( B ) U2OS ( left panel ) , RPE1 - hTERT ( middle panel ) , HeLa ( right panel ) were monitored for their capacity to form IR - induced BRCA1 foci . For each , following conditions were used : U2OS were transfected with a nontargeting siRNA ( siCtrl ) or an siRNA targeting human POGZ ( siPOGZ - 1 or - 2 ) ; RPE1 - hTERT were transduced a control shRNA ( shCtrl ) or a shRNA directed against human POGZ ( shPOGZ - 1 or - 2 ) ; HeLa were expressing a non - targeting sgRNA ( sgCtrl ) or a sgRNA targeting human POGZ sub - cloned ( POGZ ∆ - 1 or - 2 ) . were exposed to 1 Gy before being pulsed with Edu for 1hr were recovered 1h post - exposure to IR . were fixed , stained , imaged via confocal microscopy . Data are total number of ΒRCA1 foci in EdU + represented as a bar graph showing mean ± SD ( n = 3 biological replicates , with at least 100 analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s . * P < 0 . 005 , * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS (left panel), RPE1-hTERT (middle panel), and HeLa cells (right panel) were monitored for their capacity to form IR-induced BRCA1 foci. For each cell line, the following conditions were used: U2OS cells were transfected with a nontargeting siRNA (siCtrl) or an siRNA targeting human POGZ (siPOGZ-1 or -2); RPE1-hTERT cells were transduced a control shRNA (shCtrl) or a shRNA directed against human POGZ (shPOGZ-1 or -2); HeLa cells were expressing a non-targeting sgRNA (sgCtrl) or a sgRNA targeting human POGZ and sub-cloned (POGZ∆-1 or -2). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post-exposure to IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.001.


( E ) U2OS stably expressing mCherry - LacR - FokI were transduced with indicated shRNA subsequently transfected with indicated siRNA . 24h post - transfection , damage was induced using Shield - 1 4 - OHT . Immunofluorescence against BRCA1 was subsequently performed to monitor its accumulation at sites of damage by confocal microscopy . Data are represented as a box - - whisker ( 10 - 90 percentile ) where signal at mCherry focus was normalized to background . At least 50 per condition were counted . Significance was determined by two - way ANOVA followed by a Dunnett ' s . * P < 0 . 0001 .

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

(E) U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated shRNA and subsequently transfected with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield-1 and 4-OHT. Immunofluorescence against BRCA1 was subsequently performed to monitor its accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 50 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.0001.


( G ) U2OS stably expressing mCherry - LacR - FokI were transduced with indicated siRNA . 24h post - transfection , damage was induced using Shield - 1 4 - OHT . Immunofluorescence against indicated HP1 isoform was subsequently performed to monitor its respective accumulation at sites of damage by confocal microscopy . Data are represented as a box - - whisker ( 10 - 90 percentile ) where signal at mCherry focus was normalized to background . At least 75 per condition were counted . Significance was determined by two - way ANOVA followed by a Dunnett ' s . * P < 0 . 005 , * * P < 0 . 0001 .

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

(G) U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield-1 and 4-OHT. Immunofluorescence against the indicated HP1 isoform was subsequently performed to monitor its respective accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear background. At least 75 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.0001.


( C ) HeLa transfected with indicated construct were monitored for their capacity to form IR - induced BRCA1 foci . HeLa where POGZ has been targeted by CRISPR technology ( POGZ ∆ - 1 ) in control HeLa ( sgCtrl ) were transfected by an empty Flag vector ( EV ) or a sgRNA - resistant FLAG - tagged POGZ cDNA construct corresponding to indicated rescue mutant ( full - , FL ; POGZ801 - 848 , HPZ ) . were exposed to 1 Gy before being pulsed with Edu for 1hr were recovered 1h post - exposure to IR . were fixed , stained , imaged via confocal microscopy . Data are total number of ΒRCA1 foci in EdU + represented as a bar graph showing mean ± SD ( n = 3 biological replicates , with at least 100 analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s . * P < 0 . 05 , * * P < 0 . 0001 . ( D ) Representative used for in ( C ) . Scale bar = 5 μm .

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

(C) HeLa cells transfected with the indicated construct were monitored for their capacity to form IR-induced BRCA1 foci. HeLa cells where POGZ has been targeted by CRISPR technology (POGZ∆-1) and in control HeLa cells (sgCtrl) were transfected by an empty Flag vector (EV) or a sgRNA-resistant FLAG-tagged POGZ cDNA construct corresponding to indicated rescue mutant (full-length, FL; POGZ801-848, HPZ). Cells were exposed to 1 Gy before being pulsed with Edu for 1hr and were recovered 1h post-exposure to IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.0001. (D) Representative images used for quantification in (C). Scale bar = 5 μm.


