Figure 3 ( B ) Quantitative analysis of AEP positive cells and C / EBPβ positive cells , respectively . The density of both AEP positive cells and C / EBPβ positive cells were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . Representative data of five samples , data are shown as mean ± SEM . * P = 0 . 0390 ( * P < 0 . 05 ) , * * * P = 0 . 0002 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA . ( C ) Quantitative analysis of AT8 positive cells ( left ) and TauN368 positive cells ( right ) , respectively . The density of both AT8 positive cells and TauN368 positive cells were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . n = 5 in each group , data are shown as mean ± SEM . * P = 0 . 0194 ( * P < 0 . 05 ) , * * * P = 0 . 0001 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA . ( D ) Quantitative analysis of Aβ positive cells ( left ) and APPC586 positive cells ( right ) , respectively . The fluorescence intensity of Aβ positive cells and the density APPC586 positive cells were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . Representative data of five samples , data are shown as mean ± SEM . * P = 0 . 0107 ( * P < 0 . 05 ) , * * P = 0 . 0048 ( 3xTg vs 3xTg AEP KO ) , * * P = 0 . 0044 ( 3xTg vs WT ) ( * * P < 0 . 01 ) , * * * P = 0 . 0006 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 , one - way ANOVA . ( E ) Quantitative analysis of AT8 positive cells ( upper ) and T22 positive cells ( lower ) , respectively . The fluorescence intensity of both AT8 positive cells and T22 positive cells were significantly decreased through knocking down C / EBPβ or knocking out AEP in mice . Representative data of five samples , data are shown as mean ± SEM . * * P = 0 . 0014 ( 3xTg vs 3xTg C / EBPβ + / - ) , * * P = 0 . 0030 ( 3xTg vs 3xTg AEP KO ) ( * * P < 0 . 01 ) , * * * P = 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA . ( G ) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining . ( Scale bar : 5 μm ) . ( H ) Quantitative analysis of the synaptic densities in DSS - treated mice . Representative data of five samples , Data are shown as mean ± SEM . * * * * P < 0 . 0001 , one - way ANOVA . ( I ) Quantitative analysis of the spine density . Representative data of five samples , Data are shown as mean ± SEM . * * * * P < 0 . 0001 , one - way ANOVA .

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

Figure 3(B) Quantitative analysis of AEP positive cells and C/EBPβ positive cells, respectively. The density of both AEP positive cells and C/EBPβ positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. *P =0.0390 (*P < 0.05), ***P =0.0002 (***P < 0.001), ****P < 0.0001, one-way ANOVA. (C) Quantitative analysis of AT8 positive cells (left) and TauN368 positive cells (right), respectively. The density of both AT8 positive cells and TauN368 positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. n=5 in each group, data are shown as mean± SEM. *P =0.0194 (*P< 0.05), ***P =0.0001 (***P < 0.001), ****P < 0.0001, one-way ANOVA. (D) Quantitative analysis of Aβ positive cells (left) and APPC586 positive cells (right), respectively. The fluorescence intensity of Aβ positive cells and the density APPC586 positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. *P=0.0107 (*P < 0.05), **P=0.0048 (3xTg vs 3xTg AEP KO), **P=0.0044 (3xTg vs WT) (**P< 0.01), ***P=0.0006 (***P< 0.001), ****P < 0.0001, one-way ANOVA. (E) Quantitative analysis of AT8 positive cells (upper) and T22 positive cells (lower), respectively. The fluorescence intensity of both AT8 positive cells and T22 positive cells were significantly decreased through knocking down C/EBPβ or knocking out AEP in mice. Representative data of five samples, data are shown as mean± SEM. **P=0.0014 (3xTg vs 3xTg C/EBPβ+/-), **P=0.0030 (3xTg vs 3xTg AEP KO) (**P<0.01), ***P=0.001, ****P<0.0001, one-way ANOVA.(G) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining. (Scale bar: 5 μm). (H) Quantitative analysis of the synaptic densities in DSS-treated mice. Representative data of five samples, Data are shown as mean ± SEM. ****P < 0.0001, one-way ANOVA. (I) Quantitative analysis of the spine density. Representative data of five samples, Data are shown as mean ± SEM. ****P < 0.0001, one-way ANOVA.


