( C ) Immunofluorescent staining of cleaved APPC586 ( red ) and Aβ ( green ) in cerebral cortex of brains from colonic - injected PBS , colonic - injected AD brain extracts , and pre - Vagotomy colonic injected AD brain extracts 3xTg mice . ( D ) Quantitative analysis of cleaved APP C586 positive cells and Aβ positive cells , respectively . The density of both cleaved APP C586 positive cells and Aβ positive cells were significantly increased by colonic - injection of AD brain extracts and decreased through vagotomy before colonic - injection . Representative data of three samples , data are shown as mean ± SEM . * * * * P < 0 . 0001 , one - way ANOVA .

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(C) Immunofluorescent staining of cleaved APPC586 (red) and Aβ (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. (D) Quantitative analysis of cleaved APP C586 positive cells and Aβ positive cells, respectively. The density of both cleaved APP C586 positive cells and Aβ positive cells were significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. ****P<0.0001, one-way ANOVA.


( E ) Immunofluorescent staining of T22 ( red ) and HT7 ( green ) in cerebral cortex of brains from colonic - injected PBS , colonic - injected AD brain extracts , and pre - Vagotomy colonic injected AD brain extracts 3xTg mice . ( F ) Quantitative analysis of T22 positive cells . The density of T22 positive cells was significantly increased by colonic - injection of AD brain extracts and decreased through vagotomy before colonic - injection . Representative data of three samples , data are shown as mean ± SEM . * * * P = 0 . 0007 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA .

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(E) Immunofluorescent staining of T22 (red) and HT7 (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. (F) Quantitative analysis of T22 positive cells. The density of T22 positive cells was significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. ***P=0.0007 (***P < 0.001), ****P<0.0001, one-way ANOVA.


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 )

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


( B ) FACS plots depict 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 ) .

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


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

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


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

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

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


E . FACS plots illustrate gating 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 .

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

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


F . FACS plots illustrate 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 .

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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 .

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

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


A . Representative FACS profiles of HSPC 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 .

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 .

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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 .

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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 .

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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 .

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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 .

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

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 ) .

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(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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

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

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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


F . Relative ATP content in indicated HSPC subsets determined using an ATP 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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


I . ROS 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 ) 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 .

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.


Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2 , 4 and 6 after BAC treatment . The intestinal extracts from the sham - treated guts were used as controls . ( A ) Morphology of the ENCC - derived neurospheres on days 2 and 4 . ( B - C ) The proliferation of ENCC - derived neurospheres is represented by neurosphere diameter ( B ) and change in diameter on day 4 compared with day 2 ( C ) .

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Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (A) Morphology of the ENCC-derived neurospheres on days 2 and 4. (B-C) The proliferation of ENCC-derived neurospheres is represented by neurosphere diameter (B) and change in diameter on day 4 compared with day 2 (C).


Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2 , 4 and 6 after BAC treatment . The intestinal extracts from the sham - treated guts were used as controls . ( D ) ENCC viability assessed by CCK - 8 assay .

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Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (D) ENCC viability assessed by CCK-8 assay.


Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2 , 4 and 6 after BAC treatment . The intestinal extracts from the sham - treated guts were used as controls . ( E ) ENCCs number analyzed by nuclear staining with DAPI .

Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (E) ENCCs number analyzed by nuclear staining with DAPI.


Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2 , 4 and 6 after BAC treatment . The intestinal extracts from the sham - treated guts were used as controls . ( F ) Migration of ENCCs evaluated by transwell plate assay and stain with crystal violet ,

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

Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (F) Migration of ENCCs evaluated by transwell plate assay and stain with crystal violet,


Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2 , 4 and 6 after BAC treatment . The intestinal extracts from the sham - treated guts were used as controls . ( G ) ENCC apoptosis determined by AnnexinV - FITC / PI stain and flow cytometry .

Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (G) ENCC apoptosis determined by AnnexinV-FITC/PI stain and flow cytometry.


Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats . For hypoganglionosis , fecal samples from the narrow segments ( NS , BAC - treated , hypoganglionic ) and expanded segments ( ES , proximal to narrow segments , with abnormal ganglion ) colon are analyzed , respectively . ( A - D ) Cluster analysis at the ( A ) phylum and ( C ) genus levels . A column accumulation diagram of the top 10 most abundant ( B ) phyla and ( D ) genera .

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

Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. (A-D) Cluster analysis at the (A) phylum and (C) genus levels. A column accumulation diagram of the top 10 most abundant (B) phyla and (D) genera.


Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats . For hypoganglionosis , fecal samples from the narrow segments ( NS , BAC - treated , hypoganglionic ) and expanded segments ( ES , proximal to narrow segments , with abnormal ganglion ) colon are analyzed , respectively . Inter - group diversity analysis to compare the differences in ( H ) intestinal microbiota The species indicated in red are linked to SCFAs production .

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

Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. Inter-group diversity analysis to compare the differences in (H) intestinal microbiota The species indicated in red are linked to SCFAs production.


Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats . For hypoganglionosis , fecal samples from the narrow segments ( NS , BAC - treated , hypoganglionic ) and expanded segments ( ES , proximal to narrow segments , with abnormal ganglion ) colon are analyzed , respectively . metabolic mass spectrometry to compare the differences ( I ) SCFAs metabolites between the hypoganglionosis and sham rats .

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Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. metabolic mass spectrometry to compare the differences (I) SCFAs metabolites between the hypoganglionosis and sham rats.


Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats . For hypoganglionosis , fecal samples from the narrow segments ( NS , BAC - treated , hypoganglionic ) and expanded segments ( ES , proximal to narrow segments , with abnormal ganglion ) colon are analyzed , respectively . ( J ) Representative HE staining and quantification to assess the intestinal mucosal injury .

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

Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. (J) Representative HE staining and quantification to assess the intestinal mucosal injury.


Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats . For hypoganglionosis , fecal samples from the narrow segments ( NS , BAC - treated , hypoganglionic ) and expanded segments ( ES , proximal to narrow segments , with abnormal ganglion ) colon are analyzed , respectively . ( K ) Representative IHC stain and quantification of CRP and TNF - α levels .

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

Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. (K) Representative IHC stain and quantification of CRP and TNF-α levels.


Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats . For hypoganglionosis , fecal samples from the narrow segments ( NS , BAC - treated , hypoganglionic ) and expanded segments ( ES , proximal to narrow segments , with abnormal ganglion ) colon are analyzed , respectively . ( L ) Representative immunofluorescence stain and quantification of β - catenin levels . Nuclei were stained blue with DAPI .

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

Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. (L) Representative immunofluorescence stain and quantification of β-catenin levels. Nuclei were stained blue with DAPI.


Comparison of ENCC - derived neurospheres treated with the intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) and sham rat . The diameter of ENCC - derived neurospheres on day 2 ( A ) .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. The diameter of ENCC-derived neurospheres on day 2 (A).


Comparison of ENCC - derived neurospheres treated with the intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) and sham rat . ENCC viability assessed by CCK - 8 assay ( B ) .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. ENCC viability assessed by CCK-8 assay (B).


Comparison of ENCC - derived neurospheres treated with the intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) and sham rat . ( C - D ) Migration of ENCCs using transwell plate assay and stain with crystal violet ( C ) , scale bar , 200 µm . ( D ) Quantification of C . ( E - F ) ENCC apoptosis determined by AnnexinV - FITC / PI stain and flow cytometry ( E ) . ( F ) Quantification of E .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. (C-D) Migration of ENCCs using transwell plate assay and stain with crystal violet (C), scale bar, 200 µm. (D) Quantification of C. (E-F) ENCC apoptosis determined by AnnexinV-FITC/PI stain and flow cytometry (E). (F) Quantification of E.


Comparison of ENCC - derived neurospheres treated with the intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) and sham rat . ( G ) Graphical protocol and weight change in the hypoganglionosis : untreated and treated with ENCCs only , or ENCCs + FMT .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. (G) Graphical protocol and weight change in the hypoganglionosis: untreated and treated with ENCCs only, or ENCCs+FMT.


