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

O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O 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-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O

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


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

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O O O O O

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


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

O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-TISSUE O B-TISSUE O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-ORGANISM O

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


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

O O B-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-TISSUE O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-ORGANISM O

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


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

O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-ORGANISM O

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


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

O O B-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-TISSUE O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-ORGANISM O

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


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

O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-ORGANISM O

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


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

O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O B-GENEPROD O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O 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-EXP_ASSAY O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O O B-CELL_TYPE B-EXP_ASSAY O O O B-GENEPROD B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-EXP_ASSAY O O O B-TISSUE O O O B-GENEPROD O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O O O B-TISSUE I-TISSUE O B-TISSUE O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-TISSUE O O O O O O O O

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


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

O O O O O B-CELL_TYPE O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-TISSUE O O O O O O O O

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


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

O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O B-ORGANISM O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O B-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O B-ORGANISM B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O B-CELL_TYPE O O O B-EXP_ASSAY O O B-TISSUE B-CELL_TYPE O O O B-ORGANISM O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY B-CELL_TYPE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-CELL_TYPE O B-TISSUE B-CELL_TYPE O O O O B-GENEPROD O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-ORGANISM O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_TYPE O O O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-SUBCELLULAR O O O O O O B-ORGANISM O O O O O O O O O

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


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

O O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O B-EXP_ASSAY O B-TISSUE I-TISSUE B-CELL_TYPE O O O O B-ORGANISM O O 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-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O B-TISSUE I-TISSUE B-CELL_TYPE O O O O B-ORGANISM O O 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-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O B-TISSUE I-TISSUE B-CELL_TYPE O O O O B-ORGANISM O O 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-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


F RT - qPCR fold - change induction of PUPPIES - uns , PUPPIES - prom , PUPPIES - fusion , shDOG1 , lgDOG1 in puppies - ox relative to WT ( blue dashed ) , in dry ( grey ) imbibed seeds in presence of 100 mM NaCl ( red ) . Significance from two - Student ' s t - for comparing dry seeds of WT puppies - ox is represented with grey asterisks , salt - imbibed seeds of WT puppies - ox are represented with red asterisks . Black asterisks represent significance from two - way ANOVA with Tukey ' s multiple comparisons 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 mean ± SD . n = 4 biological replicates .

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F 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 lgDOG1 on nascent from seeds imbibed in 100 mM NaCl from puppies - ox relative to WT . Nascent were normalized to UBC21 . Bars error bars represent mean ± SD . Points represent biological replicates . Statistical significance from two - Student ' s t - . ∗ p - value < 0 . 05 .

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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.


A z - stack max - projection image of smFISH for DOG1 . " Inferno " colour scale is used for of from Quasar670 fluorophore ( DOG1 ) . blue colour shows from DAPI ( nuclei ) . Arrowheads point to foci corresponding to transcription sites ( TS ) . scale bar is 20 μm . B Distribution of DOG1 foci per in WT . blue vertical dashed indicates average .

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A 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. C


G z - stack max - projection from seeds imbibed in 100 mM NaCl of WT ( left ) versus puppies - ox ( right ) . Arrowheads point to foci corresponding to TS .

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G 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.


A - E profile of Pol II during DOG1 transcription . Plots show 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 gene . Pol II dynamics is analyzed separately for each region sequenced in exon 1 ( A ) , exon 2 ( B ) , exon 3 ( C ) , intron 1 ( D ) , intron 2 ( E ) . x - axis shows in bp from DOG1 . A red colour fill highlights regions where Pol II density is higher in puppies - ox . A blue colour fill highlights regions where Pol II density is higher in WT . For each plot , coloured tiles represent fold - change ( puppies - ox / WT ) on a logarithmic scale . Greyscale tiles represent result of a statistical for difference between genotypes from two - Student ' s t - after Bonferroni correction . Tiles correspond to a 25 bp bin each . Vertical dashed lines connect exon boundaries on plots with schematics of DOG1 gene with grey boxes representing exons black lines representing introns .

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A-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 beta isoforms unspliced intron 1 DOG1 isoforms in puppies - ox , puppies - 1 relative to WT , in seeds treated with 100 mM NaCl .

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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.