Figure 4 ( E ) Gastrointestinal permeability barrier defect as determined by FITC - dextran translocation in pre - vagotomy DSS - treated , DSS - treated and vehicle - treated 3xTg mice . Data represent the mean ± SEM ; representative data of three samples ; * P = 0 . 0126 ( * P < 0 . 001 ) , * * * P = 0 . 0002 ( * * * P < 0 . 001 ) compared with control , one - way ANOVA . ( F ) Pro - inflammatory cytokines IL - 6 , IL1 - β , and TNFα concentrations in colon lysates of vehicle - treated 3xTg mice , DSS - treated single side vagotomy 3xTg and DSS - treated 3xTg , respectively . Representative data of three samples ; data are shown as mean ± SEM . * P = 0 . 0372 ( * P < 0 . 05 ) , * * P = 0 . 0050 ( * * P < 0 . 01 ) , * * * P = 0 . 0008 ( * * * P < 0 . 001 ) , * * * * P < 0 . 0001 compared with control , two - way ANOVA . ( H ) Representative electron microscopy of the synaptic structures in hippocampus . Red stars indicate the synapses . ( Scale bar : 1 μm ) . ( I ) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining . ( Scale bar : 5 μm ) . ( J ) Quantitative analysis of the synaptic densities in DSS - treated mice . Representative data of five samples , data are shown as mean ± SEM . * * * * P < 0 . 0001 , unpaired t tests . ( K ) Quantitative analysis of the spine density . Representative data of five samples , data are shown as mean ± SEM . * * * P = 0 . 0001 ( * * * P < 0 . 001 ) , unpair t tests .

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

Figure 4(E) Gastrointestinal permeability barrier defect as determined by FITC-dextran translocation in pre-vagotomy DSS-treated, DSS-treated and vehicle-treated 3xTg mice. Data represent the mean ± SEM; representative data of three samples; *P=0.0126 (*P < 0.001), ***P=0.0002 (***P < 0.001) compared with control, one-way ANOVA.(F) Pro-inflammatory cytokines IL-6, IL1-β, and TNFα concentrations in colon lysates of vehicle-treated 3xTg mice, DSS-treated single side vagotomy 3xTg and DSS-treated 3xTg, respectively. Representative data of three samples; data are shown as mean ± SEM. *P=0.0372 (*P < 0.05), **P=0.0050 (**P < 0.01), ***P=0.0008 (***P <0.001), ****P<0.0001 compared with control, two-way ANOVA.(H) Representative electron microscopy of the synaptic structures in hippocampus. Red stars indicate the synapses. (Scale bar: 1 μm). (I) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining. (Scale bar: 5 μm). (J) Quantitative analysis of the synaptic densities in DSS-treated mice. Representative data of five samples, data are shown as mean ± SEM. ****P < 0.0001, unpaired t tests. (K) Quantitative analysis of the spine density. Representative data of five samples, data are shown as mean ± SEM. ***P =0.0001 (***P < 0.001), unpair t tests.


Figure 5 ( A & B ) Cued and Contextual Fear - conditioning tests . Data represent the mean ± SEM of n = 6 - 8 mice per group ; * * P = 0 . 0067 ( 3xTg + DSS vs 3xTg ctrl ) , * * P = 0 . 0059 ( 3xTg + DSS vs 3xTg R - vagotomy + DSS ) ( * * P < 0 . 01 ) , * * * P = 0 . 0009 ( * * * P < 0 . 001 ) , two - way ANOVA ( A ) ; * * P = 0 . 0086 ( 3xTg + DSS vs 3xTg ctrl ) , * * P = 0 . 0085 ( 3xTg + DSS vs 3xTg R - vagotomy + DSS ) ( * * P < 0 . 01 ) , * * * P = 0 . 0005 ( * * * P < 0 . 001 ) , one - way ANOVA ( B ) . ( D ) Survival rate curve of the experimental mice during the experimental paradigm .

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

Figure 5(A&B) Cued and Contextual Fear-conditioning tests. Data represent the mean ± SEM of n = 6-8 mice per group; **P=0.0067 (3xTg+DSS vs 3xTg ctrl), **P=0.0059 (3xTg+DSS vs 3xTg R-vagotomy+DSS) (**P< 0.01), ***P=0.0009 (***P < 0.001), two-way ANOVA (A); **P=0.0086 (3xTg+DSS vs 3xTg ctrl), **P=0.0085 (3xTg+DSS vs 3xTg R-vagotomy+DSS) (**P< 0.01), ***P=0.0005 (***P < 0.001), one-way ANOVA (B).(D) Survival rate curve of the experimental mice during the experimental paradigm.


Figure 6 ( B ) Immunofluorescent staining showed ATTO 550 - labeled Aβ PFFs and Tau N368 PFFs spread into different brain regions in WT mice 3 months after colonic injection . Scale bar : 20 μm . ( C ) Immunohistochemistry staining of human Aβ propagating along the vagus nerve from WT mice colonic - injected with PBS , AD brain extracts or 5xFAD brain extracts , using human specific Aβ antibody ( BAN50 ) . Black arrows indicate the human Aβ in mouse vagus nerve . ( D ) Immunohistochemistry staining of human Tau propagating along the vagus nerve from WT mice colonic - injected with PBS or AD brain extracts , using human Tau specific antibody ( HT7 ) . Black arrows indicate the human Tau in mouse vagus nerve .

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

Figure 6(B) Immunofluorescent staining showed ATTO 550-labeled Aβ PFFs and Tau N368 PFFs spread into different brain regions in WT mice 3 months after colonic injection. Scale bar: 20 μm.(C) Immunohistochemistry staining of human Aβ propagating along the vagus nerve from WT mice colonic-injected with PBS, AD brain extracts or 5xFAD brain extracts, using human specific Aβ antibody (BAN50). Black arrows indicate the human Aβ in mouse vagus nerve. (D) Immunohistochemistry staining of human Tau propagating along the vagus nerve from WT mice colonic-injected with PBS or AD brain extracts, using human Tau specific antibody (HT7). Black arrows indicate the human Tau in mouse vagus nerve.