( E ) Similar as in ( C ) except that γ - H2AX foci were monitored at indicated time points by confocal microscopy . Data are total number of γ - H2AX foci in EdU + represented as a bar graph showing mean ± SD ( n = 3 biological replicates , with at least 100 analyzed for each time point ) . Significance was determined by two - way ANOVA followed by a Dunnett ' s . * P < 0 . 05 . ( F ) Representative used for in ( E ) . Scale bar = 5 μm .

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

(E) Similar as in (C) except that γ-H2AX foci were monitored at the indicated time points by confocal microscopy. Data are the total number of γ-H2AX foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05. (F) Representative images used for quantification in (E). Scale bar = 5 μm.


Observed genotypic distribution of offspring of heterozygous Pogz + / ∆ crosses at specified embryonic day . Each litter is considered a biological replicate . Data are represented as a bar graph showing mean ± ( n for each embryonic day is specified ) . ( D )

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

Observed genotypic distribution of offspring of heterozygous Pogz+/∆ crosses at specified embryonic day. Each litter is considered a biological replicate. Data are represented as a bar graph showing the mean ± SEM (n for each embryonic day is specified). (D)


( E ) mass of wild - type ( WT ) or Pogz + / ∆ mice was monitored weekly for 4 post - weaning . Data are represented as a bar graph showing mean ± each mouse is represented by a round dot ( WT ) or a square ( Pogz + / ∆ ) . At least 4 mice per genotype was monitored . Significance was determined by two - way ANOVA followed by a Sidak ' s . * P < 0 . 05 .

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

(E) The body mass of male wild-type (WT) or Pogz+/∆ mice was monitored weekly for 4 weeks post-weaning. Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a round dot (WT) or a square (Pogz+/∆ ). At least 4 mice per genotype was monitored. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05.


( H ) of travelled ( left panel ) average speed ( right panel ) of each mouse ( n = 6 mice per genotype ) in open field . Data are represented as a bar graph showing mean ± each mouse is represented by a a round dot ( WT ) or a square ( Pogz + / ∆ ) . Significance was determined by unpaired two - t - . * P < 0 . 005 .

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

(H) Quantification of the distance travelled (left panel) and the average speed (right panel) of each mouse (n=6 mice per genotype) in the open field. Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a a round dot (WT) or a square (Pogz+/∆ ). Significance was determined by unpaired two-tailed t-test. *P<0.005.


( L ) Plasma was isolated from cardiac punctures of wild - type ( WT ) or Pogz + / ∆ mice ( 8 ) assessed for circulating of specified immunoglobulin isotypes . Each mouse is represented by a round dot ( WT ) or a square ( Pogz + / ∆ ) ( n = 6 mice per genotype ) . Significance was determined by two - way ANOVA followed by a Bonferroni ' s . * P < 0 . 005 .

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

(L) Plasma was isolated from cardiac punctures of wild-type (WT) or Pogz+/∆ mice (8 weeks) and assessed for circulating levels of specified immunoglobulin isotypes. Each mouse is represented by a round dot (WT) or a square (Pogz+/∆ ) (n=6 mice per genotype). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.005.


( A ) Wild - type ( WT ) Pogz + / ∆ mice were subjected to a lethal dose of ionizing radiation ( 8 . 5 Gy ) before recovering in sterile conditions being assessed for their sensitivity to IR . Data are represented as a Kaplan - Meier survival curve of each genotype ( ( n = 6 mice per genotype ) . Significance was determined by log - rank ( Mantel - Cox ) . * P < 0 . 005 .

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

(A) Wild-type (WT) and Pogz+/∆ mice were subjected to a lethal dose of ionizing radiation (8.5 Gy) before recovering in sterile conditions and being assessed for their sensitivity to IR. Data are represented as a Kaplan-Meier survival curve of each genotype ((n=6 mice per genotype). Significance was determined by log-rank (Mantel-Cox) test. *P<0.005.


