistical significance was determined wit one-way ANOVA followed by Tukey"s multiple comparisons test in (E). n.s. = not signif


Mice were superinfected as described in Fig 3A ( A ) Frequency of IFNγ + IL - 17 + immune following intracellular cytokine flow cytometry Data information : Data are representative of at least two independent experiments with 3 mice per group . data are represented as mean ± S . E . M . Statistical significanc was determined by unpaired student ' s T -

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Mice were superinfected as described in Fig 3A (A) Frequency of IFNγ+ and IL-17+ immune cells following intracellular cytokine staining and flow cytometry Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M. Statistical significanc was determined by unpaired student's T-test


Mice were superinfected as described in Fig 3A ( B ) Representative flow cytometry plots of total IL - 17 + lymphocyte subsets on day 3 post - secondary infected lungs following intracellular cytokine . Numbers in plots indicate frequency of each IL - 17 + subset over total numbers of respective - types Data information : Data are representative of at least two independent experiments with 3 mice per group . data are represented as mean ± S . E . M

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Mice were superinfected as described in Fig 3A (B) Representative flow cytometry plots of total and IL-17+ lymphocyte subsets on day 3 post-secondary bacterial infected lungs following intracellular cytokine staining. Numbers in plots indicate the frequency of each IL-17+ cell subset over total numbers of respective cell-types Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M


Mice were superinfected as described in Fig 3A ( C ) Frequency over total IL - 17 + absolute numbers of each IL - 17 producing lymphocyte subset on days 0 , 1 3 post - secondary superinfected lungs as assessed by flow cytometry of intracellular cytokine stained Data information : Data are representative of at least two independent experiments with 3 mice per group . data are represented as mean ± S . E . M . Statistical significanc i ( C ) was determined by unpaired student ' s T -

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Mice were superinfected as described in Fig 3A (C) Frequency over total IL-17+ cells and absolute numbers of each IL-17 producing lymphocyte subset on days 0, 1 and 3 post-secondary bacterial superinfected lungs as assessed by flow cytometry of intracellular cytokine stained cells Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M. Statistical significanc i (C) was determined by unpaired student's T-test


Clustered of whole chemical genomics screen ; each subsection belongs to an ' s KO library . Grey indicate missing values .

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Clustered heatmap of the whole chemical genomics screen; each subsection belongs to an individual strain's KO library. Grey cells indicate missing values.


S - score scatterplots for each pairwise comparison , highlighting conserved ( black points ) , significant changes ( red points ) gene - condition relationships for which no call can reliably be made ( grey points ) . " r " , pearson ' s r value .

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S-score scatterplots for each pairwise strain comparison, highlighting conserved phenotypes (black points), significant changes (red points) and gene-condition relationships for which no call can reliably be made (grey points). "r", pearson's r value.


Fraction of deletion in each conserved with other stains in pairwise , three - way four - way comparisons . Error bars represent standard deviation for pairwise three - way comparisons . Only a four - way comparison is possible for each so no error bars are represented in these cases .

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Fraction of deletion phenotypes in each strain conserved with other stains in pairwise, three-way and four-way comparisons. Error bars represent standard deviation for all pairwise and three-way comparisons. Only a four-way comparison is possible for each strain so no error bars are represented in these cases.


Gene exclusiveness : a measure of each gene ' s propensity to change its chemical genomics profile across strains . top 10 genes ' names are reported .

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Gene exclusiveness: a measure of each gene's propensity to change its chemical genomics profile across strains. The top 10 genes' names are reported.


Barplots reporting average number of gain sick that are specific to each across pairwise comparisons .

Barplots reporting the average number of gain and sick phenotypes that are specific to each strain across all pairwise comparisons.


Wild type fitness of each relative to S288C across same conditions as in panel A ; each dot represents a specific replicate where colony sizes were measured .

Wild type fitness of each strain relative to S288C across the same conditions as in panel A; each dot represents a specific replicate where colony sizes were measured.


Relationship between wild type fitness relative to S288C number of conditionally essential genes relative to S288C ; each dot represents a - condition replicate as in panel B . conditions maltose , glycerol , NaCl are highlighted .

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Relationship between the wild type fitness relative to S288C and the number of conditionally essential genes relative to S288C; each dot represents a strain-condition replicate as in panel B. The conditions maltose, glycerol, and NaCl are highlighted.


Changes in gene deletion for growth on osmotic stress conditions . top contains genes belonging to HOG pathway , while bottom one those genes whose growth varies most between Y55 YPS . Significant growth are marked with " - " .

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Changes in gene deletion phenotypes for growth on osmotic stress conditions. The top heatmap contains genes belonging to the HOG pathway, while the bottom one those genes whose growth phenotypes varies the most between Y55 and YPS. Significant growth phenotypes are marked with "-".


Changes in deletion for growth on glycerol maltose . top contains MAL genes , while bottom one those genes whose growth vary most between S288C other three strains . Significant growth are marked with " - " .

Changes in deletion phenotypes for growth on glycerol and maltose. The top heatmap contains the MAL genes, while the bottom one those genes whose growth phenotypes vary the most between S288C and the other three strains. Significant growth phenotypes are marked with "-".


S - score of yeast natural isolates across 34 conditions that were also used in KO screening . B ) Number of variants significantly associated ( p - value < 1E - 6 ) with phenotypic variation in each growth condition . C ) Number of associated variants that overlap ( i . e . are in a 3kbp window ) with a conditionally essential gene in same condition , in any of four KO libraries .

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S-score heatmap of the yeast natural isolates across 34 conditions that were also used in the KO screening. B) Number of variants significantly associated (p-value < 1E-6) with phenotypic variation in each growth condition. C) Number of associated variants that overlap (i.e. are in a 3kbp window) with a conditionally essential gene in the same condition, in any of the four KO libraries.


Manhattan plots showing examples of overlaps between associated variants KO screening . top plot shows associations between variants growth in natural isolates as a function of - log10 of association p - value , while four bottom plots show strength of KO across four yeast strains , as a function of - log10 of corrected S - score p - value . Sections shaded in grey indicate overlap between associations KO data . Position in yeast chromosome is reported in kilobase units .

Manhattan plots showing examples of overlaps between associated variants and the KO screening. The top plot shows the associations between variants and growth in the natural isolates as a function of the -log10 of the association p-value, while the four bottom plots show the strength of the KO phenotypes across the four yeast strains, as a function of the -log10 of the corrected S-score p-value. Sections shaded in grey indicate the overlap between associations and KO data. Position in the yeast chromosome is reported in kilobase units.


C . RADX interacts with ssDNA . Extracts of U2OS stably expressing GFP - RADX WT or mutants were incubated with biotin - coupled ssDNA oligo immobilized on Streptavidin beads , washed extensively immunoblotted with GFP RPA1 antibodies .

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C. RADX interacts with ssDNA. Extracts of U2OS cells stably expressing GFP-RADX WT or mutants were incubated with biotin-coupled ssDNA oligo immobilized on Streptavidin beads, washed extensively and immunoblotted with GFP and RPA1 antibodies.


D . ssDNA - bound Streptavidin beads incubated with extracts as in ( C ) were washed with buffer containing indicated salt concentrations prior to immunoblotting .

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D. ssDNA-bound Streptavidin beads incubated with cell extracts as in (C) were washed with buffer containing indicated salt concentrations prior to immunoblotting.


E . Lysates of untransfected HCT116 were incubated with immobilized ssDNA or dsDNA probes as in ( C ) immunoblotted with RADX RPA1 antibodies .