Figure 7 ( A ) Immunofluorescent staining of AEP and p - C / EBP β in cerebral cortex of brains of WT mice 6 months after colonic inoculation with either Aβ PFFs or Tau N368 PFFs . Scale bar : 20 μm . White arrows indicate that AEP and p - C / EBP β signals ( green ) were colocalized with ATTO 550 signals ( red ) , respectively .

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

Figure 7(A) Immunofluorescent staining of AEP and p-C/EBP β in cerebral cortex of brains of WT mice 6 months after colonic inoculation with either Aβ PFFs or Tau N368 PFFs. Scale bar: 20 μm. White arrows indicate that AEP and p-C/EBP β signals (green) were colocalized with ATTO 550 signals (red), respectively.


Figure 8 ( A ) Immunofluorescent staining of AEP ( red ) and C / EBP β ( green ) in CA1 region from hippocampus of brains from colonic - injected PBS , colonic - injected AD brain extracts , and pre - Vagotomy colonic injected AD brain extracts 3xTg mice . Scale bar : 20μm . ( B ) Quantitative analysis of AEP positive cells and C / EBP β positive cells , respectively . The density of both AEP and C / EBP β 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 . 016 ( * * P < 0 . 01 ) , * * * * P < 0 . 0001 , one - way ANOVA . ( 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 .

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

Figure 8(A) Immunofluorescent staining of AEP (red) and C/EBP β (green) in CA1 region from hippocampus of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. Scale bar: 20μm. (B) Quantitative analysis of AEP positive cells and C/EBP β positive cells, respectively. The density of both AEP and C/EBP β 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 =0.016 (**P < 0.01), ****P<0.0001, one-way ANOVA. (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.


Figure 1A . Quantitative real - time PCR analysis of PKCδ mRNA levels in FACS sorted Lin - , LT - HSC , ST - HSC , MPP , L - S - K + , GMP , CMP , MEP and CLP subsets from C56BL / 6 wild - type ( 6 - to 9 - week old ) mice bone marrow . Levels of PKCδ expression were normalized to an internal control gene ( β - actin ) . Expression of PKCδ is shown relative to Lineage negative ( Lin - ) cells whose expression was arbitrarily set to 1 ( n = 4 mice analyzed for each population ) . Data information : All data are presented as mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 , and * * * p < 0 . 001 by with one - way ANOVAs with Sidak ' s multiple comparisons test ( A ) ( B ) FACS plots depict the percentage of live , Lin - Sca1 + c - Kit + ( LSK ) ( left panel ) , and primitive CD34 - Flt3 - , CD34 + Flt3 - and CD34 + Flt3 + HSPCs among LSK cells ( right panel ) . ( C ) Bar charts show the average frequency ( left ) and absolute number ( right ) ± SEM of the indicated populations , analyzed from 2 femurs and 2 tibias per mouse ( n = 10 mice analyzed for each genotype ) . All data are presented as mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 , and * * * p < 0 . 001 by with one - way ANOVAs with two - tailed Student ' s unpaired t - test analysis for comparison of WT to PKCδ KO mice . D . FACS plots show percentages of ' SLAM - code ' based stem and progenitor cells in the LSK population . Bar graphs show the average frequency ( left ) and absolute numbers ( right ) ± SEM of LT - HSC per 2 femurs and 2 tibias . ( n = 10 mice analyzed for each genotype ) . Data information : All data are presented as mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 , and * * * p < 0 . 001 by with one - way ANOVAs with two - tailed Student ' s unpaired t - test analysis for comparison of WT to PKCδ KO mice . E . FACS plots illustrate gating and frequencies of myeloid progenitors ( MP ) , granulocyte - monocyte progenitor ( GMP ) , common myeloid progenitor ( CMP ) , and megakaryocyte - erythroid progenitor ( MEP ) population . Bar graphs show the frequencies ( left ) and absolute numbers ( right ) of L - S - K + MP and GMP , CMP and MEP populations in the BM of WT ( n = 12 ) and PKCδ KO ( n = 12 ) . Data information : All data are presented as mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 , and * * * p < 0 . 001 by with one - way ANOVAs with two - tailed Student ' s unpaired t - test analysis for comparison of WT to PKCδ KO mice . F . FACS plots illustrate the gating and percentages of common lymphoid progenitors ( CLP ) . Bar graph shows the absolute number of CLPs per 2 femurs and 2 tibias ( n = 5 mice for each genotype ) . Data information : All data are presented as mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 , and * * * p < 0 . 001 by with one - way ANOVAs with two - tailed Student ' s unpaired t - test analysis for comparison of WT to PKCδ KO mice . G . FACS plots illustrate the percentages of uncommitted lymphoid progenitors ( Lin - IL - 7Rα + Flt3 + Ly - 6D - ) and B lymphoid progenitors ( BLP ) . Bar graph represents the average frequencies of CLPs , BLP and uncommitted lymphoid progenitors in total BM ( n = 5 mice per genotype ) . Data information : All data are presented as mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 , and * * * p < 0 . 001 by with one - way ANOVAs with two - tailed Student ' s unpaired t - test analysis for comparison of WT to PKCδ KO mice . H . Limiting dilution analysis ( LDA ) demonstrates an increased frequency of long - term repopulating HSCs in PKCδ KO BM ( solid red lines ) compared with WT BM ( black solid lines ) . Engraftment data shown at 14 - weeks post - BMT . Plots depict the percentages of recipient mice containing less than 1 % CD45 . 2 + blood nucleated cells . Dotted lines represent the 95 % confidence interval of the same ( p = 0 . 0005 ) . Data information : All data are presented as mean ± SEM . Overall test for differences in stem cell frequencies between WT to PKCδ KO mice was determined with likelihood ratio test of single - hit model ( H ) .