B . in vitro synthesized [ 35S ] Met - labeled single - cysteine Bax were activated targeted to mitochondria that were pretreated with NEM to block sulfhydryls of mitochondrial . resulting mitochondria were isolated oxidized by CuPhe for 30 min . NEM EDTA were then added to stop oxidation . For " 0 min " controls , NEM EDTA were added before addition of CuPhe . resulting samples were analyzed by phosphor - imaging after non - reducing or reducing SDS - PAGE ( see Appendix Figure S2A ) .

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B. 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 radioactive single - cysteine Bax pair or double - cysteine Bax were prepared analyzed as in ( B ) .

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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 radioactive single - cysteine Bax pair or double - cysteine Bax were prepared as in ( B ) . After an aliquot was withdrawn as input , another aliquot was extracted by Na2CO3 ( pH 11 . 5 ) centrifuged through a sucrose cushion to separate integral in pellet from soluble peripheral in supernatant . input , pellet supernatant were analyzed by non - reducing SDS - PAGE phosphor - imaging . In a parallel control experiment , pellet supernatant were analyzed by reducing SDS - PAGE immunoblotting with an antibody specific to PDHE1α , a soluble mitochondrial matrix .

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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.


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

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B. 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.


A - B . Oxidized mitochondria with radioactive single - or double - cysteine Bax , or single - cysteine Bax pairs were prepared analyzed as in Figure 1B .

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A-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 radioactive double - cysteine Bax were prepared , subjected to Na2CO3 extraction analyzed as in Figure 1D .

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C. Oxidized mitochondria with the radioactive double-cysteine Bax protein were prepared, subjected to Na2CO3 extraction and analyzed as in Figure 1D.


B D . radioactive single - or double - cysteine Bax with or without indicated mutations were activated targeted to mitochondria that were either untreated ( mito ) or pretreated with NEM ( NEM - mito ) , then oxidized analyzed as in Figure 1B . standards , monomers disulfide - linked dimers of Bax are indicated as in Figure 1B . n = 2 for mutants .

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B and D. The radioactive single- or double-cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria that were either untreated (mito) or pretreated with NEM (NEM-mito), then oxidized and analyzed as in Figure 1B. Protein standards, and monomers and disulfide-linked dimers of the Bax proteins are indicated as in Figure 1B. n = 2 for all mutants.


C . in vitro synthesized [ 35S ] Met - labeled Bax E69C with or without additional mutations ( G108E / or S184V ) were activated by in vitro synthesized [ 35S ] Met - labeled tBid L105C targeted to Bax - / - / Bak - / - mitochondria that were pretreated with NEM . resulting mitochondria were isolated oxidized by CuPhe for 0 or 30 min . resulting " 0 min " samples ( 1 equivalent each ) " 30 min " samples ( 1 equivalent each ) were analyzed by non - reducing SDS - PAGE phosphor - imaging . remaining " 30 min " samples ( 4 equivalent each ) were immunoprecipitated ( IP ) by either Bax - or tBid - specific antibody , then analyzed by non - reducing SDS - PAGE phosphor - imaging . identities of four major products , indicated on right side of image , were on their Mr recognition by respective antibody . n = 2 .

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C. The in vitro synthesized [35S]Met-labeled Bax E69C protein with or without the additional mutations (G108E and/or S184V) were activated by the in vitro synthesized [35S]Met-labeled tBid L105C protein and targeted to the Bax-/-/Bak-/- mitochondria that were pretreated with NEM. The resulting mitochondria were isolated and oxidized by CuPhe for 0 or 30 min. The resulting "0 min" samples (1 equivalent each) and "30 min" samples (1 equivalent each) were analyzed by non-reducing SDS-PAGE and phosphor-imaging. The remaining "30 min" samples (4 equivalent each) were immunoprecipitated (IP) by either Bax- or tBid-specific antibody, and then analyzed by non-reducing SDS-PAGE and phosphor-imaging. The identities of the four major products, indicated on the right side of the image, were based on their Mr and recognition by the respective antibody. n = 2.