Comparison of ENCC - derived neurospheres treated with the intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) and sham rat . ( H ) Laparoscopic analysis indicating that the same segments were treated with BAC in all the groups .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. (H) Laparoscopic analysis indicating that the same segments were treated with BAC in all the groups.


Comparison of ENCC - derived neurospheres treated with the intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) and sham rat . ( I ) Delay in distal colonic transit time .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. (I) Delay in distal colonic transit time.


Comparison of ENCC - derived neurospheres treated with the intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) and sham rat . Comparison of neuronal markers . ( J ) Immunofluorescence stain and number of PGP9 . 5 + cells . Nuclei were stained blue with DAPI ; Triangles indicate the PGP9 . 5 + cell .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. Comparison of neuronal markers. (J) Immunofluorescence stain and number of PGP9.5+ cells. Nuclei were stained blue with DAPI; Triangles indicate the PGP9.5+ cell.


Comparison of ENCC - derived neurospheres treated with the intestinal extract from hypoganglionosis rats , hypoganglionosis treated with Fecal microbiota transplantation ( FMT ) and sham rat . Comparison of neuronal markers . ( K ) Representative immunofluorescence images of Tuj1 and GFP localization at the injection and adjacent sides . Asterisks and triangles indicate the Tuj1 + / GFP + cells and Tuj1 + / GFP - cells , respectively . Nuclei were stained blue with DAPI . ( L - M ) Number of Tuj1 + , Tuj1 + / GFP + cells ( L ) and Tuj1 + / GFP - cells ( M ) .

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Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. Comparison of neuronal markers. (K) Representative immunofluorescence images of Tuj1 and GFP localization at the injection and adjacent sides. Asterisks and triangles indicate the Tuj1+/GFP+ cells and Tuj1+/GFP- cells, respectively. Nuclei were stained blue with DAPI. (L-M) Number of Tuj1+, Tuj1+/GFP+ cells (L) and Tuj1+/GFP- cells(M).


Comparison of fecal microbial species , short - chain fatty acids and inflammation between hypoganglionosis : untreated ( Hypoganglionosis ) or treated with broad - range antibiotics ( , Antibiotic ) , broad - range antibiotics followed by FMT ( FMT ) and sham groups . ( A - D ) Cluster analysis at the ( A ) phylum and ( C ) genus . Column accumulation diagrams of the top abundant microbiota at the ( B ) phylum and ( D ) genus levels .

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Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups. (A-D) Cluster analysis at the (A) phylum and (C) genus. Column accumulation diagrams of the top abundant microbiota at the (B) phylum and (D) genus levels.


Comparison of fecal microbial species , short - chain fatty acids and inflammation between hypoganglionosis : untreated ( Hypoganglionosis ) or treated with broad - range antibiotics ( , Antibiotic ) , broad - range antibiotics followed by FMT ( FMT ) and sham groups . ( G ) SCFAs subsets in the feces and intestinal extracts , analyzed by targeted mass spectrometry .

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Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups. (G) SCFAs subsets in the feces and intestinal extracts, analyzed by targeted mass spectrometry.


Comparison of fecal microbial species , short - chain fatty acids and inflammation between hypoganglionosis : untreated ( Hypoganglionosis ) or treated with broad - range antibiotics ( , Antibiotic ) , broad - range antibiotics followed by FMT ( FMT ) and sham groups . ( H ) IL - 10 , IL - 17 , and TNF - α levels in serum and intestinal extracts , assessed by ELISA .

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Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups. (H) IL-10, IL-17, and TNF-α levels in serum and intestinal extracts, assessed by ELISA.


Comparison of fecal microbial species , short - chain fatty acids and inflammation between hypoganglionosis : untreated ( Hypoganglionosis ) or treated with broad - range antibiotics ( , Antibiotic ) , broad - range antibiotics followed by FMT ( FMT ) and sham groups . Correlation analysis of acetate , propionate or butyrate with IL - 17 in the intestinal extracts .

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

Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups. Correlation analysis of acetate, propionate or butyrate with IL-17 in the intestinal extracts.