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

Figure 1A. Quantitative real-time PCR analysis of PKCδ mRNA levels in FACS sorted Lin-, LT-HSC, ST-HSC, MPP, L-S-K+, GMP, CMP, MEP and CLP subsets from C56BL/6 wild-type (6-to 9-week old) mice bone marrow. Levels of PKCδ expression were normalized to an internal control gene (β-actin). Expression of PKCδ is shown relative to Lineage negative (Lin-) cells whose expression was arbitrarily set to 1 (n=4 mice analyzed for each population). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with Sidak's multiple comparisons test (A)(B) FACS plots depict the percentage of live, Lin-Sca1+c-Kit+ (LSK) (left panel), and primitive CD34-Flt3-, CD34+Flt3- and CD34+Flt3+ HSPCs among LSK cells (right panel).(C) Bar charts show the average frequency (left) and absolute number (right) ± SEM of the indicated populations, analyzed from 2 femurs and 2 tibias per mouse (n=10 mice analyzed for each genotype). All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.D. FACS plots show percentages of 'SLAM-code' based stem and progenitor cells in the LSK population. Bar graphs show the average frequency (left) and absolute numbers (right) ± SEM of LT-HSC per 2 femurs and 2 tibias. (n=10 mice analyzed for each genotype). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.E. FACS plots illustrate gating and frequencies of myeloid progenitors (MP), granulocyte-monocyte progenitor (GMP), common myeloid progenitor (CMP), and megakaryocyte-erythroid progenitor (MEP) population. Bar graphs show the frequencies (left) and absolute numbers (right) of L-S-K+ MP and GMP, CMP and MEP populations in the BM of WT (n=12) and PKCδ KO (n=12). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.F. FACS plots illustrate the gating and percentages of common lymphoid progenitors (CLP). Bar graph shows the absolute number of CLPs per 2 femurs and 2 tibias (n=5 mice for each genotype). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.G. FACS plots illustrate the percentages of uncommitted lymphoid progenitors (Lin- IL-7Rα+Flt3+Ly-6D-) and B lymphoid progenitors (BLP). Bar graph represents the average frequencies of CLPs, BLP and uncommitted lymphoid progenitors in total BM (n=5 mice per genotype). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.H. Limiting dilution analysis (LDA) demonstrates an increased frequency of long-term repopulating HSCs in PKCδ KO BM (solid red lines) compared with WT BM (black solid lines). Engraftment data shown at 14-weeks post-BMT. Plots depict the percentages of recipient mice containing less than 1% CD45.2+ blood nucleated cells. Dotted lines represent the 95% confidence interval of the same (p=0.0005). Data information: All data are presented as mean±SEM. Overall test for differences in stem cell frequencies between WT to PKCδ KO mice was determined with likelihood ratio test of single-hit model (H).


Figure 2A . Representative FACS profiles of HSPC cell cycle analysis using combinatorial staining for Ki67 and Hoechst 33342 . Bar charts depict the average percentage of cells in each phase of the cell cycle for each LSK subset from WT ( n = 6 ) and PKCδ KO ( n = 7 ) mice . Data compiled from two independent experiments . Data information : All data are presented as mean ± SEM , * p < 0 . 05 and * * p < 0 . 001 , with significance determined by two - tailed Student ' s unpaired t - test analysis . B . FACS profiles of indicated BM subsets from WT and PKCδ KO mice 20hr after BrdU injection . Data information : All data are presented as mean ± SEM , * p < 0 . 05 and * * p < 0 . 001 , with significance determined by two - tailed Student ' s unpaired t - test analysis . C . Average percentages of cells in each phase of the cell cycle phases for each of the indicated HSPC subsets from WT and PKCδ KO mice . Data are pooled from two independent experiments ( totaling n = 6 - 7 mice per genotype ) . Data information : All data are presented as mean ± SEM , * p < 0 . 05 and * * p < 0 . 001 , with significance determined by two - tailed Student ' s unpaired t - test analysis . D . Representative FACS plots and summary of FACS data analyzing the frequency of apoptotic HSPC cells using co - staining for AnnexinV and 7 - AAD ( WT and PKCδ − / − ( n = 7 per genotype ) . Data information : All data are presented as mean ± SEM , * p < 0 . 05 and * * p < 0 . 001 , with significance determined by two - tailed Student ' s unpaired t - test analysis .