A . in vitro synthesized radioactive Bax , each with a single cysteine positioned in α9 , were activated targeted to mitochondria , labeled with IASD as in Figure 2B . IASD - labeled unlabeled Bax were resolved using gradient SDS - PAGE , detected by phosphor - imaging indicated by triangles arrows , respectively . For mutants , n = 2 , except for A183C , n = 3 . B - C . IASD labeling data in ( A ) similar data from independent replicates were quantified to derive or burial indices shown in Appendix Figure S5 , from which relative ( B ) or ( C ) burial indices were obtained as described in Figure 2C - D , shown . D . structure of α9 ( extracted from NMR structure of Bax monomer , PDB entry 1F16 ) is shown with residues that are buried in MOM or exposed to aqueous milieu , as IASD labeling data in ( A - C ) suggested , presented in yellow or cyan stick form , respectively .

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A. The in vitro synthesized radioactive Bax proteins, each with a single cysteine positioned in α9, were activated and targeted to the mitochondria, and labeled with IASD as in Figure 2B. The IASD-labeled and unlabeled Bax proteins were resolved using gradient SDS-PAGE, detected by phosphor-imaging and indicated by triangles and arrows, respectively. For all mutants, n = 2, except for A183C, n = 3.B-C. The IASD labeling data in (A) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices shown in Appendix Figure S5, from which the relative membrane (B) or protein (C) burial indices were obtained as described in Figure 2C-D, and shown.D. The structure of α9 (extracted from the NMR structure of Bax monomer, PDB entry 1F16) is shown with the residues that are buried in the MOM or exposed to the aqueous milieu, as the IASD labeling data in (A-C) suggested, presented in yellow or cyan stick form, respectively.


B D . oxidized mitochondria with radioactive single - cysteine Bax were prepared analyzed as in Figure 1B .

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B and D. The oxidized mitochondria with radioactive single-cysteine Bax proteins were prepared and analyzed as in Figure 1B.


C E . oxidized mitochondria with radioactive single - cysteine Bax were prepared , subjected to Na2CO3 extraction , analyzed as in Figure 1D .

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C and E. The oxidized mitochondria with the radioactive single-cysteine Bax proteins were prepared, subjected to Na2CO3 extraction, and analyzed as in Figure 1D.


B - D . in vitro synthesized radioactive single or double - cysteine Bax with or without indicated mutations were activated targeted to mitochondria , oxidized , analyzed as in Figure 1B . standards , monomers disulfide - linked dimers of Bax are indicated as in Figure 1B . n = 2 for mutants .

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B-D. The in vitro synthesized radioactive single or double-cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria, oxidized, and analyzed as in Figure 1B. Protein standards, and monomers and disulfide-linked dimers of the Bax proteins are indicated as in Figure 1B. n = 2 for all mutants.


A . oxidized mitochondria with radioactive single , double , or triple - cysteine Bax were prepared analyzed by non - reducing or reducing SDS - PAGE as in Figure 1B . standards , Bax monomers disulfide - linked Bax homodimers are also indicated as in Figure 1B . Closed triangles indicate disulfide - linked higher order Bax homo - oligomers . Open triangles indicate a background band that could not be reduced , hence is not a disulfide - linked .

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A. The oxidized mitochondria with radioactive single, double, or triple-cysteine Bax proteins were prepared and analyzed by non-reducing or reducing SDS-PAGE as in Figure 1B. Protein standards, Bax monomers and disulfide-linked Bax homodimers are also indicated as in Figure 1B. Closed triangles indicate disulfide-linked higher order Bax homo-oligomers. Open triangles indicate a background band that could not be reduced, and hence is not a disulfide-linked complex.


B . BMH - treated mitochondria with radioactive single - double - cysteine Bax were prepared analyzed as in Figure EV3G , except that closed triangles indicate BMH - linked Bax oligomers .

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B. The BMH-treated mitochondria with the radioactive single- and double-cysteine Bax proteins were prepared and analyzed as in Figure EV3G, except that closed triangles indicate the BMH-linked Bax oligomers.


A - C . single or double - cysteine Bax with or without indicated mutations were synthesized in vitro , activated by tBid targeted to Bax - / - / Bak - / - mitochondria . Cytochrome c release from mitochondria was measured using ELISA . dots are fractions of cytochrome c release from two independent replicates after they were corrected as described in Figure EV1B , lines are means . amount of Bax mutants bound to mitochondria in was determined results are shown in Appendix Figure S1 .

