Figure 4A . 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 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 DNA fibers in ( A ) ( bars , mean ; 200 fibers analyzed 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 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 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 DNA fibers in ( F ) ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) . H . New origin firing among DNA fibers in ( F ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. 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 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 DNA fibers in (A) (bars, mean; 200 fibers analyzed 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 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 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 DNA fibers in (F) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).H. New origin firing among DNA fibers in (F) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


Figure 5B . 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 cell lines . 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 cells exposed or not to HU ( n = 6 independent experiments ) . See also Fig EV5B . 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 cells were treated with indicated doses of HU ( mean ± SEM ; n = 3 independent experiments ) . H . As in ( F ) , using U2OS and U2OS / Super - RPA cells ( mean ± SEM ; n = 3 independent experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B. 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 cell lines.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 cells exposed or not to HU (n=6 independent experiments). See also Fig EV5B.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 cells were treated with indicated doses of HU (mean±SEM;n=3 independent experiments).H. As in (F), using U2OS and U2OS/Super-RPA cells (mean±SEM;n=3 independent experiments).


Figure 1A 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 μmF 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A 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 μmF 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).


Figure 2B 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 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 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 .

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Figure 2B 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 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 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.


Figure 3B 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B 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 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 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.


Figure 4A , 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 μmD 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A, 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 μmD 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 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.


Figure 5C 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 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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5C 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 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


Figure 6A 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A 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 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 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 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.


Figure 7A 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 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 ) 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A 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 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) 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 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.


Figure 1A . 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 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 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 ' 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 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 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. 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 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 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' 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 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 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 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 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).


Figure 2A . 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. 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 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 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 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.


Figure 3B . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. 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. 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.


Figure 4B , 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 ) . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B,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).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 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 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.


Figure 5B . 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 . 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 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 ) 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B. 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. 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 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) 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).


Figure 6A . 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 ) ; 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 ) . 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 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. 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); 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). 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 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 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 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 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 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.


Figure 7Quantitative 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 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 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 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 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 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 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 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 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 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 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 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 ) 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Quantitative 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 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 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 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 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 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 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 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 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 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 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 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) 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.


Figure 1A 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 . CC 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A 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. CC 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


Figure 2A 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 .

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Figure 2A 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.


Figure 3Germination 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 plottedF RT - qPCR fold - change induction of PUPPIES - uns , PUPPIES - prom , PUPPIES - fusion , shDOG1 , and lgDOG1 in puppies - ox relative to WT ( blue dashed line ) , in dry ( light grey ) and imbibed seeds in the presence of 100 mM NaCl ( red ) . Significance from the two - tailed Student ' s t - test for comparing dry seeds of WT and puppies - ox is represented with light grey asterisks , and salt - imbibed seeds of WT and puppies - ox are represented with red asterisks . Black asterisks represent significance from two - way ANOVA with Tukey ' s multiple comparisons test for dry seeds vs imbibed seeds . ns p - value > 0 . 05 , ∗ p - value < 0 . 05 , ∗ ∗ p - value < 0 . 01 , ∗ ∗ ∗ p - value < 0 . 001 . Error bars represent the mean ± SD . n = 4 biological replicates . G RT - qPCR with primers for shDOG1 and lgDOG1 on nascent RNA from seeds imbibed in 100 mM NaCl from puppies - ox relative to WT . Nascent RNA levels were normalized to UBC21 . Bars and error bars represent the mean ± SD . Points represent biological replicates . Statistical significance from two - tailed Student ' s t - test . ∗ p - value < 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Germination 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 plottedF RT-qPCR fold-change induction of PUPPIES-uns, PUPPIES-prom, PUPPIES-fusion, shDOG1, and lgDOG1 in puppies-ox relative to WT (blue dashed line), in dry (light grey) and imbibed seeds in the presence of 100 mM NaCl (red). Significance from the two-tailed Student's t-test for comparing dry seeds of WT and puppies-ox is represented with light grey asterisks, and salt-imbibed seeds of WT and puppies-ox are represented with red asterisks. Black asterisks represent significance from two-way ANOVA with Tukey's multiple comparisons test for dry seeds vs imbibed seeds. ns p-value > 0.05, ∗ p-value < 0.05, ∗∗ p-value < 0.01, ∗∗∗ p-value < 0.001. Error bars represent the mean ± SD. n = 4 biological replicates.G RT-qPCR with primers for shDOG1 and lgDOG1 on nascent RNA from seeds imbibed in 100 mM NaCl from puppies-ox relative to WT. Nascent RNA levels were normalized to UBC21. Bars and error bars represent the mean ± SD. Points represent biological replicates. Statistical significance from two-tailed Student's t-test. ∗ p-value < 0.05.