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

Figure 2A. Representative FACS profiles of HSPC cell cycle analysis using combinatorial staining for Ki67 and Hoechst 33342. Bar charts depict the average percentage of cells in each phase of the cell cycle for each LSK subset from WT (n=6) and PKCδ KO (n=7) mice. Data compiled from two independent experiments. Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.B. FACS profiles of indicated BM subsets from WT and PKCδ KO mice 20hr after BrdU injection. Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.C. Average percentages of cells in each phase of the cell cycle phases for each of the indicated HSPC subsets from WT and PKCδ KO mice. Data are pooled from two independent experiments (totaling n=6-7 mice per genotype). Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.D. Representative FACS plots and summary of FACS data analyzing the frequency of apoptotic HSPC cells using co-staining for AnnexinV and 7-AAD (WT and PKCδ−/− (n=7 per genotype). Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.


Figure 3B . Percent of total donor - derived , hematopoietic cells ( CD45 . 2 + ) , B - cells ( B220 + ) , myeloid cells ( CD11b + Gr1 + ) , and T cells ( CD3 + ) in the peripheral blood ( PB ) of recipient mice , as determined by FACS at the indicated time points . The statistical significance of differences was determined using two - ANOVAs with Holm - Sidak ' s multiple comparisons tests ( n = 8 recipients per genotype in each experiment ) . Data information : All data are presented as mean ± SD . * p < 0 . 05 , * * p < 0 . 001 , with significance determined by repeated measures of two - way ANOVA analysis with Sidak ' s multiple comparison tests for comparison of recipients of WT and PKCδ KO marrow at each time point and for each cell population . C . Percent donor - derived HSPCs in the BM of secondary ( left ) and tertiary ( right ) recipients of WT or PKCδ − / − marrow . Data are compiled from two independent experiments ( n = 8 recipients per genotype ) . Data information : All data are presented as mean ± SD . * p < 0 . 05 , * * p < 0 . 001 , with significance determined by repeated measures of two - way ANOVA analysis with Sidak ' s multiple comparison tests for comparison of recipients of WT and PKCδ KO marrow at each time point and for each cell population .

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

Figure 3B. Percent of total donor-derived, hematopoietic cells (CD45.2+), B-cells (B220+), myeloid cells (CD11b+Gr1+), and T cells (CD3+) in the peripheral blood (PB) of recipient mice, as determined by FACS at the indicated time points. The statistical significance of differences was determined using two-ANOVAs with Holm-Sidak's multiple comparisons tests (n=8 recipients per genotype in each experiment). Data information: All data are presented as mean±SD. *p<0.05, **p<0.001, with significance determined by repeated measures of two-way ANOVA analysis with Sidak's multiple comparison tests for comparison of recipients of WT and PKCδ KO marrow at each time point and for each cell population.C. Percent donor-derived HSPCs in the BM of secondary (left) and tertiary (right) recipients of WT or PKCδ−/− marrow. Data are compiled from two independent experiments (n=8 recipients per genotype). Data information: All data are presented as mean±SD. *p<0.05, **p<0.001, with significance determined by repeated measures of two-way ANOVA analysis with Sidak's multiple comparison tests for comparison of recipients of WT and PKCδ KO marrow at each time point and for each cell population.


Figure 4B , C Representative FACS plots of PKCδ fl / fl and cKO BM 4 - 8 weeks ( B ) , and 20 - 24 - weeks ( C ) after pIpC treatment . Numbers in plot indicate the absolute numbers of the indicated subset within the gated population ( n = 8 - 9 mice per genotype and time point ) . Data information : Data presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 005 and * * * p < 0 . 001 by two - tailed Student ' s unpaired t - test analysis . ( E ) FACS plots show contribution of donor - derived ( CD45 . 2 + ) HSPCs in recipient BM ( E ) ( total of n = 7 - 8 mice per genotype ) . Calculated absolute numbers of donor - derived HSPC subsets in transplanted mice ( F ) . ( total of n = 7 - 8 mice per genotype ) . Data information : Data presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 005 and * * * p < 0 . 001 by two - tailed Student ' s unpaired t - test analysis . G . Average percentage BrdU + HPSCs ( 3 days after BrdU injection into mice reconstituted 24 weeks previously with control or cKO marrow and injected 16 weeks previously with pIpC among donor - derived LSK cells in the control and cKO chimeras BM . Data information : Data presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 005 and * * * p < 0 . 001 by two - tailed Student ' s unpaired t - test analysis . H . Percentages of apoptotic cells ( AnnexinV + / 7 - AAD - ) in donor - derived LSK cells in control and cKO chimera mouse BM at 24 weeks after transplant and 16 weeks after pIpC injection . Data are compiled from two independent experiments ( total of n = 7 - 8 mice per genotype ) . Data information : Data presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 005 and * * * p < 0 . 001 by two - tailed Student ' s unpaired t - test analysis .