Phenotype analysis of ENCCs treated with SCFAs . ( A ) ENCC viability assessed by CCK - 8 assay . Data are normalized to the NC group .

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Phenotype analysis of ENCCs treated with SCFAs. (A) ENCC viability assessed by CCK-8 assay. Data are normalized to the NC group.


Phenotype analysis of ENCCs treated with SCFAs . ( B - C ) Migration of ENCCs evaluated using transwell plate assay and stain with crystal violet ( B ) ; Scale bars represent 200 µm . ( C ) Quantitative analysis of B .

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Phenotype analysis of ENCCs treated with SCFAs. (B-C) Migration of ENCCs evaluated using transwell plate assay and stain with crystal violet (B); Scale bars represent 200 µm. (C) Quantitative analysis of B.


Phenotype analysis of ENCCs treated with SCFAs . Analysis of different IL - 17 concentrations on ENCC viability ( D )

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Phenotype analysis of ENCCs treated with SCFAs. Analysis of different IL-17 concentrations on ENCC viability (D)


Phenotype analysis of ENCCs treated with SCFAs . Analysis of different IL - 17 concentrations 50 ng / mL IL - 17 on ENCC migration ( E , F )

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Phenotype analysis of ENCCs treated with SCFAs. Analysis of different IL-17 concentrations 50 ng/mL IL-17 on ENCC migration (E, F)


Phenotype analysis of ENCCs treated with SCFAs . ( G - H ) ENCC apoptosis determined by AnnexinV - FITC / PI stain and flow cytometry ( G ) . ( H ) . Quantification of G .

Phenotype analysis of ENCCs treated with SCFAs. (G-H) ENCC apoptosis determined by AnnexinV-FITC/PI stain and flow cytometry (G). (H). Quantification of G.


( B ) Delay in distal colonic transit time .

(B) Delay in distal colonic transit time.


( C - D ) Representative immunofluorescent images ( C ) and ( D ) number of PGP9 . 5 + cells . Nuclei were stained blue with DAPI ; Triangles indicate the PGP9 . 5 + cell .

O O O O O O O O O O O O O O O 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-D) Representative immunofluorescent images (C) and (D) number of PGP9.5+ cells. Nuclei were stained blue with DAPI; Triangles indicate the PGP9.5+ cell.


( E ) Representative immunofluorescent images of Tuj1 and GFP localization at the injection and adjacent sides . Asterisks and triangles indicate the Tuj1 + / GFP + cells and Tuj1 + / GFP - cells , respectively .

(E) Representative immunofluorescent images of Tuj1 and GFP localization at the injection and adjacent sides. Asterisks and triangles indicate the Tuj1+/GFP+ cells and Tuj1+/GFP- cells, respectively.


( F - G ) Number of Tuj1 + and Tuj1 + / GFP + cells ( F ) and Tuj1 + / GFP - cells ( G ) .

(F-G) Number of Tuj1+ and Tuj1+/GFP+ cells (F) and Tuj1+/GFP- cells(G).


Volcano diagram shows differentially expressed genes ( DEGs ) in ENCCs after SCFAs and IL - 17A intervention , with up - regulated and down - regulated genes shown in yellow and blue , respectively ( | log fold change | ≥ 0 . 5 and adjusted p < 0 . 05 ) .

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Volcano diagram shows differentially expressed genes (DEGs) in ENCCs after SCFAs and IL-17A intervention, with up-regulated and down-regulated genes shown in yellow and blue, respectively (|log fold change|≥0.5 and adjusted p<0.05).


( E ) ENCC viability assessed by CCK - 8 assay after intervention with MEK1 / 2 inhibitor ( U0126 , 50 μM ) , p38 inhibitor ( SB203580 , 1 μM ) , JNK inhibitor ( SP600125 , 50 μM ) and MER5 inhibitor ( BIX 02189 , 10 μM ) .

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(E) ENCC viability assessed by CCK-8 assay after intervention with MEK1/2 inhibitor (U0126, 50 μM), p38 inhibitor (SB203580, 1 μM), JNK inhibitor (SP600125, 50 μM) and MER5 inhibitor (BIX 02189, 10 μM).