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A-C. The single or double-cysteine Bax proteins with or without the indicated mutations were synthesized in vitro, activated by tBid protein and targeted to the Bax-/-/Bak-/- mitochondria. Cytochrome c release from the mitochondria was measured using ELISA. The dots are the fractions of cytochrome c release from two independent replicates after they were corrected as described in Figure EV1B, and the lines are the means. The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1.


A . cytochrome c release by indicated Bax mutants was measured in two independent replicates , corrected shown as in Figure 9 . amount of Bax mutants bound to mitochondria in was determined results are shown in Appendix Figure S1 .

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A. The cytochrome c release by the indicated Bax mutants was measured in two independent replicates, corrected and shown as in Figure 9. The amount of the Bax mutants bound to the mitochondria in the assay was determined and the results are shown in Appendix Figure S1.


B . Purified wild - type ( WT ) or mutant ( G179I or T182I ) Bax of indicated was incubated with Bax - / - / Bak - / - mitochondria in absence or presence of tBid . released from those remaining in mitochondria were separated analyzed by SDS - PAGE immunoblotting with antibody specific to either cytochrome c or Smac . n = 2 .

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B. Purified wild-type (WT) or mutant (G179I or T182I) Bax protein of the indicated concentration was incubated with the Bax-/-/Bak-/- mitochondria in the absence or presence of tBid protein. The proteins released from and those remaining in the mitochondria were separated and analyzed by SDS-PAGE and immunoblotting with antibody specific to either cytochrome c or Smac. n = 2.


C . Purified wild - type or mutant Bax of indicated alone or together with purified cBid was incubated with Bax - / - / Bak - / - mitochondria containing Smac - mCherry . Release of Smac - mCherry was detected by measuring in supernatant after mitochondria were pelleted by centrifugation . dots are fractions of Smac - mCherry release from three independent replicates , lines are means .

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C. Purified wild-type or mutant Bax protein of the indicated concentration alone or together with purified cBid was incubated with the Bax-/-/Bak-/- mitochondria containing Smac-mCherry protein. Release of Smac-mCherry was detected by measuring the fluorescence in the supernatant after the mitochondria were pelleted by centrifugation. The dots are the fractions of Smac-mCherry release from three independent replicates, and the lines are the means.


D . in vitro synthesized radioactive Bax with indicated mutations were targeted to mitochondria in absence or presence of purified tBid , then labeled with IASD , as in Figure 2B . IASD - labeled unlabeled Bax were resolved using either gradient SDS - PAGE or IEF , detected by phosphor - imaging , indicated by triangles arrows , respectively . n = 3 for A178C , G179I ; 2 for other mutants . E . IASD labeling data in ( D ) similar data from independent replicates were quantified to derive burial indices shown in Appendix Figure S7A , from which relative burial indices were obtained as described in Figure 2C , shown .

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D. The in vitro synthesized radioactive Bax proteins with indicated mutations were targeted to the mitochondria in the absence or presence of purified tBid protein, and then labeled with IASD, as in Figure 2B. The IASD-labeled and unlabeled Bax proteins were resolved using either gradient SDS-PAGE or IEF, detected by phosphor-imaging, and indicated by triangles and arrows, respectively. n = 3 for A178C,G179I; 2 for other mutants.E. The IASD labeling data in (D) and the similar data from the independent replicates were quantified to derive the membrane burial indices shown in Appendix Figure S7A, from which the relative membrane burial indices were obtained as described in Figure 2C, and shown.


Representative IF of Sox2 ( green ) Ki67 ( red ) on P7 CLE transit amplifying regions counterstained with DAPI ( blue ) on a sagittal section . Dotted lines , basement ; yellow arrowheads mark approximate boundaries . SCs , ; TACs , transit amplifying ; Ant , Anterior ; Post , Posterior .