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

Figure 4B,C Representative FACS plots of PKCδ fl/fl and cKO BM 4-8 weeks (B), and 20-24-weeks (C) after pIpC treatment. Numbers in plot indicate the absolute numbers of the indicated subset within the gated population (n=8-9 mice per genotype and time point). Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.(E) FACS plots show contribution of donor-derived (CD45.2+) HSPCs in recipient BM (E) (total of n=7-8 mice per genotype).Calculated absolute numbers of donor-derived HSPC subsets in transplanted mice (F). (total of n=7-8 mice per genotype). Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.G. Average percentage BrdU+ HPSCs (3 days after BrdU injection into mice reconstituted 24 weeks previously with control or cKO marrow and injected 16 weeks previously with pIpC among donor-derived LSK cells in the control and cKO chimeras BM. Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.H. Percentages of apoptotic cells (AnnexinV+/7-AAD-) in donor-derived LSK cells in control and cKO chimera mouse BM at 24 weeks after transplant and 16 weeks after pIpC injection. Data are compiled from two independent experiments (total of n=7-8 mice per genotype). Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.


Figure 5B . Kinetics of LSK cell recovery . Representative FACS profiles of BM LSK cells from WT and cKO mice at the indicated time points after 5 - FU treatment . Numbers in the contour plots indicate the percentages of LSK subsets among Lin - live cells . C . Average absolute numbers of BM LSK cells at day13 and day28 after 5 - FU treatment . Data are pooled from two independent experiments . ( total of n = 6 - 8 mice per time point and genotype ) . Data information : Data presented as mean ± SEM . The statistical significance was assessed using repeated - measures two - way ANOVAs with Sidak ' s multiple comparisons tests ( C ) * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 . D . Quantification of apoptotic HSPCs at the indicted time points after 5 - FU treatment of control and cKO mice ( n = 6 per genotype ) . Data information : Data presented as mean ± SEM . The statistical significance was assessed using repeated - measures two - way ANOVAs by two - tailed Student ' s unpaired t - test analysis ( D and E ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 . E . Representative FACS plots showing analysis of in vivo BrdU incorporation assays . Mice were received 2mg of BrdU ( i . p . ) at d13 ( left ) or d28 ( right ) after 5 - FU treatment . 20hrs later , BrdU incorporation in BM LSK cells was assessed using anti - BrdU antibodies . Bar chart at right indicates percent of BrdU + LSK cells at d13 and d28 after 5 - FU treatment ( n = 6 per genotype and time point ) . Data information : Data presented as mean ± SEM . The statistical significance was assessed using repeated - measures two - way ANOVAs with two - tailed Student ' s unpaired t - test analysis ( D and E ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 . F . Kaplan - Meier survival cures of control ( n = 12 ) and cKO ( n = 11 ) mice after sequential 5 - FU treatment . Mice were treated with 5 - FU ( 150mg / kg , i . p . ) once each week for three weeks . Arrows indicate time points of 5 - FU injection . Data information : Data presented as mean ± SEM . Survival data ( F ) were analyzed using a log - rank nonparametric test ( Mantel - Cox test ) ( p = 0 . 0141 ) .

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

Figure 5B. Kinetics of LSK cell recovery. Representative FACS profiles of BM LSK cells from WT and cKO mice at the indicated time points after 5-FU treatment. Numbers in the contour plots indicate the percentages of LSK subsets among Lin- live cells. C. Average absolute numbers of BM LSK cells at day13 and day28 after 5-FU treatment. Data are pooled from two independent experiments. (total of n=6-8 mice per time point and genotype). Data information: Data presented as mean ± SEM. The statistical significance was assessed using repeated-measures two-way ANOVAs with Sidak's multiple comparisons tests (C) *p<0.05, **p<0.01 and ***p<0.001.D. Quantification of apoptotic HSPCs at the indicted time points after 5-FU treatment of control and cKO mice (n=6 per genotype). Data information: Data presented as mean ± SEM. The statistical significance was assessed using repeated-measures two-way ANOVAs by two-tailed Student's unpaired t-test analysis (D and E). *p<0.05, **p<0.01 and ***p<0.001.E. Representative FACS plots showing analysis of in vivo BrdU incorporation assays. Mice were received 2mg of BrdU (i.p.) at d13 (left) or d28 (right) after 5-FU treatment. 20hrs later, BrdU incorporation in BM LSK cells was assessed using anti-BrdU antibodies. Bar chart at right indicates percent of BrdU+ LSK cells at d13 and d28 after 5-FU treatment (n=6 per genotype and time point). Data information: Data presented as mean ± SEM. The statistical significance was assessed using repeated-measures two-way ANOVAs with two-tailed Student's unpaired t-test analysis (D and E). *p<0.05, **p<0.01 and ***p<0.001.F. Kaplan-Meier survival cures of control (n=12) and cKO (n=11) mice after sequential 5-FU treatment. Mice were treated with 5-FU (150mg/kg, i.p.) once each week for three weeks. Arrows indicate time points of 5-FU injection. Data information: Data presented as mean ± SEM. Survival data (F) were analyzed using a log-rank nonparametric test (Mantel-Cox test) (p=0.0141).