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E. Lysates of untransfected HCT116 cells were incubated with immobilized ssDNA or dsDNA probes as in (C) and immunoblotted with RADX and RPA1 antibodies.


F . Immunoblot of RADX expression in human lines . See also Fig EV1B .

F. Immunoblot analysis of RADX expression in human cell lines. See also Fig EV1B.


G . Representative from in situ proximity ligation assays ( PLAs ) in BrdU - labeled U2OS GFP / GFP - RADX lines ( Fig EV1E , F ) using GFP BrdU antibodies under native conditions . Scale bar , 10 μm .

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G. Representative images from in situ proximity ligation assays (PLAs) in BrdU-labeled U2OS and GFP/GFP-RADX cell lines (Fig EV1E,F) using GFP and BrdU antibodies under native conditions. Scale bar, 10 μm.


I . Soluble - enriched fractions of U2OS lines stably expressing GFP - RADX alleles were immunoblotted with indicated antibodies .

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I. Soluble and chromatin-enriched fractions of U2OS cell lines stably expressing GFP-RADX alleles were immunoblotted with indicated antibodies.


A . Immunoblot of fractions of U2OS transfected with indicated siRNAs treated or not with HU .

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A. Immunoblot analysis of chromatin fractions of U2OS cells transfected with indicated siRNAs and treated or not with HU.


B . U2OS treated with HU in presence or absence of ATR inhibitor ( ATRi ) were fractionated immunoblotted with indicated antibodies . Relative RADX on were quantified normalized to histone H3 . See also Fig EV2A .

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B. U2OS cells treated with HU in the presence or absence of ATR inhibitor (ATRi) were fractionated and immunoblotted with indicated antibodies. Relative RADX levels on chromatin were quantified and normalized to histone H3. See also Fig EV2A.


C . Representative of U2OS stably expressing indicated GFP - RADX alleles that were subjected to microirradiation , fixed with methanol / acetone immediately afterwards immunostained with γH2AX antibody .

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C. Representative images of U2OS cells stably expressing indicated GFP-RADX alleles that were subjected to laser microirradiation, fixed with methanol/acetone immediately afterwards and immunostained with γH2AX antibody.


D . As in ( C ) , except were fixed with paraformaldehyde 20 min after damage infliction immunostained with PCNA antibody .

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D. As in (C), except cells were fixed with paraformaldehyde 20 min after DNA damage infliction and immunostained with PCNA antibody.


E . Representative from PLAs with GFP RPA2 antibodies in U2OS U2OS / GFP - RADX lines labeled with EdU for 30 min then fixed or exposed to HU or CPT for 4 h before fixation .

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E. Representative images from PLAs with GFP and RPA2 antibodies in U2OS and U2OS/GFP-RADX cell lines labeled with EdU for 30 min and then fixed or exposed to HU or CPT for 4 h before fixation.


A . Immunoblot of HCT116 transfected with indicated RADX siRNAs RADX knockout lines ( RADXΔ ) .

A. Immunoblot analysis of HCT116 cells transfected with indicated RADX siRNAs and RADX knockout cell lines (RADXΔ).


C . in ( A ) were labeled with consecutive pulses of CldU IdU as shown in ( B ) . Replication fork speeds were calculated as of labeled track divided by time ( bars , mean ; n = 400 , pooled from two independent experiments , per condition ) .

C. Cells in (A) were labeled with consecutive pulses of CldU and IdU as shown in (B). Replication fork speeds were calculated as length of labeled track divided by pulse time (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


D . Bidirectional replication fork symmetry was calculated as of shorter divided by longer tracks from ( C ) . Concordance is 100 % , representing equal rates of bidirectional elongation for both daughter forks ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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D. Bidirectional replication fork symmetry was calculated as percentage of shorter divided by longer tracks from (C). Concordance is 100%, representing equal rates of bidirectional elongation for both daughter forks (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


E . Proportion of stalled forks ( CldU - only tracks ) among from ( C ) ( bars , mean ; 200 analyzed per condition ; n = 2 independent experiments ) .

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E. Proportion of stalled forks (CldU-only tracks) among DNA fibers from (C) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


F . Proportion of new origins ( IdU - only tracks ) among in ( C ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

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F. Proportion of new origins (IdU-only tracks) among DNA fibers in (C) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


H . Proportion of stalled forks ( CldU - only tracks ) among from labeled as in ( G ) ( bars , mean ; 200 analyzed per condition ; n = 2 independent experiments ) .

H. Proportion of stalled forks (CldU-only tracks) among DNA fibers from cells labeled as in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


I . Proportion of restarted forks ( CldU - IdU - positive tracks ) among in ( G ) ( bars , mean ; 200 analyzed per condition ; n = 2 independent experiments ) .

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I. Proportion of restarted forks (CldU- and IdU-positive tracks) among DNA fibers in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


A . Replication fork speeds in U2OS or U2OS / GFP - RADX transfected with control ( − ) or RADX ( # 6 ) siRNA targeting 3 ' UTR labeled with CldU IdU as in Fig 3B ( bars , mean ; n = 400 , pooled from two independent experiments , per condition ) .

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A. Replication fork speeds in U2OS or U2OS/GFP-RADX cells transfected with control (−) or RADX(#6) siRNA targeting the 3'UTR and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


B . Fork symmetry in treated as in ( A ) ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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B. Fork symmetry in cells treated as in (A) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


C . Proportion of stalled forks ( CldU - only tracks ) among in ( A ) ( bars , mean ; 200 analyzed per condition ; n = 2 independent experiments ) .

C. Proportion of stalled forks (CldU-only tracks) among DNA fibers in (A) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


D . Proportion of new origins ( IdU - only tracks ) among in ( A ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

D. Proportion of new origins (IdU-only tracks) among DNA fibers in (A) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


E . Parental U2OS derivative lines stably expressing GFP - RADX alleles or three RPA isoforms at near - endogenous ( U2OS / Super - RPA ) ( Fig EV4A ) were transfected with control ( − ) or RADX siRNA . Replication fork speeds were determined as in ( A ) ( bars , mean ; n = 400 , pooled from two independent experiments , per condition ) .

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E. Parental U2OS and derivative lines stably expressing GFP-RADX alleles or all three RPA isoforms at near-endogenous levels (U2OS/Super-RPA) (Fig EV4A) were transfected with control (−) or RADX siRNA. Replication fork speeds were determined as in (A) (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


F . Replication fork speeds in HCT116 with indicated genotypes transfected with control ( − ) , RPA1 ( 0 . 2 nM [ 17 ] ) or RAD51 siRNAs ( Fig EV4B ) labeled with CldU IdU as in Fig 3B ( bars , mean ; n = 400 , pooled from two independent experiments , per condition ) .

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F. Replication fork speeds in HCT116 cells with indicated genotypes transfected with control (−), RPA1 (0.2 nM concentration [17]) or RAD51 siRNAs (Fig EV4B) and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


G . Fork symmetry among in ( F ) ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

G. Fork symmetry among DNA fibers in (F) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


H . New origin firing among in ( F ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

H. New origin firing among DNA fibers in (F) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


B . Replication fork degradation ( IdU / CldU ) in U2OS transfected with indicated siRNAs processed as in ( A ) ( bars , mean ; n = 200 , pooled from two independent experiments , per condition ) .

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B. Replication fork degradation (IdU/CldU ratio) in U2OS cells transfected with indicated siRNAs and processed as in (A) (bars, mean; n=200 fibers, pooled from two independent experiments, per condition).


C . As in ( B ) , except fork degradation was analyzed in U2OS / GFP - RADX lines .