( D ) MEFs were monitored for their sensitivity to radiomimetic drug phleomycin using SRB . Immortalized MEFS obtained from indicated genotype were treated with increasing concentrations of phleomycin for 1hr , replenished with fresh medium incubated for 4 days before being processed for SRB assays . Data are represented as a bar graph showing relative mean ± , each replicate being representing as a round dot ( WT ) or a square ( Pogz + / ∆ ) . Significance was determined by two - way ANOVA followed by a Bonferroni ' s . * P < 0 . 005 , * * P < 0 . 0005 .

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

(D) MEFs were monitored for their sensitivity to the radiomimetic drug phleomycin using the SRB assay. Immortalized MEFS obtained from the indicated genotype were treated with increasing concentrations of phleomycin for 1hr, replenished with fresh medium and incubated for 4 days before being processed for SRB assays. Data are represented as a bar graph showing the relative mean ± SEM, each replicate being representing as a round dot (WT) or a square (Pogz+/∆ ). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.005, **P<0.0005.


( H ) Stimulated CD43 - negative splenocytes were loaded on slides via Cytospin processed for γ - H2AX immunofluorescence . were fixed , stained , imaged via confocal microscopy . Data are of in a field of view with indicated γ - H2AX foci are represented as a bar graph showing mean ± . At least 100 per genotype were counted . Significance was determined by two - way ANOVA followed by a Sidak ' s . * P < 0 . 05 , * * P < 0 . 005 .

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

(H) Stimulated CD43-negative splenocytes were loaded on slides via Cytospin and processed for γ-H2AX immunofluorescence. Cells were fixed, stained, and imaged via confocal microscopy. Data are the percentage of cells in a field of view with indicated γ-H2AX foci and are represented as a bar graph showing the mean ± SEM. At least 100 cells per genotype were counted. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05, **P<0.005.


A Volcano plot representing statistical of fold enrichment of after affinity purification in HEPES epes buffer of ABCE1 - TAP followed by label - free ( LFQ ) using liquid chromatography - tandem mass spectrometry ( LC - MS / MS ) . above curved lines show a statistically significant enrichment according to t value .

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

A Volcano plot representing the statistical analysis of the fold enrichment of proteins after affinity purification in HEPES epes buffer of ABCE1-TAP followed by label-free quantification (LFQ) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins above the curved lines show a statistically significant enrichment according to the t test value.


B , C Sucrose density gradient UV - profile after in vitro splitting assays ( B ) relative abundance of 80S subunits as calculated from triplicates displayed as mean ± SD . ( C ) ; SF = splitting factors including Dom34 , Hbs1 , ABCE1 eIF6 ; ( + ) = 4 - fold molar excess of eIF3j ; ( + + ) = 20 - fold molar excess of eIF3j .

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

B, C Sucrose density gradient UV-profile after in vitro splitting assays (B) and relative abundance of 80S and subunits as calculated from triplicates and displayed as mean ± SD. (C); SF = splitting factors including Dom34, Hbs1, ABCE1 and eIF6; (+) = 4-fold molar excess of eIF3j; (++) = 20-fold molar excess of eIF3j.


( A ) Top 2 significantly overrepresented expression patterns for ERS UP ERS DOWN genes following PHx . Data show changes in expression at 4 , 10 , 48h 1 week after PHx ( 0h ) for genes comprised within each model profile of dynamic expression identified by .

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

(A) Top 2 significantly overrepresented expression patterns for ERS UP and ERS DOWN genes following PHx. Data show changes in expression at 4, 10, 48h and 1 week after PHx (0h) for genes comprised within each model profile of dynamic expression identified by STEM.


( C ) Bagplots showing breadth of transcriptomic changes for indicated datasets . Genes were positioned on their basal expression in control conditions their FC ( Log2 ) in indicated ( patho ) physiological context . blue is " bag " ( 50 % of data points around median , which is indicated by a red cross ) while blue delimits " loop " Red dots are outliers .

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

(C) Bagplots showing the breadth of transcriptomic changes for the indicated datasets. Genes were positioned based on their basal expression levels in the control conditions and their FC (Log2) in the indicated (patho)physiological context. The dark blue area is the "bag" (50% of the data points around the median, which is indicated by a red cross) while the light blue area delimits the "loop" Red dots are outliers.


( D - E ) Box plots showing normalized expression in liver ( D ) liver - specificity index ( E ) of LIVER - ID genes , UBQ genes other genes . Liver - specificity index was calculated as difference of normalized expression in liver ( 2 replicates ) mean of normalized expression in control tissues ( 2 replicates per tissue ) using data from BioGPS is reported as Log2 . Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . One - way ANOVA with Welch ' s correction Dunnett ' s Modified Tukey - Kramer pairwise multiple comparison was used to assess statistical significance , * P < 0 . 05 .