Figure 6A . MitoStress test revealed increased oxygen consumption rates ( OCR ) in PKCδ - deleted LSKs . BM LSK cells were sorted from WT and PKCδ cKO mice 12 - weeks after pIpC - treatment . OCR was measured at basal level and after sequential loading of ATP synthase inhibitor Oligomycin ( 350nm ) , mitochondrial uncoupler , FCCP ( 10μM ) , and electron transport chain inhibitor , Rotenone ( 1μM ) using Seahorse XF24 extracellular flux analyzer . Data are from 3 independent experiments ( n = 9 mice ) . In each experiment , bone marrow cells from identical genotypes ( n = 3 mice per genotype ) were pooled and used for LSK cell sorting . Data information : The statistical significance of difference was assessed using two - tailed Student ' s unpaired t - test analysis . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . ( B ) Basal OCR was measured before inhibitors treatment ( left ) ; and the Maximal OCR capacity after FCCP treatment ( right ) . ( n = 9 per genotype , from 3 independent experiments ) . Data information : The statistical significance of difference was assessed using two - tailed Student ' s unpaired t - test analysis . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . ( C ) . The Spare respiratory capacity ( SRC ) of LSKs was calculated ( n = 9 per genotype , from 3 independent experiments ) . Data information : The statistical significance of difference was assessed using two - tailed Student ' s unpaired t - test analysis . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . D , E . ( D ) Glycolysis Stress test . Extracellular acidification rates ( ECAR ) of LSKs were measured at basal conditions ( unbuffered assay medium without glucose ) and after sequential loading of glucose ( 7 . 5mM ) , Oligomycin ( 350nm ) , and 2 - deoxyglucose ( 50mM ; 2DG , a glucose analog ) . ( E ) . AUC of baseline ECAR levels ( left ) , and glycolytic capacity ( maximal ECAR ) was calculated from the data shown in panel D ( n = 7 - 8 per genotype , from 3 independent experiments ) . Data information : The statistical significance of difference was assessed using two - tailed Student ' s unpaired t - test analysis . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . F . Relative ATP content in indicated HSPC subsets determined using an ATP assay kit ( n = 6 mice per genotype ) . Data information : The statistical significance of difference was assessed using two - tailed Student ' s unpaired t - test analysis . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . G . Representative FACS histograms of MitoTracker Green fluorescence intensity on indicated HSPC subsets from control and cKO mice . Relative MitoTracker Green mean fluorescence intensity ( MFI ) is quantified below for n = 6 mice of each genotype . Data information : The statistical significance of difference was assessed using two - tailed Student ' s unpaired t - test analysis . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . H . Representative electron microscopy images ( 4 , 800x magnification ) of control and PKCδ cKO HSPCs . Arrows indicate the mitochondria enriched regions ( n = 3 mice per genotype ) . Scale bar represents 500nm . I . ROS levels in control and PKCδ cKO HSPCs as measured by FACS based CM - H2DCFDA staining . Bar graph at right shows the relative CM - H2DCFDA MFI . Data compiled from three independent experiments ( n = 6 - 8 per genotype ) . Data information : The statistical significance of difference was assessed using two - tailed Student ' s unpaired t - test analysis . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 .

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

Figure 6A. MitoStress test revealed increased oxygen consumption rates (OCR) in PKCδ-deleted LSKs. BM LSK cells were sorted from WT and PKCδ cKO mice 12-weeks after pIpC-treatment. OCR was measured at basal level and after sequential loading of ATP synthase inhibitor Oligomycin (350nm), mitochondrial uncoupler, FCCP (10μM), and electron transport chain inhibitor, Rotenone (1μM) using Seahorse XF24 extracellular flux analyzer. Data are from 3 independent experiments (n=9 mice). In each experiment, bone marrow cells from identical genotypes (n=3 mice per genotype) were pooled and used for LSK cell sorting. Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.(B) Basal OCR was measured before inhibitors treatment (left); and the Maximal OCR capacity after FCCP treatment (right). (n=9 per genotype, from 3 independent experiments). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.(C). The Spare respiratory capacity (SRC) of LSKs was calculated (n=9 per genotype, from 3 independent experiments). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.D, E. (D) Glycolysis Stress test. Extracellular acidification rates (ECAR) of LSKs were measured at basal conditions (unbuffered assay medium without glucose) and after sequential loading of glucose (7.5mM), Oligomycin (350nm), and 2-deoxyglucose (50mM; 2DG, a glucose analog). (E). AUC of baseline ECAR levels (left), and glycolytic capacity (maximal ECAR) was calculated from the data shown in panel D (n=7-8 per genotype, from 3 independent experiments). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.F. Relative ATP content in indicated HSPC subsets determined using an ATP assay kit (n=6 mice per genotype). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.G. Representative FACS histograms of MitoTracker Green fluorescence intensity on indicated HSPC subsets from control and cKO mice. Relative MitoTracker Green mean fluorescence intensity (MFI) is quantified below for n=6 mice of each genotype. Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.H. Representative electron microscopy images (4,800x magnification) of control and PKCδ cKO HSPCs. Arrows indicate the mitochondria enriched regions (n=3 mice per genotype). Scale bar represents 500nm.I. ROS levels in control and PKCδ cKO HSPCs as measured by FACS based CM-H2DCFDA staining. Bar graph at right shows the relative CM-H2DCFDA MFI. Data compiled from three independent experiments (n=6-8 per genotype). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.