C. As in (B), except fork degradation was analyzed in U2OS/GFP-RADX cell lines.


D . U2OS or U2OS / GFP - RADX transfected with indicated siRNAs exposed to HU for 4 h were co - immunostained with RPA1 γ - H2AX antibodies analyzed by QIBC . Proportion of displaying maximal RPA loading accompanied by H2AX hyperphosphorylation ( Fig EV5A ) , reflecting replication fork catastrophe [ 17 ] , is indicated ( bars , mean ; n = 5 independent experiments ( ≥ 3000 analyzed per condition ) ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , unpaired t - ) .

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D. U2OS or U2OS/GFP-RADX cells transfected with indicated siRNAs and exposed to HU for 4 h were co-immunostained with RPA1 and γ-H2AX antibodies and analyzed by QIBC. Proportion of cells displaying maximal RPA chromatin loading accompanied by H2AX hyperphosphorylation (Fig EV5A), reflecting replication fork catastrophe [17], is indicated (bars, mean; n=5 independent experiments (≥3000 cells analyzed per condition); *P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001, unpaired t-test).


E . As in ( D ) , but using U2OS or U2OS / Super - RPA exposed or not to HU ( n = 6 independent experiments ) . See also Fig EV5B .

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E. As in (D), but using U2OS or U2OS/Super-RPA cells exposed or not to HU (n=6 independent experiments). See also Fig EV5B.


F . Clonogenic survival of U2OS transfected with indicated siRNAs subjected to different CPT doses for 24 h ( mean ± ; n = 3 independent experiments ) . LQ ( Linear Quadratic ) model was fitted to fractional survival data , using non - linear least square method . Overlap between confidence intervals of fitting coefficients was used to evaluate statistical difference between survival after siRADX siCTRL treatment ( * No overlap of 95 % confidence interval with siCTRL ) .

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F. Clonogenic survival of U2OS cells transfected with indicated siRNAs and subjected to different CPT doses for 24 h (mean±SEM;n=3 independent experiments). LQ (Linear Quadratic) model was fitted to the fractional survival data, using non-linear least square method. Overlap between confidence intervals of the fitting coefficients was used to evaluate the statistical difference between cell survival after siRADX and siCTRL treatment (*No overlap of 95% confidence interval with siCTRL).


G . As in ( F ) , except were treated with indicated doses of HU ( mean ± ; n = 3 independent experiments ) .

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G. As in (F), except cells were treated with indicated doses of HU (mean±SEM;n=3 independent experiments).


H . As in ( F ) , using U2OS U2OS / Super - RPA ( mean ± ; n = 3 independent experiments ) .

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H. As in (F), using U2OS and U2OS/Super-RPA cells (mean±SEM;n=3 independent experiments).


A Turbidity bright - field microscope of indicated groups ( left ) quantitative of turbidity of samples were measured by absorbance at 600 nm ( right ) . 10 μM cGAS , 5 μM G3BP1 , 5 μM BSA 100 nM dsDNA ( 60 bp ) were used in these assays . n = 3 biological replicates . Data information : Representative are shown Error bars , mean with s . d . , * P < 0 . 05 , * * * * P < 0 . 0001 , two - t - . NS , non - significant . Scale bars , 10

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A Turbidity and bright-field microscope images of indicated groups (left) and the quantitative analysis of turbidity of samples were measured by absorbance at 600 nm (right). 10 μM cGAS, 5 μM G3BP1, 5 μM BSA and 100 nM dsDNA (60 bp) were used in these assays. n = 3 biological replicates. Data information: Representative images are shown Error bars, mean with s.d. , *P < 0.05, ****P < 0.0001, two-tailed t-test. NS, non-significant. Scale bars, 10 μm


F FRAP of - induced droplets of cGAS ( 1 μM dsDNA , 10 μM cGAS - mCherry ) , yellow dotted circle indicated region of photobleaching . G FRAP curve of ( F ) . n = 3 biological replicates . H FRAP of cGAS - G3BP1 droplets . 10 μM cGAS - mCherry 5 μM G3BP1 were used . yellow dotted circle indicated region of photobleaching . I FRAP curve of ( H ) . n = 3 biological replicates . Data information : Representative are shown ( A , Error bars , mean with s . d . , * P < 0 . 05 , * * * * P < 0 . 0001 , two - t - . NS , non - significant . FRAP , recovery after photobleaching . Scale bars , 2 μm ( F H ) .

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F FRAP analysis of DNA-induced droplets of cGAS (1 μM dsDNA, 10 μM cGAS-mCherry), the yellow dotted circle indicated the region of photobleaching. G FRAP curve of (F). n = 3 biological replicates. H FRAP analysis of cGAS-G3BP1 droplets. 10 μM cGAS-mCherry and 5 μM G3BP1 were used. The yellow dotted circle indicated the region of photobleaching. I FRAP curve of (H). n = 3 biological replicates. Data information: Representative images are shown (A, Error bars, mean with s.d. , *P < 0.05, ****P < 0.0001, two-tailed t-test. NS, non-significant. FRAP, Fluorescence recovery after photobleaching. Scale bars, 2 μm (F and H).


B Immunofluorescent of cGAS in both WT G3BP1 - / - U937 . 3D were reconstituted with Leica LAS X software . C Quantitative of total cGAS number ( left ) volume ( right ) per of ( B ) . n = 51 . D Data information : Representative are shown Scale bars , 5 μm . Hoechst ( blue ) , . Error bars , mean with s . d , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - t - . NS , non - significant . WT , wild - type .

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B Immunofluorescent staining of cGAS in both WT and G3BP1-/- U937 cells. 3D images were reconstituted with Leica LAS X software. C Quantitative analysis of total cGAS puncta number (left) and volume (right) per cell of (B). n = 51 cells. D Data information: Representative images are shown Scale bars, 5 μm. Hoechst (blue), nuclear staining. Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type.


F ELISA of secreted IFN - β in HT - - treated WT or G3BP1 - / - U937 . n = 3 biological replicates . Data information : Error bars , mean with s . d , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - t - . NS , non - significant . WT , wild - type . HT - , Herring testes .

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F ELISA analysis of the secreted IFN-β in HT-DNA-treated WT or G3BP1-/- U937 cells. n = 3 biological replicates. Data information: Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. HT-DNA, Herring testes DNA.


G cGAMP production in ISD - treated WT or G3BP1 - / - U937 were analyzed by LC - MS / MRM . ND , not detected . n = 3 technical replicates . Data information : Error bars , mean with s . d , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - t - . NS , non - significant . WT , wild - type . ISD , interferon stimulatory .

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G cGAMP production in ISD-treated WT or G3BP1-/- U937 cells were analyzed by LC-MS/MRM. ND, not detected. n = 3 technical replicates. Data information: Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. ISD, interferon stimulatory DNA.


B distribution on cGAS - G3BP1 droplets . A quantitative of a representative cGAS - G3BP1 droplet is shown . Along on merged image , of both G3BP1 cGAS channels were recorded . Data information : Representative are shown Scale bars , 10 μm 45 μM cGAS - mCherry , 10 μM G3BP1 - mEGFP were used in assays .

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B Fluorescence intensity distribution on cGAS-G3BP1 droplets. A quantitative analysis of a representative cGAS-G3BP1 droplet is shown. Along the white line on the merged image, the fluorescence intensity of both G3BP1 and cGAS channels were recorded. Data information: Representative images are shown Scale bars, 10 μm 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP were used in the assays.


