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 .

O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-TISSUE 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 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 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 .

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

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


A . Representative FACS profiles of HSPC 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 .

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

O O B-EXP_ASSAY O O O B-TISSUE O 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 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 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 .

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

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 . 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 .

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

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


C . 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 O O O O O B-CELL_TYPE O O B-TISSUE O 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 O O O O B-GENEPROD O B-TISSUE O O O O O O O O O O

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


B , C Representative FACS plots of PKCδ fl / fl 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 .

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

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 ± SEM . * p < 0 . 05 , * * p < 0 . 005 and * * * p < 0 . 001 by two - tailed Student ' s unpaired t - test analysis .

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 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 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 .

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

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 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 .

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

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 . 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 .

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

O O O B-EXP_ASSAY O 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 O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE 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 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 ) 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 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 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 O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O 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 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 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 .

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

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 ) ; 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 .

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 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 ) . 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 .

O 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 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 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 .

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

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 .

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

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

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 .

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

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 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 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 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 .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O 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 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 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 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 .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 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 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 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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

; 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 .

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

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 .

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

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 .

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

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 .

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

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 , 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 .

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

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 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 .

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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 ' 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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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 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

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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 , 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 .

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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 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 .

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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 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 .

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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 images 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 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 .

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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 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 .

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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 . 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 ) .

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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 the radioactive single - cysteine Bax protein pair or double - cysteine Bax protein were prepared and 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 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 .

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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 . 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 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 B-SUBCELLULAR O O O B-SUBCELLULAR 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 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 B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 the radioactive single - or double - cysteine Bax proteins , or the single - cysteine Bax protein pairs were prepared and 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 the radioactive double - cysteine Bax protein were prepared , subjected to Na2CO3 extraction and 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 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 .

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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 . 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 .

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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 . 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 .

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

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 and D . The oxidized mitochondria with radioactive single - cysteine Bax proteins were prepared and 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 and E . The oxidized mitochondria with the radioactive single - cysteine Bax proteins were prepared , subjected to Na2CO3 extraction , and 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 . 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 .

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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 . 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 .

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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 . 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 .

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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 . 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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.


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 .

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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 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 ) .

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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 the stem cells and transit amplifying cells based on AcTub immunostaining .

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


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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


3D reconstruction showing the association of Prom1 ( green ) with AcTub - labeled ( red ) primary cilia in stem cell and transit amplifying cell regions . Note that the expression of Prom1 is not limited primary cilium but also to microvilli . A representative example of Prom1 association with one primary cilium at the stem cell to transit amplifying cell transition region . Green channel transparency was set up to 70 % .

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3D reconstruction showing the association of Prom1 (green) with AcTub-labeled (red) primary cilia in stem cell and transit amplifying cell regions. Note that the expression of Prom1 is not limited primary cilium but also to microvilli. A representative example of Prom1 association with one primary cilium at the stem cell to transit amplifying cell transition region. Green channel transparency was set up to 70%.


Representative IF staining of Prom1 using antibodies directed either its extracellular loop ( clone 13A4 , green ) or cytoplasmic C - terminal end ( Biorbyt , Orb129549 , red ) on transit amplifying cell regions of the WT vs . Prom1 KO mice . Samples are counterstained with DAPI ( blue ) . Note the lack of Prom1 labeling in Prom1 KO mice .

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Representative IF staining of Prom1 using antibodies directed either its extracellular loop (clone 13A4, green) or cytoplasmic C-terminal end (Biorbyt, Orb129549, red) on transit amplifying cell regions of the WT vs. Prom1 KO mice. Samples are counterstained with DAPI (blue). Note the lack of Prom1 labeling in Prom1 KO mice.


The mRNA profiling on shRNA - meditated Prom1 knockdown ( 3 different shRNAs were used , marked as NO . 1 , 2 and 3 ) in cultured CLESCs . qRT - PCR results are in arbitrary values after normalization for GapDH .