Figure 7Quantitative RT - PCR analysis of selected ( A ) HSPC transcriptional regulators expression in bone marrow HSPCs ( n = 4 mice per genotype ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . Quantitative RT - PCR analysis of selected ( B ) cell cycle and apoptosis regulators expression in bone marrow HSPCs ( n = 4 mice per genotype ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . Quantitative RT - PCR analysis of selected ( C ) known HSPC metabolic regulators expression in bone marrow HSPCs ( n = 4 mice per genotype ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . Intracellular flow cytometry analysis of Cyclin D1 protein ( D ) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . Intracellular flow cytometry analysis of pro - survival regulators Mcl1 , Blc2 , and Bim protein ( E ) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . representative FACS histograms and bar graphs show relative levels of pAKT ( Ser473 ) relative to WT controls ( F ) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . representative FACS histograms and bar graphs show relative levels of pFoxO3a ( Ser253 ) ( G ) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . representative FACS histograms and bar graphs show relative levels of Total cMyc protein ( H ) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . ; representative FACS histograms and bar graphs show relative levels of pRb ( Ser807 / 811 ) ( I ) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . representative FACS histograms and bar graphs show total PPARγ protein levels relative to WT controls ( J ) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . Relative expression levels of total PPARδ protein in HSPCs ( K ) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . Activation of pmTOR ( Ser2448 ) ( L ) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 . Activation of pS6 ( Ser235 / 236 ) and ( Ser240 / 244 ) ( M ) . Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls ( n = 4 - 6 mice per genotype per target ) . Data information : The statistical significance of difference was assessed using Two - tailed Student ' s unpaired t - test analysis for comparison of control and PKCδ cKO HSPCs . All data are presented as mean ± SEM . , * p < 0 . 05 , * * p < 0 . 001 .

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

Figure 7Quantitative RT-PCR analysis of selected (A) HSPC transcriptional regulators expression in bone marrow HSPCs (n=4 mice per genotype). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.Quantitative RT-PCR analysis of selected (B) cell cycle and apoptosis regulators expression in bone marrow HSPCs (n=4 mice per genotype). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.Quantitative RT-PCR analysis of selected (C) known HSPC metabolic regulators expression in bone marrow HSPCs (n=4 mice per genotype). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.Intracellular flow cytometry analysis of Cyclin D1 protein (D) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.Intracellular flow cytometry analysis of pro-survival regulators Mcl1, Blc2, and Bim protein (E) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.representative FACS histograms and bar graphs show relative levels of pAKT (Ser473) relative to WT controls (F) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.representative FACS histograms and bar graphs show relative levels of pFoxO3a (Ser253) (G) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.representative FACS histograms and bar graphs show relative levels of Total cMyc protein (H) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.; representative FACS histograms and bar graphs show relative levels of pRb (Ser807/811) (I) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.representative FACS histograms and bar graphs show total PPARγ protein levels relative to WT controls (J) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.Relative expression levels of total PPARδ protein in HSPCs (K) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.Activation of pmTOR (Ser2448) (L) Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.Activation of pS6 (Ser235/236) and (Ser240/244) (M). Data shown in bar graphs are mean fluorescence intensity of indicated protein expression in PKCδ cKO HSPCs subsets relative to WT controls (n=4-6 mice per genotype per target). Data information: The statistical significance of difference was assessed using Two-tailed Student's unpaired t-test analysis for comparison of control and PKCδ cKO HSPCs. All data are presented as mean± SEM., *p<0.05, **p<0.001.


Figure 1Comparison 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 . 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 , 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 .

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

Figure 1Comparison 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.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,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.


Figure 2Comparison 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Comparison 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.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.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.


Figure 3Comparison 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 ) . 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 . 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 . 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 ) .

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

Figure 3Comparison 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).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.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.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).


Figure 4Comparison 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Comparison 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.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.


Figure 5Phenotype 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 . 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 ) 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 .

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Figure 5Phenotype 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.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)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.


Figure 6 ( 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 . ( 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 ) .

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Figure 6(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.(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).


Figure 7Volcano 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 ) . 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 . 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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Figure 7Volcano 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).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.migration of ENCCs evaluated using transwell plate assay show that MEK1/2 inhibitor U0126 suppressed SCFA-induced ENCC proliferation and migration.


Figure 2A . 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 .

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

Figure 2A. 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.