D cGAS , G3BP1 Cy5 - dsDNA were incubated . A quantitative of a representative cGAS - droplet is shown . Along on merged image , of G3BP1 , cGAS dsDNA channels were recorded . Data information : Representative are shown Scale bars , 10 μm 45 μM cGAS - mCherry , 10 μM G3BP1 - mEGFP , 2 μM dsDNA were used in assays .

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D cGAS, G3BP1 and Cy5-dsDNA were incubated. A quantitative analysis of a representative cGAS-DNA droplet is shown. Along the white line on the merged image, the fluorescence intensity of G3BP1, cGAS and dsDNA channels were recorded. Data information: Representative images are shown Scale bars, 10 μm 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP, 2 μM dsDNA were used in the assays.


F cGAS , G3BP1 Cy5 - dsRNA were incubated . Similar as in D was performed . Data information : Representative are shown Scale bars , 5 μm 45 μM cGAS - mCherry , 10 μM G3BP1 - mEGFP , 2 μM dsRNA were used in assays .

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F cGAS, G3BP1 and Cy5-dsRNA were incubated. Similar quantification analysis as in D was performed. Data information: Representative images are shown Scale bars, 5 μm 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP, 2 μM dsRNA were used in the assays.


A , B Recombinant cGAS ( 10 μM ) indicated amounts of Cy5 - labeled dsDNA were incubated , with or without G3BP1 ( 2 μM ) . - induced cGAS - liquid droplets were analyzed with time - lapse imaging . Data information : Representative are shown Scale bars , 10

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A, B Recombinant cGAS (10 μM) and the indicated amounts of Cy5-labeled dsDNA were incubated, with or without G3BP1 (2 μM). The DNA-induced cGAS-DNA liquid droplets were analyzed with time-lapse imaging. Data information: Representative images are shown Scale bars, 10 μm


D cGAMP production assays . Recombinant cGAS ( 10 μM ) was incubated with indicated amounts of dsDNA , with or without G3BP1 ( 10 μM ) . produced cGAMP was quantitatively analyzed by LC - MS / MRM . n = 3 technical replicates . Data information : Error bars , mean with s . d . , * * P < 0 . 01 , two - t - .

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D cGAMP production assays. Recombinant cGAS (10 μM) was incubated with the indicated amounts of dsDNA, with or without G3BP1 (10 μM). The produced cGAMP was quantitatively analyzed by LC-MS/MRM. n = 3 technical replicates. Data information: Error bars, mean with s.d. , **P < 0.01, two-tailed t-test.


G qPCR of IFNB mRNA in WT G3BP1 - / - U937 transfected with dsDNA as indicated . n = 3 technical replicates . Data information : Error bars , mean with s . d . , * * P < 0 . 01 , two - t - .

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G qPCR analysis of IFNB mRNA levels in WT and G3BP1-/- U937 cells transfected with dsDNA as indicated. n = 3 technical replicates. Data information: Error bars, mean with s.d. , **P < 0.01, two-tailed t-test.


C cGAS - G3BP1 interaction . Full - truncated G3BP1 was pulled - down using anti - Flag M2 beads . cGAS - G3BP1 interaction was further analyzed with immunoblotting ( IB ) . 2 μg recombinant full - cGAS 2 μg each of truncated G3BP1 were used in .

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C cGAS-G3BP1 interaction analysis. Full-length and truncated G3BP1 was pulled-down using anti-Flag M2 beads. The cGAS-G3BP1 interaction was further analyzed with immunoblotting (IB). 2 μg recombinant full-length cGAS and 2 μg each of truncated G3BP1 were used in the assay.


D , Time - lapse imaging of - induced LLPS of cGAS , in presence of full or truncated G3BP1 ( 2 μM ) n = 3 biological replicates . 10 μM cGAS , 100 nM dsDNA were used in Data information : Representative are shown Scale bars , 10

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D, Time-lapse imaging of DNA-induced LLPS of cGAS, in the presence of full length or truncated G3BP1 (2 μM) n = 3 biological replicates.10 μM cGAS, 100 nM dsDNA were used in the assay Data information: Representative images are shown Scale bars, 10 μm


A Bright - field microscope of indicated groups ( left ) a quantitative of total of droplets ( right ) . n = 3 biological replicates . 10 μM cGAS , 5 μM G3BP1 50 μM KCl , 50 μM MgCl2 , 50 μM ZnCl2 were used . Data information : Representative are shown Scale bars , 10 μm . Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - t - . NS , non - significant . NT , non - treated .

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A Bright-field microscope images of indicated groups (left) and a quantitative analysis of total area of droplets (right). n = 3 biological replicates. 10 μM cGAS, 5 μM G3BP1 and 50 μM KCl, 50 μM MgCl2, 50 μM ZnCl2 were used. Data information: Representative images are shown Scale bars, 10 μm. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.


C of - induced cGAS LLPS in presence of indicated reagent . n = 3 biological replicates . 10 μM cGAS , 50 μM of each ion 100 nM dsDNA were used in these assays . Data information : Representative are shown Scale bars , 10 μm . Partition coefficient was calculated as total of droplets / bulk of background Hoechst ( blue ) , . Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - t - . NS , non - significant . NT , non - treated .

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C Fluorescent images and analysis of DNA-induced cGAS LLPS in the presence of the indicated reagent. n = 3 biological replicates. 10 μM cGAS, 50 μM of each ion and 100 nM dsDNA were used in these assays. Data information: Representative images are shown Scale bars, 10 μm. The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst (blue), nuclear staining. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.


D of - induced LLPS of cGAS in presence of different amounts of ZnCl2 . n = 3 biological replicates . 10 μM cGAS 100 nM dsDNA were used in . Data information : Representative are shown Scale bars , 10 μm . Partition coefficient was calculated as total of droplets / bulk of background Hoechst ( blue ) , . Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - t - . NS , non - significant . NT , non - treated .

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D Fluorescent images and analysis of DNA-induced LLPS of cGAS in the presence of different amounts of ZnCl2. n = 3 biological replicates. 10 μM cGAS and 100 nM dsDNA were used in the assay. Data information: Representative images are shown Scale bars, 10 μm. The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst (blue), nuclear staining. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.


E of - induced LLPS of cGAS in presence of G3BP1 ( 2 μM ) / or ZnCl2 ( 20 μM ) as indicated . n = 3 biological replicates . Data information : Representative are shown Scale bars , 10 μm . Partition coefficient was calculated as total of droplets / bulk of background Hoechst ( blue ) , . Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - t - . NS , non - significant . NT , non - treated .

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E Fluorescent images and analysis of DNA-induced LLPS of cGAS in the presence of G3BP1 (2 μM) and/or ZnCl2 (20 μM) as indicated. n = 3 biological replicates. Data information: Representative images are shown Scale bars, 10 μm. The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst (blue), nuclear staining. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.


A of cGAS - G3BP1 droplets in presence or absence of 10 μM EGCG ( left ) . partition coefficient was calculated as total of droplets / bulk of background ( right ) , n = 3 biological replicates . 45 μM cGAS - mCherry 10 μM G3BP1 - mEGFP were used in these assays . Data information : Representative are shown Scale bars , 10 μm Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - t - . NS , non - significant .

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A Fluorescent images of cGAS-G3BP1 droplets in the presence or absence of 10 μM EGCG (left). The partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background (right), n = 3 biological replicates. 45 μM cGAS-mCherry and 10 μM G3BP1-mEGFP were used in these assays. Data information: Representative images are shown Scale bars, 10 μm Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.


D of - induced LLPS of cGAS , with or without G3BP1 , in absence or presence of 10 μM EGCG ( left ) . partition coefficient was calculated as in ( A ) , n = 3 biological replicates . 10 μM cGAS , 5 μM G3BP1 100 nM dsDNA were used in these assays . Data information : Representative are shown Scale bars , 10 μm Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - t - . NS , non - significant .