Diameter analyses of the ENCC - derived neurospheres ( F ) ,

Diameter analyses of the ENCC-derived neurospheres (F),


migration of ENCCs evaluated using transwell plate assay and stain with crystal violet on day 2 ( G , H ) show that MEK1 / 2 inhibitor U0126 suppressed SCFA - induced ENCC proliferation and migration .

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migration of ENCCs evaluated using transwell plate assay and stain with crystal violet on day 2 (G, H) show that MEK1/2 inhibitor U0126 suppressed SCFA-induced ENCC proliferation and migration.


migration of ENCCs evaluated using transwell plate assay show that MEK1 / 2 inhibitor U0126 suppressed SCFA - induced ENCC proliferation and migration .

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migration of ENCCs evaluated using transwell plate assay show that MEK1/2 inhibitor U0126 suppressed SCFA-induced ENCC proliferation and migration.


A . Average ribosome occupancy and their respective codon bias - derived occupancy scores across the CDS of transcript ( in 25 bins ) . Ribosome occupancy for 16 , 423 transcripts and their respective codon bias - derived occupancy were firstly binned into 25 bins and the mean occupancy was calculated for each bin . B . Cumulative distribution plots showing the distributions of correlations between ribosome occupancy and codon bias - derived occupancy . Correlations obtained from the ribosome occupancy and scrambled codon bias - derived occupancy served as the control . A Kolmogorov - Smirnov test was performed between the codon bias - derived occupancy and the control group . Data information : error bars represent the 95 % confidence intervals .

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A. Average ribosome occupancy and their respective codon bias-derived occupancy scores across the CDS of transcript (in 25 bins). Ribosome occupancy for 16,423 transcripts and their respective codon bias-derived occupancy were firstly binned into 25 bins and the mean occupancy was calculated for each bin. B. Cumulative distribution plots showing the distributions of correlations between ribosome occupancy and codon bias-derived occupancy. Correlations obtained from the ribosome occupancy and scrambled codon bias-derived occupancy served as the control. A Kolmogorov-Smirnov test was performed between the codon bias-derived occupancy and the control group. Data information: error bars represent the 95% confidence intervals.


C . Comparison of average transcript translation efficiencies ( TEs ) across their respective GC3 - content ranges . Number of transcripts within each gene optimality range is indicated above their respective points . Data information : error bars represent the 95 % confidence intervals .

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C. Comparison of average transcript translation efficiencies (TEs) across their respective GC3-content ranges. Number of transcripts within each gene optimality range is indicated above their respective points. Data information: error bars represent the 95% confidence intervals.


A . HEK293 Tet - off experiments showing the degradation of REL - OPT and REL - WT transcripts ( left ) as well as IL6 - OPT , IL6 - WT and IL6 - DE transcripts ( right ) , post - doxycycline addition . Data information : data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . The half - lives of the respective transcripts are indicated in brackets .

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A. HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (left) as well as IL6-OPT, IL6-WT and IL6-DE transcripts (right), post-doxycycline addition. Data information: data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.


B . Representative immunoblot of FLAG - tagged REL - OPT and REL - WT in HEK293T cells transfected with either empty plasmids , plasmids bearing REL - OPT or REL - WT . The immunoblot is representative of 3 independent experiments . ACTB is shown as the loading controls .

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B. Representative immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells transfected with either empty plasmids, plasmids bearing REL-OPT or REL-WT. The immunoblot is representative of 3 independent experiments. ACTB is shown as the loading controls.


C . ELISA of secreted IL6 concentrations of IL6 - OPT , IL6 - WT and IL6 - DE from HEK293T cells transfected with plasmids bearing IL6 - OPT , IL6 - WT and IL6 - DE . Data information : In ( C ) , the data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A one - way ANOVA with Tukey ' s multiple comparisons was performed between samples where , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ELISA of secreted IL6 concentrations of IL6-OPT, IL6-WT and IL6-DE from HEK293T cells transfected with plasmids bearing IL6-OPT, IL6-WT and IL6-DE. Data information: In (C), the data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A one-way ANOVA with Tukey's multiple comparisons was performed between samples where, p<0.01 (**) and p<0.001 (***).