Figure 4A z - stack max - projection image of smFISH for DOG1 RNA . The " Inferno " colour scale is used for the intensity of fluorescence from Quasar670 fluorophore ( DOG1 ) . The blue colour shows fluorescence from DAPI ( nuclei staining ) . Arrowheads point to foci corresponding to transcription sites ( TS ) . The scale bar is 20 μm . B Distribution of cytoplasmic DOG1 foci per cell in WT . The blue vertical dashed line indicates the average . CG z - stack max - projection images from seeds imbibed in 100 mM NaCl of WT ( left ) versus puppies - ox ( right ) . Arrowheads point to foci corresponding to TS .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A z-stack max-projection image of smFISH for DOG1 RNA. The "Inferno" colour scale is used for the intensity of fluorescence from Quasar670 fluorophore (DOG1). The blue colour shows fluorescence from DAPI (nuclei staining). Arrowheads point to foci corresponding to transcription sites (TS). The scale bar is 20 μm. B Distribution of cytoplasmic DOG1 foci per cell in WT. The blue vertical dashed line indicates the average. CG z-stack max-projection images from seeds imbibed in 100 mM NaCl of WT (left) versus puppies-ox (right). Arrowheads point to foci corresponding to TS.


Figure 5A - E The profile of Pol II during DOG1 transcription . Plots show the fraction of unique reads from targeted NET - Seq using rolling median ( 11 nt ) . Targeted libraries for sequencing of DOG1 locus were obtained by usage of 5 primers across the gene . Pol II dynamics is analyzed separately for each region sequenced in exon 1 ( A ) , exon 2 ( B ) , exon 3 ( C ) , intron 1 ( D ) , and intron 2 ( E ) . The x - axis shows the distance in bp from DOG1 . A light red colour fill highlights the regions where Pol II density is higher in puppies - ox . A light blue colour fill highlights the regions where Pol II density is higher in WT . For each plot , coloured tiles represent the fold - change ( puppies - ox / WT ) on a logarithmic scale . Greyscale tiles represent the result of a statistical test for the difference between genotypes from the two - tailed Student ' s t - test after Bonferroni correction . Tiles correspond to a 25 bp bin each . Vertical dashed lines connect the exon boundaries on the plots with the schematics of the DOG1 gene with grey boxes representing exons and black lines representing introns . F , G RT - qPCR measurement of alternative splicing of lgDOG1 alpha and beta isoforms and unspliced intron 1 DOG1 isoforms levels in puppies - ox , and puppies - 1 relative to WT , in seeds treated with 100 mM NaCl .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-E The profile of Pol II during DOG1 transcription. Plots show the fraction of unique reads from targeted NET-Seq using rolling median (11 nt). Targeted libraries for sequencing of DOG1 locus were obtained by usage of 5 primers across the gene. Pol II dynamics is analyzed separately for each region sequenced in exon 1 (A), exon 2 (B), exon 3 (C), intron 1 (D), and intron 2 (E). The x-axis shows the distance in bp from DOG1. A light red colour fill highlights the regions where Pol II density is higher in puppies-ox. A light blue colour fill highlights the regions where Pol II density is higher in WT. For each plot, coloured tiles represent the fold-change (puppies-ox / WT) on a logarithmic scale. Greyscale tiles represent the result of a statistical test for the difference between genotypes from the two-tailed Student's t-test after Bonferroni correction. Tiles correspond to a 25 bp bin each. Vertical dashed lines connect the exon boundaries on the plots with the schematics of the DOG1 gene with grey boxes representing exons and black lines representing introns.F, G RT-qPCR measurement of alternative splicing of lgDOG1 alpha and beta isoforms and unspliced intron 1 DOG1 isoforms levels in puppies-ox, and puppies-1 relative to WT, in seeds treated with 100 mM NaCl.