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D Fluorescent images of DNA-induced LLPS of cGAS, with or without G3BP1, in the absence or presence of 10 μM EGCG (left). The partition coefficient was calculated as in (A), n = 3 biological replicates. 10 μM cGAS, 5 μM G3BP1 and 100 nM dsDNA were used in these assays. Data information: Representative images are shown Scale bars, 10 μm Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.


E cGAMP production assays . Recombinant cGAS ( 10 μM ) G3BP1 ( 10 μM ) were incubated , in presence or absence of dsDNA ( 200 nM ) , with or without EGCG ( 20 μM ) . produced cGAMP was quantitatively analyzed by LC - MS / MRM . n = 3 technical replicates . Data information : Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - t - . NS , non - significant .

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E cGAMP production assays. Recombinant cGAS (10 μM) and G3BP1 (10 μM) were incubated, in the presence or absence of dsDNA (200 nM), with or without EGCG (20 μM). The produced cGAMP was quantitatively analyzed by LC-MS/MRM. n = 3 technical replicates. Data information: Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.


F cGAS - G3BP1 interaction . cGAS was precipitated using anti - cGAS antibodies in U937 treated with EGCG as indicated . IgG served as control . cGAS - G3BP1 interaction was further analyzed by immunoblotting . Data information : Representative are shown WCL , whole lysate . IP , immunoprecipitation .

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F cGAS-G3BP1 interaction analysis. cGAS was precipitated using anti-cGAS antibodies in U937 cells treated with EGCG as indicated. IgG served as control. The cGAS-G3BP1 interaction was further analyzed by immunoblotting. Data information: Representative images are shown WCL, whole cell lysate. IP, immunoprecipitation.


A . Quantitative real - time PCR of PKCδ mRNA in FACS sorted Lin - , LT - HSC , ST - HSC , MPP , L - S - K + , GMP , CMP , MEP CLP subsets from C56BL / 6 wild - type ( 6 - to 9 - week old ) mice bone marrow . of PKCδ expression were normalized to an internal control gene ( β - actin ) . Expression of PKCδ is shown relative to Lineage negative ( Lin - ) whose expression was arbitrarily set to 1 ( n = 4 mice analyzed for each population ) . Data information : data are presented as mean ± . * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 by with one - way ANOVAs with Sidak ' s multiple comparisons ( A )

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A. Quantitative real-time PCR analysis of PKCδ mRNA levels in FACS sorted Lin-, LT-HSC, ST-HSC, MPP, L-S-K+, GMP, CMP, MEP and CLP subsets from C56BL/6 wild-type (6-to 9-week old) mice bone marrow. Levels of PKCδ expression were normalized to an internal control gene (β-actin). Expression of PKCδ is shown relative to Lineage negative (Lin-) cells whose expression was arbitrarily set to 1 (n=4 mice analyzed for each population). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with Sidak's multiple comparisons test (A)


( B ) FACS plots depict of live , Lin - Sca1 + c - Kit + ( LSK ) ( left panel ) , primitive CD34 - Flt3 - , CD34 + Flt3 - CD34 + Flt3 + HSPCs among LSK ( right panel ) .

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(B) FACS plots depict the percentage of live, Lin-Sca1+c-Kit+ (LSK) (left panel), and primitive CD34-Flt3-, CD34+Flt3- and CD34+Flt3+ HSPCs among LSK cells (right panel).


( C ) Bar charts show average frequency ( left ) absolute number ( right ) ± of indicated populations , analyzed from 2 femurs 2 tibias per mouse ( n = 10 mice analyzed for each genotype ) . data are presented as mean ± . * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 by with one - way ANOVAs with two - Student ' s unpaired t - for comparison of WT to PKCδ KO mice .

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(C) Bar charts show the average frequency (left) and absolute number (right) ± SEM of the indicated populations, analyzed from 2 femurs and 2 tibias per mouse (n=10 mice analyzed for each genotype). All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.


D . FACS plots show percentages of ' SLAM - code ' progenitor in LSK population . Bar graphs show average frequency ( left ) absolute numbers ( right ) ± of LT - HSC per 2 femurs 2 tibias . ( n = 10 mice analyzed for each genotype ) . Data information : data are presented as mean ± . * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 by with one - way ANOVAs with two - Student ' s unpaired t - for comparison of WT to PKCδ KO mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. FACS plots show percentages of 'SLAM-code' based stem and progenitor cells in the LSK population. Bar graphs show the average frequency (left) and absolute numbers (right) ± SEM of LT-HSC per 2 femurs and 2 tibias. (n=10 mice analyzed for each genotype). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.


E . FACS plots illustrate gating frequencies of myeloid progenitors ( MP ) , granulocyte - monocyte progenitor ( GMP ) , common myeloid progenitor ( CMP ) , megakaryocyte - erythroid progenitor ( MEP ) population . Bar graphs show frequencies ( left ) absolute numbers ( right ) of L - S - K + MP GMP , CMP MEP populations in BM of WT ( n = 12 ) PKCδ KO ( n = 12 ) . Data information : data are presented as mean ± . * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 by with one - way ANOVAs with two - Student ' s unpaired t - for comparison of WT to PKCδ KO mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. FACS plots illustrate gating and frequencies of myeloid progenitors (MP), granulocyte-monocyte progenitor (GMP), common myeloid progenitor (CMP), and megakaryocyte-erythroid progenitor (MEP) population. Bar graphs show the frequencies (left) and absolute numbers (right) of L-S-K+ MP and GMP, CMP and MEP populations in the BM of WT (n=12) and PKCδ KO (n=12). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.


F . FACS plots illustrate gating percentages of common lymphoid progenitors ( CLP ) . Bar graph shows absolute number of CLPs per 2 femurs 2 tibias ( n = 5 mice for each genotype ) . Data information : data are presented as mean ± . * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 by with one - way ANOVAs with two - Student ' s unpaired t - for comparison of WT to PKCδ KO mice .

F. FACS plots illustrate the gating and percentages of common lymphoid progenitors (CLP). Bar graph shows the absolute number of CLPs per 2 femurs and 2 tibias (n=5 mice for each genotype). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.


G . FACS plots illustrate percentages of uncommitted lymphoid progenitors ( Lin - IL - 7Rα + Flt3 + Ly - 6D - ) B lymphoid progenitors ( BLP ) . Bar graph represents average frequencies of CLPs , BLP uncommitted lymphoid progenitors in total BM ( n = 5 mice per genotype ) . Data information : data are presented as mean ± . * p < 0 . 05 ; * * p < 0 . 01 , * * * p < 0 . 001 by with one - way ANOVAs with two - Student ' s unpaired t - for comparison of WT to PKCδ KO mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. FACS plots illustrate the percentages of uncommitted lymphoid progenitors (Lin- IL-7Rα+Flt3+Ly-6D-) and B lymphoid progenitors (BLP). Bar graph represents the average frequencies of CLPs, BLP and uncommitted lymphoid progenitors in total BM (n=5 mice per genotype). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.


H . Limiting dilution ( LDA ) demonstrates an increased frequency of long - term repopulating HSCs in PKCδ KO BM ( solid red lines ) compared with WT BM ( black solid lines ) . Engraftment data shown at 14 - post - BMT . Plots depict percentages of recipient mice containing less than 1 % CD45 . 2 + blood nucleated . Dotted lines represent 95 % confidence interval of same ( p = 0 . 0005 ) . Data information : data are presented as mean ± . Overall for differences in frequencies between WT to PKCδ KO mice was determined with likelihood of single - hit model ( H ) .