D . Fold changes of REL - OPT and REL - WT transcript levels ( top ) relative to their abundances from fraction 1 as detected by qPCR across polysome fractions ( below ) . Data represents the mean ± SD for 3 biological replicates . Data information data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) . The half - lives of the respective transcripts are indicated in brackets .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Fold changes of REL-OPT and REL-WT transcript levels (top) relative to their abundances from fraction 1 as detected by qPCR across polysome fractions (below). Data represents the mean ± SD for 3 biological replicates. Data information data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.


HEK293 Tet - off experiments showing the degradation of REL - OPT and REL - WT transcripts under vehicle ( DMSO ) - and cycloheximide ( CHX ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

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HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts under vehicle (DMSO)- and cycloheximide (CHX)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing the degradation of IL6 - OPT , IL6 - WT and IL6 - DE transcripts ( B ) , under vehicle ( DMSO ) - and cycloheximide ( CHX ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT and IL6-DE transcripts (B), under vehicle (DMSO)- and cycloheximide (CHX)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing the degradation of REL - OPT and REL - WT transcripts ( C ) under vehicle ( PBS ) - and anisomycin ( ANI ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (C) under vehicle (PBS)- and anisomycin (ANI)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing the degradation of IL6 - OPT , IL6 - WT and IL6 - DE transcripts ( D ) , under vehicle ( PBS ) - and anisomycin ( ANI ) - treatment , post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT and IL6-DE transcripts (D), under vehicle (PBS)- and anisomycin (ANI)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing the degradation of REL - OPT , REL - OPT ( + 1 Frameshift ) and REL - WT transcripts ( E ) post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of REL-OPT, REL-OPT (+1 Frameshift) and REL-WT transcripts (E) post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


HEK293 Tet - off experiments showing the degradation of IL6 - OPT , IL6 - WT , IL6 - DE and IL6 - OPT ( + 1 Frameshift ) transcripts ( F ) post - doxycycline addition . data is representative of 3 independent experiments each with 3 replicates . The data represents the mean ± SD for 3 replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) .

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

HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT, IL6-DE and IL6-OPT (+1 Frameshift) transcripts (F) post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).


Volcano plots showing the enrichment of RBPs which bind to REL - WT relative to REL - OPT transcripts ( A ) Data information : vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate the p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 and p < 0 . 05 .

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

Volcano plots showing the enrichment of RBPs which bind to REL-WT relative to REL-OPT transcripts (A) Data information: vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.


Volcano plots showing the enrichment of RBPs which bind to , IL6 - DE relative to IL6 - WT transcripts ( B ) Data information : vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate the p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 and p < 0 . 05 .

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

Volcano plots showing the enrichment of RBPs which bind to , IL6-DE relative to IL6-WT transcripts (B) Data information: vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.


Volcano plots showing the enrichment of RBPs which bind to IL6 - WT relative to IL6 - OPT transcripts ( C ) . Data information : , vertical dotted lines indicate a 1 . 5 - fold enrichment while horizontal dashed lines indicate the p - value cut - off of 0 . 05 . Points shaded in blue indicate RBPs which have a differential fold change of more than 1 . 5 and p < 0 . 05 .

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

Volcano plots showing the enrichment of RBPs which bind to IL6-WT relative to IL6-OPT transcripts (C). Data information: , vertical dotted lines indicate a 1.5-fold enrichment while horizontal dashed lines indicate the p-value cut-off of 0.05. Points shaded in blue indicate RBPs which have a differential fold change of more than 1.5 and p<0.05.