Figure 1B . The in vitro synthesized [ 35S ] Met - labeled single - cysteine Bax proteins were activated and targeted to the mitochondria that were pretreated with NEM to block the sulfhydryls of mitochondrial proteins . The resulting mitochondria were isolated and oxidized by CuPhe for 30 min . NEM and EDTA were then added to stop the oxidation . For the " 0 min " controls , NEM and EDTA were added before the addition of CuPhe . The resulting samples were analyzed by phosphor - imaging after non - reducing or reducing SDS - PAGE ( see Appendix Figure S2A ) . C . Oxidized mitochondria with the radioactive single - cysteine Bax protein pair or double - cysteine Bax protein were prepared and analyzed as in ( B ) . D . Oxidized mitochondria with the radioactive single - cysteine Bax protein pair or double - cysteine Bax protein were prepared as in ( B ) . After an aliquot was withdrawn as input , another aliquot was extracted by Na2CO3 ( pH 11 . 5 ) and centrifuged through a sucrose cushion to separate the integral proteins in the membrane pellet from the soluble and peripheral proteins in the supernatant . The input , pellet and supernatant were analyzed by non - reducing SDS - PAGE and phosphor - imaging . In a parallel control experiment , the pellet and supernatant were analyzed by reducing SDS - PAGE and immunoblotting with an antibody specific to PDHE1α , a soluble mitochondrial matrix protein .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. The in vitro synthesized [35S]Met-labeled single-cysteine Bax proteins were activated and targeted to the mitochondria that were pretreated with NEM to block the sulfhydryls of mitochondrial proteins. The resulting mitochondria were isolated and oxidized by CuPhe for 30 min. NEM and EDTA were then added to stop the oxidation. For the "0 min" controls, NEM and EDTA were added before the addition of CuPhe. The resulting samples were analyzed by phosphor-imaging after non-reducing or reducing SDS-PAGE (see Appendix Figure S2A).C. Oxidized mitochondria with the radioactive single-cysteine Bax protein pair or double-cysteine Bax protein were prepared and analyzed as in (B).D. Oxidized mitochondria with the radioactive single-cysteine Bax protein pair or double-cysteine Bax protein were prepared as in (B). After an aliquot was withdrawn as input, another aliquot was extracted by Na2CO3 (pH 11.5) and centrifuged through a sucrose cushion to separate the integral proteins in the membrane pellet from the soluble and peripheral proteins in the supernatant. The input, pellet and supernatant were analyzed by non-reducing SDS-PAGE and phosphor-imaging. In a parallel control experiment, the pellet and supernatant were analyzed by reducing SDS-PAGE and immunoblotting with an antibody specific to PDHE1α, a soluble mitochondrial matrix protein.


Figure 2B . The in vitro synthesized radioactive Bax proteins , each with a single cysteine positioned in helix α2 , α3 , α4 or α5 , were activated and targeted to the mitochondria . The resulting mitochondria were isolated and treated with IASD in the absence or presence of CHAPS , urea , or both . After 30 min , the labeling reactions were stopped by mercaptoethanol . For the " 0 min " controls , the samples were pretreated with mercaptoethanol before addition of IASD . The IASD - labeled radioactive Bax proteins were resolved from the unlabeled ones using either isoelectric focusing ( IEF ; as indicated ) or gradient SDS - PAGE and detected by phosphor - imaging . Triangles and arrows indicate the unlabeled and IASD - labeled Bax proteins , respectively . n = 3 for V111C , and K119C ; 4 for W107C , and A117C ; 2 for other mutants . C - D . The phosphor - imaging data for IASD labeling of Bax mutants in ( B ) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices as described and shown in Appendix Figure S3 . The membrane burial indices were normalized by that of G179C in α9 , a reference for the membrane - buried residues . The resulting relative membrane burial indices are shown in ( C ) . The residues with the relative membrane burial index ≥ 40 % of that of Gly179 are considered as buried in the MOM . Similarly , the relative protein burial indices shown in ( D ) were obtained by using Y115C in α5 as a reference for the protein - buried residues . The residues with the relative protein burial index ≥ 40 % of that of Tyr115 are considered as buried in the protein or its complex .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. The in vitro synthesized radioactive Bax proteins, each with a single cysteine positioned in helix α2, α3, α4 or α5, were activated and targeted to the mitochondria. The resulting mitochondria were isolated and treated with IASD in the absence or presence of CHAPS, urea, or both. After 30 min, the labeling reactions were stopped by mercaptoethanol. For the "0 min" controls, the samples were pretreated with mercaptoethanol before addition of IASD. The IASD-labeled radioactive Bax proteins were resolved from the unlabeled ones using either isoelectric focusing (IEF; as indicated) or gradient SDS-PAGE and detected by phosphor-imaging. Triangles and arrows indicate the unlabeled and IASD-labeled Bax proteins, respectively. n = 3 for V111C, and K119C; 4 for W107C, and A117C; 2 for other mutants.C-D. The phosphor-imaging data for IASD labeling of Bax mutants in (B) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices as described and shown in Appendix Figure S3. The membrane burial indices were normalized by that of G179C in α9, a reference for the membrane-buried residues. The resulting relative membrane burial indices are shown in (C). The residues with the relative membrane burial index ≥ 40% of that of Gly179 are considered as buried in the MOM. Similarly, the relative protein burial indices shown in (D) were obtained by using Y115C in α5 as a reference for the protein-buried residues. The residues with the relative protein burial index ≥ 40% of that of Tyr115 are considered as buried in the protein or its complex.


Figure 3A - B . Oxidized mitochondria with the radioactive single - or double - cysteine Bax proteins , or the single - cysteine Bax protein pairs were prepared and analyzed as in Figure 1B . C . Oxidized mitochondria with the radioactive double - cysteine Bax protein were prepared , subjected to Na2CO3 extraction and analyzed as in Figure 1D .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-B. Oxidized mitochondria with the radioactive single- or double-cysteine Bax proteins, or the single-cysteine Bax protein pairs were prepared and analyzed as in Figure 1B.C. Oxidized mitochondria with the radioactive double-cysteine Bax protein were prepared, subjected to Na2CO3 extraction and analyzed as in Figure 1D.