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The mRNA profiling on shRNA-meditated Prom1 knockdown (3 different shRNAs were used, marked as NO.1, 2 and 3) in cultured CLESCs. qRT- PCR results are in arbitrary values after normalization for GapDH.


protein profiling on shRNA - meditated Prom1 knockdown ( 3 different shRNAs were used , marked as NO . 1 , 2 and 3 ) in cultured CLESCs . The immunoblotting was performed with anti - Prom1 C - terminal antibody . β - actin was used as loading control ( F ) . Molecular mass markers are indicated .

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protein profiling on shRNA-meditated Prom1 knockdown (3 different shRNAs were used, marked as NO.1, 2 and 3) in cultured CLESCs. The immunoblotting was performed with anti-Prom1 C-terminal antibody. β-actin was used as loading control (F). Molecular mass markers are indicated.


Representative IF staining of Prom1 ( red ) and Sox2 ( green ) in CLESCs in vitro . Sample is counterstained with DAPI ( blue ) . Arrows indicate colocalisation of Prom1 with low - medium level of Sox2 in the cell nuclei .

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Representative IF staining of Prom1 (red) and Sox2 (green) in CLESCs in vitro. Sample is counterstained with DAPI (blue). Arrows indicate colocalisation of Prom1 with low-medium level of Sox2 in the cell nuclei.


Subcellular fractionation of CLESCs into cytoplasm / membrane ( C + M ) and nuclear ( N ) fractions . Materials were analyzed by immunoblotting for Prom1 ( H ) and Arl13b ( I ) . As controls , membranes were blotted for anti - Lamin A / C ( nuclei markers ) and GapDH ( cytoplasm marker ) . Molecular mass markers are indicated . Note the presence of full - length Prom1 as well as Arl13b in nuclear fraction .

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Subcellular fractionation of CLESCs into cytoplasm/membrane (C+M) and nuclear (N) fractions. Materials were analyzed by immunoblotting for Prom1 (H) and Arl13b (I). As controls, membranes were blotted for anti-Lamin A/C (nuclei markers) and GapDH (cytoplasm marker). Molecular mass markers are indicated. Note the presence of full-length Prom1 as well as Arl13b in nuclear fraction.


Representative images ( A ) and quantitative analysis ( B ) of IF staining and 3D reconstruction on Sox2 - positive region for WT vs . Prom1 KO mice . Ant : Anterior ; Post : Posterior ( A ) . Statistics was performed using Student ' s t - test : * * , p < 0 . 01 . Data are represented as mean and standard deviation .

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Representative images (A) and quantitative analysis (B) of IF staining and 3D reconstruction on Sox2-positive region for WT vs. Prom1 KO mice. Ant: Anterior; Post: Posterior (A). Statistics was performed using Student's t-test: **, p < 0.01. Data are represented as mean and standard deviation.


Representative IF double staining of Sox 2 ( green ) and Ki67 ( red ) on the and transit amplifying cell regions of WT vs . Prom1 KO mice on frontal sections . Samples are counterstained with DAPI ( blue ) . Quantification of Ki67 + cell number in Sox2 - positive region based on IF data from WT and Prom1 KO mice ( C ) .

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Representative IF double staining of Sox 2 (green) and Ki67 (red) on the and transit amplifying cell regions of WT vs. Prom1 KO mice on frontal sections. Samples are counterstained with DAPI (blue). Quantification of Ki67+ cell number in Sox2-positive region based on IF data from WT and Prom1 KO mice (C).


Stereo images of three explants cultured with indicated recombination conditions before harvesting at day 3 . Dotted line indicates the CLE .

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Stereo images of three explants cultured with indicated recombination conditions before harvesting at day 3. Dotted line indicates the CLE.


Passaging capacity analysis of the CLESCs derived from P7 WT and Prom1 KO mice . Note the passages of cells derived from WT animals are still ongoing while those from Prom1 KO mice were arrested at the first passage . Arrows indicated cells were still under passaging while blunt bars represent cells could not be passaged and stopped at the indicated passage .

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Passaging capacity analysis of the CLESCs derived from P7 WT and Prom1 KO mice. Note the passages of cells derived from WT animals are still ongoing while those from Prom1 KO mice were arrested at the first passage. Arrows indicated cells were still under passaging while blunt bars represent cells could not be passaged and stopped at the indicated passage.