A . Cumulative distribution plots showing the difference in distribution of transcript optimality between upregulated and downregulated transcripts in K562 cells subject to ILF2 CRISPR interference targeting ILF2 . Transcript quantities are indicated in the figure legend . Data information : Wilcoxon signed rank tests were performed on the upregulated and downregulated groups against the control group . P - values are denoted ( right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cumulative distribution plots showing the difference in distribution of transcript optimality between upregulated and downregulated transcripts in K562 cells subject to ILF2 CRISPR interference targeting ILF2. Transcript quantities are indicated in the figure legend. Data information: Wilcoxon signed rank tests were performed on the upregulated and downregulated groups against the control group. P-values are denoted (right).


B , C . HEK293 Tet - off experiments showing the degradation of REL - OPT ( B ) and REL - WT ( C ) transcripts with ILF2 and ILF3 siRNA and Control ( CTR ) siRNA treatment , post - doxycycline addition . Data information : data is representative of 3 independent experiments in which the data represents the mean ± SD for 3 biological replicates . A two - way ANOVA with Holm - Sidak multiple comparisons was performed . P - values are denoted as follows : p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

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

B, C. HEK293 Tet-off experiments showing the degradation of REL-OPT (B) and REL-WT (C) transcripts with ILF2 and ILF3 siRNA and Control (CTR) siRNA treatment, post-doxycycline addition. Data information: data is representative of 3 independent experiments in which the data represents the mean ± SD for 3 biological replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**).


D . Representative immunoblot of FLAG - tagged REL - OPT and REL - WT expressed in HEK293T cells under ILF2 and ILF3 siRNA treatment . The immunoblot is representative of 3 independent experiments . ACTB is shown as loading controls .

O O O O O O O O O O O O O O O O O O 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 immunoblot of FLAG-tagged REL-OPT and REL-WT expressed in HEK293T cells under ILF2 and ILF3 siRNA treatment. The immunoblot is representative of 3 independent experiments. ACTB is shown as loading controls.


E . Representative immunoblot of FLAG - tagged REL - OPT and REL - WT in HEK293T cells co - expressed with two different isoforms of ILF2 . The immunoblot is representative of 3 independent experiments . ACTB is shown as loading controls .

O O O O O O O O O O O O O O O O O O O O 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 immunoblot of FLAG-tagged REL-OPT and REL-WT in HEK293T cells co-expressed with two different isoforms of ILF2. The immunoblot is representative of 3 independent experiments. ACTB is shown as loading controls.


a , b . The amount of FDP - lysine , which was major protein - bound acrolein , in the sera of healthy controls and PD patients was determined by an ELISA ( a . all patients , ( control n = 22 ; PD n = 94 ) b . staged by the H & Y scale , a five - steps index of PD severity . Larger number indicates more severe condition . n = 94 ; H & Y stage I ( n = 18 ) , stage II ( n = 42 ) stage III ( n = 23 ) , stage IV ( n = 7 ) , and stage V ( n = 4 ) .

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a, b. The amount of FDP-lysine, which was major protein-bound acrolein, in the sera of healthy controls and PD patients was determined by an ELISA (a. all patients, (control n= 22; PD n=94) b. staged by the H&Y scale, a five-steps index of PD severity. Larger number indicates more severe condition. n=94; H&Y stage I (n=18), stage II (n=42) stage III (n=23), stage IV (n=7), and stage V (n=4).


SH - SY5Y cells were treated with the indicated chemicals at 50 μM for 4 h ( a ) Cells were fixed and stained with an anti - LAMP2 ( green ) antibody as a lysosomal marker . Nuclei were stained with DAPI ( blue ) . Cell membrane is indicated by the white line . Clustered relative to whole lysosomes were quantified as lysosomal clustering value ( a , right ) .

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SH-SY5Y cells were treated with the indicated chemicals at 50 μM for 4 h (a) Cells were fixed and stained with an anti-LAMP2 (green) antibody as a lysosomal marker. Nuclei were stained with DAPI (blue). Cell membrane is indicated by the white line. Clustered relative to whole lysosomes were quantified as lysosomal clustering value (a, right).


e . SH - SY5Y cells were treated with acrolein for 2 h . Cell lysates were immunoblotted with the indicated antibodies .

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

e. SH-SY5Y cells were treated with acrolein for 2 h. Cell lysates were immunoblotted with the indicated antibodies.