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

(D-E) Box plots showing normalized expression in liver (D) and liver-specificity index (E) of LIVER-ID genes, UBQ genes and other genes. Liver-specificity index was calculated as the difference of normalized expression in liver (2 replicates) and mean of normalized expression in control tissues (2 replicates per tissue) using data from BioGPS and is reported as Log2. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-way ANOVA with Welch's correction and Dunnett's Modified Tukey-Kramer pairwise multiple comparison test was used to assess statistical significance, *P < 0.05.


( F ) Similar analyses using LIVER - ID genes .

(F) Similar analyses using LIVER-ID genes.


Box plots showing Log2 FC ERS / Control in MPH ( 3 independent experiments ) ( G ) for LIVER - ID genes , UBQ genes other genes . Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . One - way ANOVA with Welch ' s correction Dunnett ' s Modified Tukey - Kramer pairwise multiple comparison was used to assess statistical significance , * P < 0 . 05 .

Box plots showing Log2 FC ERS/Control in MPH (3 independent experiments) (G) for LIVER-ID genes, UBQ genes and other genes. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-way ANOVA with Welch's correction and Dunnett's Modified Tukey-Kramer pairwise multiple comparison test was used to assess statistical significance, *P < 0.05.


Box plots showing Log2 FC ERS / Control in mouse liver ( 3 mice per group ) ( H ) for LIVER - ID genes , UBQ genes other genes . Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . One - way ANOVA with Welch ' s correction Dunnett ' s Modified Tukey - Kramer pairwise multiple comparison was used to assess statistical significance , * P < 0 . 05 .

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

Box plots showing Log2 FC ERS/Control in mouse liver (3 mice per group) (H) for LIVER-ID genes, UBQ genes and other genes. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-way ANOVA with Welch's correction and Dunnett's Modified Tukey-Kramer pairwise multiple comparison test was used to assess statistical significance, *P < 0.05.


( I ) Enrichment plots from gene set enrichment analyses ( GSEA ) performed using LIVER - ID genes as gene set transcriptomic changes induced by acute ERS in MPH ( upper panel ) or mouse liver ( lower panel ) as ranked gene list . NES FDR ( as in subsequent GSEA panels ) are normalized enrichment score false discovery rate provided by GSEA software , respectively .

(I) Enrichment plots from gene set enrichment analyses (GSEA) performed using LIVER-ID genes as the gene set and transcriptomic changes induced by acute ERS in MPH (upper panel) or mouse liver (lower panel) as the ranked gene list. NES and FDR (as in all subsequent GSEA panels) are the normalized enrichment score and the false discovery rate provided by the GSEA software, respectively.


Genes repressed by ERS in MPH ( 3 independent experiments ) ( J ) were ranked on their Log2 FC ERS / Control divided into quartiles ( increased repression from Q1 to Q4 ) . fraction of LIVER - ID genes in 4 quartiles was defined is displayed relative to that obtained for Q1 arbitrarily set to 1 . Chi - square with BH correction for multiple testing was used to assess statistical significance , * P < 0 . 05 .

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

Genes repressed by ERS in MPH (3 independent experiments) (J) were ranked based on their Log2 FC ERS/Control and divided into quartiles (increased repression from Q1 to Q4). The fraction of LIVER-ID genes in the 4 quartiles was defined and is displayed relative to that obtained for Q1 arbitrarily set to 1. Chi-square test with BH correction for multiple testing was used to assess statistical significance, *P < 0.05.


Genes repressed by ERS in mouse liver ( 3 mice per group ) ( K ) were ranked on their Log2 FC ERS / Control divided into quartiles ( increased repression from Q1 to Q4 ) . fraction of LIVER - ID genes in 4 quartiles was defined is displayed relative to that obtained for Q1 arbitrarily set to 1 . Chi - square with BH correction for multiple testing was used to assess statistical significance , * P < 0 . 05 .

Genes repressed by ERS in mouse liver (3 mice per group) (K) were ranked based on their Log2 FC ERS/Control and divided into quartiles (increased repression from Q1 to Q4). The fraction of LIVER-ID genes in the 4 quartiles was defined and is displayed relative to that obtained for Q1 arbitrarily set to 1. Chi-square test with BH correction for multiple testing was used to assess statistical significance, *P < 0.05.