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Representative IF staining of Sox2 (green) and Ki67 (red) on the P7 CLE stem cell and transit amplifying cell regions and counterstained with DAPI (blue) on a sagittal section. Dotted lines, basement membrane; yellow arrowheads mark approximate stem cell boundaries. SCs, stem cells; TACs, transit amplifying cells; Ant, Anterior; Post, Posterior.


mRNA expression profiling on specific markers of ( B ) transit amplifying captured on P7 incisors CLE followed by using real - time RT - PCR ( C ) . qRT - PCR results are in arbitrary values after normalization for GapDH . Statistics was performed on triplicates using one - way ANOVA followed by Bonferroni ' s .

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The mRNA expression profiling on specific markers of stem cells (B) and transit amplifying cells captured on P7 incisors CLE followed by analysis using real-time RT-PCR (C). qRT- PCR results are in arbitrary values after normalization for GapDH. Statistics was performed on triplicates using one-way ANOVA followed by Bonferroni's test.


Representative IF double of E - Cadherin ( ECad , green , for border ) AcTub ( red , for cores of cilia ) on a sagittal section . Sample is counterstained with DAPI ( blue ) .

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Representative IF double staining of E-Cadherin (ECad, green, for cell border) and AcTub (red, for the cores of primary cilia) on a sagittal section. Sample is counterstained with DAPI (blue).


3D reconstructions of ciliary cores of transit amplifying on AcTub immunostaining .

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3D reconstructions of ciliary cores of the stem cells and transit amplifying cells based on AcTub immunostaining.


of cilia ( F ) volume ( G ) on 3D reconstruction ( E ) comparison between those in transit amplifying regions . Numbers of quantified cilia were indicated ( n ) . Statistics was performed using one - way ANOVA : error bars were defined in Statistical section : real - time RT - PCR results were presented using style of mean standard deviation .

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Quantification of cilia length (F) and volume (G) based on 3D reconstruction (E) and comparison between those in stem cell and transit amplifying cell regions. Numbers of quantified cilia were indicated (n). Statistics was performed using one-way ANOVA: All error bars were defined in Statistical Analysis section: All quantification and real-time RT-PCR results were presented using style of mean and standard deviation.


Tamoxifen induced IFT88 deletion strategy ( top panel ) representative of Sox2 ( green ) Ki67 ( red ) immunostaining on CLE regions of indicated mouse strains ( bottom panels ) . Dotted lines , basement . Note significant reduction of both Sox2 Ki67 immunolabeling upon deletion of IFT88 .

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Tamoxifen induced IFT88 deletion strategy (top panel) and representative images of Sox2 (green) and Ki67 (red) immunostaining on CLE regions of the indicated mouse strains (bottom panels). Dotted lines, basement membrane. Note the significant reduction of both Sox2 and Ki67 immunolabeling upon deletion of IFT88.


3D reconstruction of Sox2 - positive regions on its immunolabeling of indicated mouse strains ( I ) their volume comparison between mouse strains as indicated ( J ) . Statistics was performed using Student ' s t - : * * , p < 0 . 01 . Data are represented as mean standard deviation .

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3D reconstruction of Sox2-positive regions based on its immunolabeling of the indicated mouse strains (I) and their volume comparison between mouse strains as indicated (J). Statistics was performed using Student's t-test: **, p < 0.01. Data are represented as mean and standard deviation.


Representative IF double of ADP - ribosylation factor - like 13B ( Arl13b , red , K ) or histone deacetylase 6 ( Hdac6 , red , L ) with AcTub ( green ) as a marker of cilium following to transit amplifying transition .

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Representative IF double staining of ADP-ribosylation factor-like protein 13B (Arl13b, red, K) or histone deacetylase 6 (Hdac6, red, L) with AcTub (green) as a marker of the primary cilium core following stem cell to transit amplifying cell transition.


Representative IF of Prom1 using specific antibody clone 13A4 targeting extracellular loop ( green ) on transit amplifying regions of lower incisor CLE at P7 . Sample is counterstained with DAPI ( blue ) . Dotted lines , basement . SCs , ; TACs , transit amplifying ; Ams , ameloblasts .

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Representative IF staining of Prom1 using specific antibody clone 13A4 targeting extracellular loop (green) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Sample is counterstained with DAPI (blue). Dotted lines, basement membrane. SCs, stem cells; TACs, transit amplifying cells; Ams, ameloblasts.