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

H. Limiting dilution analysis (LDA) demonstrates an increased frequency of long-term repopulating HSCs in PKCδ KO BM (solid red lines) compared with WT BM (black solid lines). Engraftment data shown at 14-weeks post-BMT. Plots depict the percentages of recipient mice containing less than 1% CD45.2+ blood nucleated cells. Dotted lines represent the 95% confidence interval of the same (p=0.0005). Data information: All data are presented as mean±SEM. Overall test for differences in stem cell frequencies between WT to PKCδ KO mice was determined with likelihood ratio test of single-hit model (H).


A . Representative FACS profiles of HSPC cycle using combinatorial for Ki67 Hoechst 33342 . Bar charts depict average of in each phase of cycle for each LSK subset from WT ( n = 6 ) PKCδ KO ( n = 7 ) mice . Data compiled from two independent experiments . Data information : data are presented as mean ± , * p < 0 . 05 * * p < 0 . 001 , with significance determined by two - Student ' s unpaired t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FACS profiles of HSPC cell cycle analysis using combinatorial staining for Ki67 and Hoechst 33342. Bar charts depict the average percentage of cells in each phase of the cell cycle for each LSK subset from WT (n=6) and PKCδ KO (n=7) mice. Data compiled from two independent experiments. Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.


B . FACS profiles of indicated BM subsets from WT PKCδ KO mice 20hr after BrdU injection . Data information : data are presented as mean ± , * p < 0 . 05 * * p < 0 . 001 , with significance determined by two - Student ' s unpaired t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. FACS profiles of indicated BM subsets from WT and PKCδ KO mice 20hr after BrdU injection. Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.


C . Average percentages of in each phase of cycle phases for each of indicated HSPC subsets from WT PKCδ KO mice . Data are pooled from two independent experiments ( totaling n = 6 - 7 mice per genotype ) . Data information : data are presented as mean ± , * p < 0 . 05 * * p < 0 . 001 , with significance determined by two - Student ' s unpaired t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Average percentages of cells in each phase of the cell cycle phases for each of the indicated HSPC subsets from WT and PKCδ KO mice. Data are pooled from two independent experiments (totaling n=6-7 mice per genotype). Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.


D . Representative FACS plots summary of FACS data analyzing frequency of apoptotic HSPC using co - for AnnexinV 7 - AAD ( WT PKCδ − / − ( n = 7 per genotype ) . Data information : data are presented as mean ± , * p < 0 . 05 * * p < 0 . 001 , with significance determined by two - Student ' s unpaired t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative FACS plots and summary of FACS data analyzing the frequency of apoptotic HSPC cells using co-staining for AnnexinV and 7-AAD (WT and PKCδ−/− (n=7 per genotype). Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.


B . of total donor - derived , hematopoietic ( CD45 . 2 + ) , B - ( B220 + ) , myeloid ( CD11b + Gr1 + ) , T ( CD3 + ) in peripheral blood ( PB ) of recipient mice , as determined by FACS at indicated time points . statistical significance of differences was determined using two - ANOVAs with Holm - Sidak ' s multiple comparisons ( n = 8 recipients per genotype in each experiment ) . Data information : data are presented as mean ± SD . * p < 0 . 05 , * * p < 0 . 001 , with significance determined by repeated measures of two - way ANOVA with Sidak ' s multiple comparison for comparison of recipients of WT PKCδ KO marrow at each time point for each population .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Percent of total donor-derived, hematopoietic cells (CD45.2+), B-cells (B220+), myeloid cells (CD11b+Gr1+), and T cells (CD3+) in the peripheral blood (PB) of recipient mice, as determined by FACS at the indicated time points. The statistical significance of differences was determined using two-ANOVAs with Holm-Sidak's multiple comparisons tests (n=8 recipients per genotype in each experiment). Data information: All data are presented as mean±SD. *p<0.05, **p<0.001, with significance determined by repeated measures of two-way ANOVA analysis with Sidak's multiple comparison tests for comparison of recipients of WT and PKCδ KO marrow at each time point and for each cell population.


C . donor - derived HSPCs in BM of secondary ( left ) tertiary ( right ) recipients of WT or PKCδ − / − marrow . Data are compiled from two independent experiments ( n = 8 recipients per genotype ) . Data information : data are presented as mean ± SD . * p < 0 . 05 , * * p < 0 . 001 , with significance determined by repeated measures of two - way ANOVA with Sidak ' s multiple comparison for comparison of recipients of WT PKCδ KO marrow at each time point for each population .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Percent donor-derived HSPCs in the BM of secondary (left) and tertiary (right) recipients of WT or PKCδ−/− marrow. Data are compiled from two independent experiments (n=8 recipients per genotype). Data information: All data are presented as mean±SD. *p<0.05, **p<0.001, with significance determined by repeated measures of two-way ANOVA analysis with Sidak's multiple comparison tests for comparison of recipients of WT and PKCδ KO marrow at each time point and for each cell population.


B , C Representative FACS plots of PKCδ fl / fl cKO BM 4 - 8 ( B ) , 20 - 24 - ( C ) after pIpC treatment . Numbers in plot indicate absolute numbers of indicated subset within gated population ( n = 8 - 9 mice per genotype time point ) . Data information : Data presented as mean ± . * p < 0 . 05 , * * p < 0 . 005 * * * p < 0 . 001 by two - Student ' s unpaired t - .

O O O O 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

B,C Representative FACS plots of PKCδ fl/fl and cKO BM 4-8 weeks (B), and 20-24-weeks (C) after pIpC treatment. Numbers in plot indicate the absolute numbers of the indicated subset within the gated population (n=8-9 mice per genotype and time point). Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.


( E ) FACS plots show contribution of donor - derived ( CD45 . 2 + ) HSPCs in recipient BM ( E ) ( total of n = 7 - 8 mice per genotype ) .

O O O O O O O O O O O O O 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) FACS plots show contribution of donor-derived (CD45.2+) HSPCs in recipient BM (E) (total of n=7-8 mice per genotype).


Calculated absolute numbers of donor - derived HSPC subsets in transplanted mice ( F ) . ( total of n = 7 - 8 mice per genotype ) . Data information : Data presented as mean ± . * p < 0 . 05 , * * p < 0 . 005 * * * p < 0 . 001 by two - Student ' s unpaired t - .

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

Calculated absolute numbers of donor-derived HSPC subsets in transplanted mice (F). (total of n=7-8 mice per genotype). Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.


G . Average BrdU + HPSCs ( 3 days after BrdU injection into mice reconstituted 24 previously with control or cKO marrow injected 16 previously with pIpC among donor - derived LSK in control cKO chimeras BM . Data information : Data presented as mean ± . * p < 0 . 05 , * * p < 0 . 005 * * * p < 0 . 001 by two - Student ' s unpaired t - .

G. Average percentage BrdU+ HPSCs (3 days after BrdU injection into mice reconstituted 24 weeks previously with control or cKO marrow and injected 16 weeks previously with pIpC among donor-derived LSK cells in the control and cKO chimeras BM. Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.


H . Percentages of apoptotic ( AnnexinV + / 7 - AAD - ) in donor - derived LSK in control cKO chimera mouse BM at 24 after transplant 16 after pIpC injection . Data are compiled from two independent experiments ( total of n = 7 - 8 mice per genotype ) . Data information : Data presented as mean ± . * p < 0 . 05 , * * p < 0 . 005 * * * p < 0 . 001 by two - Student ' s unpaired t - .