RT - qPCR analyses of selected ERS UP LIVER - ID genes monitoring expression changes induced by acute ERS in MPH ( 3 to 9 independent experiments ) ( L ) bar graphs show means ± SD ( standard deviations ) . One - sample t - with BH correction for multiple testing was used to determine if mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 .

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

RT-qPCR analyses of selected ERS UP and LIVER-ID genes monitoring expression changes induced by acute ERS in MPH (3 to 9 independent experiments) (L) The bar graphs show means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.


RT - qPCR analyses of selected ERS UP LIVER - ID genes monitoring expression changes induced by acute ERS in mouse liver ( 5 to 7 mice per group ) ( M ) . bar graphs show means ± SD ( standard deviations ) . One - sample t - with BH correction for multiple testing was used to determine if mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 .

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

RT-qPCR analyses of selected ERS UP and LIVER-ID genes monitoring expression changes induced by acute ERS in mouse liver (5 to 7 mice per group) (M). The bar graphs show means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.


( N ) Heatmaps showing normalized expression of ERS UP ERS DOWN genes from MPH ( upper panel ) or mouse liver ( lower panel ) in mouse liver at indicated stages of development .

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

(N) Heatmaps showing normalized expression of ERS UP and ERS DOWN genes from MPH (upper panel) or mouse liver (lower panel) in mouse liver at the indicated stages of development.


( O ) Average expression of ERS DOWN genes from MPH in single - from hepatobiliary lineage . hepatoblast - to - hepatocyte hepatoblast - to - cholangiocyte differentiation paths are indicated with arrows .

(O) Average expression of ERS DOWN genes from MPH in single-cells from the hepatobiliary lineage. The hepatoblast-to-hepatocyte and hepatoblast-to-cholangiocyte differentiation paths are indicated with arrows.


( A ) Comparison of transcriptomics ( data from 3 independent experiments ) H3K27ac ChIP - seq ( data from 3 independent experiments ) from MPH . Genes were assigned to H3K27ac regions number of ERS DOWN genes is indicated relative to number of ERS UP genes for 3 categories of H3K27ac regions . Fisher ' s exact with BH correction for multiple testing was used to assess statistical significance , * P < 0 . 05 , # P < 0 . 05 .

(A) Comparison of transcriptomics (data from 3 independent experiments) and H3K27ac ChIP-seq (data from 3 independent experiments) from MPH. Genes were assigned to H3K27ac regions The number of ERS DOWN genes is indicated relative to the number of ERS UP genes for the 3 categories of H3K27ac regions. Fisher's exact test with BH correction for multiple testing was used to assess statistical significance, *P < 0.05, #P < 0.05.


( B ) number of LIVER - ID UBQ genes is indicated relative to number of other genes for 3 categories of H3K27ac regions . H3K27ac ChIP - seq data were obtained from 3 independent MPH experiments . Chi - square with BH correction for multiple testing was used to assess statistical significance , # P < 0 . 05 .

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(B) The number of LIVER-ID and UBQ genes is indicated relative to the number of other genes for the 3 categories of H3K27ac regions. The H3K27ac ChIP-seq data were obtained from 3 independent MPH experiments. Chi-square test with BH correction for multiple testing was used to assess statistical significance, #P < 0.05.


( C ) Multidimensional scaling ( MDS ) was performed transcriptional regulator co - recruitment was depicted using density plots for regions with increased ( a ) , decreased ( b ) or unchanged ( c ) H3K27ac in MPH upon acute ERS . boxed ( d ) represents a subset of transcriptional regulators ( TR ) with a high degree of co - binding in H3K27ac DOWN regions . LIVER - ID TFs are depicted in red .

(C) Multidimensional scaling (MDS) was performed and transcriptional regulator co-recruitment was depicted using density plots for regions with increased (a), decreased (b) or unchanged (c) H3K27ac levels in MPH upon acute ERS. The boxed area (d) represents a subset of transcriptional regulators (TR) with a high degree of co-binding in H3K27ac DOWN regions. LIVER-ID TFs are depicted in red.


( D ) Heatmaps showing of enhancers overlapping H3K27ac down regions . Enhancers were first split into those bound or not by a given LIVER - ID TF ( right ) then on co - binding of additional LIVER - ID TFs ( defining 3 subgroups with 0 - 2 , 3 - 5 or 6 - 8 co - bound additional LIVER - ID TFs ) .

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(D) Heatmaps showing the percentage of enhancers overlapping H3K27ac down regions. Enhancers were first split into those bound or not by a given LIVER-ID TF (right) and then based on co-binding of additional LIVER-ID TFs (defining 3 subgroups with 0-2, 3-5 or 6-8 co-bound additional LIVER-ID TFs).