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

H. Percentages of apoptotic cells (AnnexinV+/7-AAD-) in donor-derived LSK cells in control and cKO chimera mouse BM at 24 weeks after transplant and 16 weeks after pIpC injection. Data are compiled from two independent experiments (total of n=7-8 mice per genotype). Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.


B . Kinetics of LSK recovery . Representative FACS profiles of BM LSK from WT cKO mice at indicated time points after 5 - FU treatment . Numbers in contour plots indicate percentages of LSK subsets among Lin - live . C . Average absolute numbers of BM LSK at day13 day28 after 5 - FU treatment . Data are pooled from two independent experiments . ( total of n = 6 - 8 mice per time point genotype ) . Data information : Data presented as mean ± . statistical significance was assessed using repeated - measures two - way ANOVAs with Sidak ' s multiple comparisons ( C ) * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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 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

B. Kinetics of LSK cell recovery. Representative FACS profiles of BM LSK cells from WT and cKO mice at the indicated time points after 5-FU treatment. Numbers in the contour plots indicate the percentages of LSK subsets among Lin- live cells. C. Average absolute numbers of BM LSK cells at day13 and day28 after 5-FU treatment. Data are pooled from two independent experiments. (total of n=6-8 mice per time point and genotype). Data information: Data presented as mean ± SEM. The statistical significance was assessed using repeated-measures two-way ANOVAs with Sidak's multiple comparisons tests (C) *p<0.05, **p<0.01 and ***p<0.001.


D . of apoptotic HSPCs at indicted time points after 5 - FU treatment of control cKO mice ( n = 6 per genotype ) . Data information : Data presented as mean ± . statistical significance was assessed using repeated - measures two - way ANOVAs by two - Student ' s unpaired t - ( D E ) . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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

D. Quantification of apoptotic HSPCs at the indicted time points after 5-FU treatment of control and cKO mice (n=6 per genotype). Data information: Data presented as mean ± SEM. The statistical significance was assessed using repeated-measures two-way ANOVAs by two-tailed Student's unpaired t-test analysis (D and E). *p<0.05, **p<0.01 and ***p<0.001.


E . Representative FACS plots showing of in vivo BrdU incorporation assays . Mice were received 2mg of BrdU ( i . p . ) at d13 ( left ) or d28 ( right ) after 5 - FU treatment . 20hrs later , BrdU incorporation in BM LSK was assessed using anti - BrdU antibodies . Bar chart at right indicates of BrdU + LSK at d13 d28 after 5 - FU treatment ( n = 6 per genotype time point ) . Data information : Data presented as mean ± . statistical significance was assessed using repeated - measures two - way ANOVAs with two - Student ' s unpaired t - ( D E ) . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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

E. Representative FACS plots showing analysis of in vivo BrdU incorporation assays. Mice were received 2mg of BrdU (i.p.) at d13 (left) or d28 (right) after 5-FU treatment. 20hrs later, BrdU incorporation in BM LSK cells was assessed using anti-BrdU antibodies. Bar chart at right indicates percent of BrdU+ LSK cells at d13 and d28 after 5-FU treatment (n=6 per genotype and time point). Data information: Data presented as mean ± SEM. The statistical significance was assessed using repeated-measures two-way ANOVAs with two-tailed Student's unpaired t-test analysis (D and E). *p<0.05, **p<0.01 and ***p<0.001.


F . Kaplan - Meier survival cures of control ( n = 12 ) cKO ( n = 11 ) mice after sequential 5 - FU treatment . Mice were treated with 5 - FU ( 150mg / kg , i . p . ) once each week for three . Arrows indicate time points of 5 - FU injection . Data information : Data presented as mean ± . Survival data ( F ) were analyzed using a log - rank nonparametric ( Mantel - Cox ) ( p = 0 . 0141 ) .

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

F. Kaplan-Meier survival cures of control (n=12) and cKO (n=11) mice after sequential 5-FU treatment. Mice were treated with 5-FU (150mg/kg, i.p.) once each week for three weeks. Arrows indicate time points of 5-FU injection. Data information: Data presented as mean ± SEM. Survival data (F) were analyzed using a log-rank nonparametric test (Mantel-Cox test) (p=0.0141).


A . MitoStress revealed increased oxygen consumption rates ( OCR ) in PKCδ - deleted LSKs . BM LSK were sorted from WT PKCδ cKO mice 12 - after pIpC - treatment . OCR was measured at basal level after sequential loading of ATP synthase inhibitor Oligomycin ( 350nm ) , mitochondrial uncoupler , FCCP ( 10μM ) , electron transport chain inhibitor , Rotenone ( 1μM ) using Seahorse XF24 extracellular flux analyzer . Data are from 3 independent experiments ( n = 9 mice ) . In each experiment , bone marrow from identical genotypes ( n = 3 mice per genotype ) were pooled used for LSK sorting . Data information : statistical significance of difference was assessed using two - Student ' s unpaired t - . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

O O O O O 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. MitoStress test revealed increased oxygen consumption rates (OCR) in PKCδ-deleted LSKs. BM LSK cells were sorted from WT and PKCδ cKO mice 12-weeks after pIpC-treatment. OCR was measured at basal level and after sequential loading of ATP synthase inhibitor Oligomycin (350nm), mitochondrial uncoupler, FCCP (10μM), and electron transport chain inhibitor, Rotenone (1μM) using Seahorse XF24 extracellular flux analyzer. Data are from 3 independent experiments (n=9 mice). In each experiment, bone marrow cells from identical genotypes (n=3 mice per genotype) were pooled and used for LSK cell sorting. Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.


( B ) Basal OCR was measured before inhibitors treatment ( left ) ; Maximal OCR capacity after FCCP treatment ( right ) . ( n = 9 per genotype , from 3 independent experiments ) . Data information : statistical significance of difference was assessed using two - Student ' s unpaired t - . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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

(B) Basal OCR was measured before inhibitors treatment (left); and the Maximal OCR capacity after FCCP treatment (right). (n=9 per genotype, from 3 independent experiments). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.


( C ) . Spare respiratory capacity ( SRC ) of LSKs was calculated ( n = 9 per genotype , from 3 independent experiments ) . Data information : statistical significance of difference was assessed using two - Student ' s unpaired t - . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). The Spare respiratory capacity (SRC) of LSKs was calculated (n=9 per genotype, from 3 independent experiments). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.


D , E . ( D ) Glycolysis Stress . Extracellular acidification rates ( ECAR ) of LSKs were measured at basal conditions ( unbuffered medium without glucose ) after sequential loading of glucose ( 7 . 5mM ) , Oligomycin ( 350nm ) , 2 - deoxyglucose ( 50mM ; 2DG , a glucose analog ) . ( E ) . AUC of baseline ECAR ( left ) , glycolytic capacity ( maximal ECAR ) was calculated from data shown in panel D ( n = 7 - 8 per genotype , from 3 independent experiments ) . Data information : statistical significance of difference was assessed using two - Student ' s unpaired t - . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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D, E. (D) Glycolysis Stress test. Extracellular acidification rates (ECAR) of LSKs were measured at basal conditions (unbuffered assay medium without glucose) and after sequential loading of glucose (7.5mM), Oligomycin (350nm), and 2-deoxyglucose (50mM; 2DG, a glucose analog). (E). AUC of baseline ECAR levels (left), and glycolytic capacity (maximal ECAR) was calculated from the data shown in panel D (n=7-8 per genotype, from 3 independent experiments). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.


F . Relative ATP content in indicated HSPC subsets determined using an ATP kit ( n = 6 mice per genotype ) . Data information : statistical significance of difference was assessed using two - Student ' s unpaired t - . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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F. Relative ATP content in indicated HSPC subsets determined using an ATP assay kit (n=6 mice per genotype). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.


G . Representative FACS histograms of MitoTracker Green on indicated HSPC subsets from control cKO mice . Relative MitoTracker Green mean ( MFI ) is quantified below for n = 6 mice of each genotype . Data information : statistical significance of difference was assessed using two - Student ' s unpaired t - . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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G. Representative FACS histograms of MitoTracker Green fluorescence intensity on indicated HSPC subsets from control and cKO mice. Relative MitoTracker Green mean fluorescence intensity (MFI) is quantified below for n=6 mice of each genotype. Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.


H . Representative electron microscopy ( 4 , 800x magnification ) of control PKCδ cKO HSPCs . Arrows indicate mitochondria enriched regions ( n = 3 mice per genotype ) . Scale bar represents 500nm .

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H. Representative electron microscopy images (4,800x magnification) of control and PKCδ cKO HSPCs. Arrows indicate the mitochondria enriched regions (n=3 mice per genotype). Scale bar represents 500nm.


I . ROS in control PKCδ cKO HSPCs as measured by FACS CM - H2DCFDA . Bar graph at right shows relative CM - H2DCFDA MFI . Data compiled from three independent experiments ( n = 6 - 8 per genotype ) . Data information : statistical significance of difference was assessed using two - Student ' s unpaired t - . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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I. ROS levels in control and PKCδ cKO HSPCs as measured by FACS based CM-H2DCFDA staining. Bar graph at right shows the relative CM-H2DCFDA MFI. Data compiled from three independent experiments (n=6-8 per genotype). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.


Quantitative RT - PCR of selected ( A ) HSPC transcriptional regulators expression in bone marrow HSPCs ( n = 4 mice per genotype ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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Quantitative RT-PCR analysis of selected (A) HSPC transcriptional regulators expression in bone marrow HSPCs (n=4 mice per genotype). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


Quantitative RT - PCR of selected ( B ) cycle apoptosis regulators expression in bone marrow HSPCs ( n = 4 mice per genotype ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

Quantitative RT-PCR analysis of selected (B) cell cycle and apoptosis regulators expression in bone marrow HSPCs (n=4 mice per genotype). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


Quantitative RT - PCR of selected ( C ) known HSPC metabolic regulators expression in bone marrow HSPCs ( n = 4 mice per genotype ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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Quantitative RT-PCR analysis of selected (C) known HSPC metabolic regulators expression in bone marrow HSPCs (n=4 mice per genotype). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


Intracellular flow cytometry of Cyclin D1 ( D ) Data shown in bar graphs are mean of indicated expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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Intracellular flow cytometry analysis of Cyclin D1 protein (D) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


Intracellular flow cytometry of pro - survival regulators Mcl1 , Blc2 , Bim ( E ) Data shown in bar graphs are mean of indicated expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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Intracellular flow cytometry analysis of pro-survival regulators Mcl1, Blc2, and Bim protein (E) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


representative FACS histograms bar graphs show relative of pAKT ( Ser473 ) relative to WT controls ( F ) Data shown in bar graphs are mean of indicated expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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representative FACS histograms and bar graphs show relative levels of pAKT (Ser473) relative to WT controls (F) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


representative FACS histograms bar graphs show relative of pFoxO3a ( Ser253 ) ( G ) Data shown in bar graphs are mean of indicated expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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representative FACS histograms and bar graphs show relative levels of pFoxO3a (Ser253) (G) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


representative FACS histograms bar graphs show relative of Total cMyc ( H ) Data shown in bar graphs are mean of indicated expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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representative FACS histograms and bar graphs show relative levels of Total cMyc protein (H) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


; representative FACS histograms bar graphs show relative of pRb ( Ser807 / 811 ) ( I ) Data shown in bar graphs are mean of indicated expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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; representative FACS histograms and bar graphs show relative levels of pRb (Ser807/811) (I) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


representative FACS histograms bar graphs show total PPARγ relative to WT controls ( J ) Data shown in bar graphs are mean of indicated expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

representative FACS histograms and bar graphs show total PPARγ protein levels relative to WT controls (J) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


Relative expression of total PPARδ in HSPCs ( K ) Data shown in bar graphs are mean of indicated expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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Relative expression levels of total PPARδ protein in HSPCs (K) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


Activation of pmTOR ( Ser2448 ) ( L ) Data shown in bar graphs are mean of indicated expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

Activation of pmTOR (Ser2448) (L) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


Activation of pS6 ( Ser235 / 236 ) ( Ser240 / 244 ) ( M ) . Data shown in bar graphs are mean of indicated expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : statistical significance of difference was assessed using Two - Student ' s unpaired t - for comparison of control PKCδ cKO HSPCs . data are presented as mean ± . , * p < 0 . 05 , * * p < 0 . 001 .

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Activation of pS6 (Ser235/236) and (Ser240/244) (M). Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


A Germination of dog1 - 3 ( knock - out ) , WT , dog1 - 5 ( DOG1 upregulation ) seeds in media supplemented with NaCl for indicated number of days after stratification ( DAS ) . Scale bar represents 5 mm . B Germination rate in 100 mM NaCl 7 days after stratification for seeds of different genotypes . Lines represent fitted curves with a 95 % confidence interval ( grey ) . ∗ p - value < 0 . 05 from two - Student ' s t - . Data for WT is same as plotted in Fig 3C .

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A Germination of dog1-3 (knock-out), WT, and dog1-5 (DOG1 upregulation) seeds in media supplemented with NaCl for the indicated number of days after stratification (DAS). Scale bar represents 5 mm. B Germination rate in 100 mM NaCl 7 days after stratification for seeds of different genotypes. Lines represent the fitted curves with a 95% confidence interval (grey area). ∗ p-value < 0.05 from two-tailed Student's t-test. Data for WT is the same as plotted in Fig 3C. C


C shDOG1 expression normalized to UBC21 ( AT5G25760 ) in dog1 - 3 dog1 - 5 relative to WT . D shDOG1 lgDOG1 expression normalized to UBC21 in seeds treated with 100 mM NaCl relative to mock .

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C shDOG1 expression levels normalized to UBC21 (AT5G25760) in dog1-3 and dog1-5 relative to WT. D shDOG1 and lgDOG1 expression levels normalized to UBC21 in seeds treated with 100 mM NaCl relative to mock. E


A 3 ' - seq read coverage on DOG1 locus . Black colour is used for coverage within scale on right - side ( 0 to 500 ) , red is used for coverage within scale on left - side ( 0 to 50 ) . Above is a schematic representation of annotated transcripts from DOG1 locus chromosome coordinates .

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A 3'RNA-seq read coverage on DOG1 locus. Black colour is used for coverage within the scale on the right-hand side (0 to 500), and red is used for coverage within the scale on the left-hand side (0 to 50). Above is a schematic representation of the annotated transcripts from the DOG1 locus and the chromosome coordinates.


Germination time - course in 100 mM NaCl after stratification for puppies - 1 ( C ) relative to WT . Lines represent fitted curves with a 95 % confidence interval ( grey ) , dots represent data points , ∗ p - value < 0 . 05 from two - Student ' s t - . ( C ) Data for WT is same as

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Germination time-course in 100 mM NaCl after stratification for puppies-1 (C) relative to WT. Lines represent fitted curves with a 95% confidence interval (grey area), dots represent data points, ∗ p-value < 0.05 from the two-tailed Student's t-test. (C) Data for WT is the same as plotted