( E ) Enrichment plots from GSEA performed using LIVER - ID TFs as gene set transcriptomic changes induced by acute ERS in MPH ( upper panel ) or mouse liver ( lower panel ) as ranked gene list .

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(E) Enrichment plots from GSEA performed using LIVER-ID TFs as the gene set and transcriptomic changes induced by acute ERS in MPH (upper panel) or mouse liver (lower panel) as the ranked gene list.


( F ) RT - qPCR analyses of selected LIVER - ID TFs monitoring expression changes induced by acute ERS in MPH ( 4 to 7 independent experiments ) . bar graph shows means ± SD ( standard deviations ) . One - sample t - with BH correction for multiple testing was used to determine if mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 .

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(F) RT-qPCR analyses of selected LIVER-ID TFs monitoring expression changes induced by acute ERS in MPH (4 to 7 independent experiments). The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.


( G ) RT - qPCR analyses of selected ERS UP LIVER - ID TF genes monitoring expression changes induced by 4h ERS in MPH pre - treated or not for 30min with 5mM PBA ( 3 independent experiments ) . Mean Log2 FC ERS / Control are shown . bar graph shows means ± SD ( standard deviations ) . Two - way ANOVA with Bonferroni ' s post - hoc was used to assess statistical significance , * P < 0 . 05 .

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(G) RT-qPCR analyses of selected ERS UP and LIVER-ID TF genes monitoring expression changes induced by 4h ERS in MPH pre-treated or not for 30min with 5mM PBA (3 independent experiments). Mean Log2 FC ERS/Control are shown. The bar graph shows means ± SD (standard deviations). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance, *P < 0.05.


( H - I ) extracts ( H ) fractions ( I ) from MPH were subjected to Western blot with antibodies against HNF4A , NR1H4 / FXR , FOXA2 / HNF3B or DDIT3 / CHOP . LMNA or histone H3 were used as loading controls . Results obtained from 3 independent biological replicates are shown .

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(H-I) Nuclear extracts (H) and chromatin fractions (I) from MPH were subjected to Western blot with antibodies against HNF4A, NR1H4/FXR, FOXA2/HNF3B or DDIT3/CHOP. LMNA or histone H3 were used as loading controls. Results obtained from 3 independent biological replicates are shown.


( A ) RT - qPCR analyses of Hlf , Tef , Dbp Nfil3 expression monitoring changes induced by acute ERS in MPH ( 3 to 5 independent experiments ) ( left panel ) or mouse liver ( 5 mice per group ) ( right panel ) . bar graphs show means ± SD ( standard deviations ) . One - sample t - with BH correction for multiple testing was used to determine if mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 .

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(A) RT-qPCR analyses of Hlf, Tef, Dbp and Nfil3 expression monitoring changes induced by acute ERS in MPH (3 to 5 independent experiments) (left panel) or mouse liver (5 mice per group) (right panel). The bar graphs show means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.


( B ) Total extracts from MPH ( left panel ) or mouse liver ( right panel ) were subjected to Western blot with an antibody against NFIL3 . ACTB was used as loading control .

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(B) Total protein extracts from MPH (left panel) or mouse liver (right panel) were subjected to Western blot with an antibody against NFIL3. ACTB was used as loading control.


( C ) fractions from MPH ( upper panel ) or mouse liver ( lower panel ) were subjected to Western blot with an antibody against NFIL3 . Histone H3 was used as loading control .

(C) Chromatin fractions from MPH (upper panel) or mouse liver (lower panel) were subjected to Western blot with an antibody against NFIL3. Histone H3 was used as loading control.


( D ) Enrichment scores from GSEA performed using LIVER - ID genes repressed by acute ERS in MPH ( MPH ERS DOWN ) as gene set liver transcriptomic changes induced by acute ERS / or deletion of Nfil3 ( NFIL3 KO ) as ranked gene lists were integrated corrected for multiple testing using BubbleGUM tool . For NFIL3 KO ERS vs WT ERS comparison , Enrichment genes ( i . e . subset of genes that contributes most to enrichment result ) were subjected to functional enrichment analyses using ToppGene Suite . top ranked KEGG Pathway with its Bonferroni - corrected p - value is shown .

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(D) Enrichment scores from GSEA performed using LIVER-ID genes repressed by acute ERS in MPH (MPH ERS DOWN) as the gene set and liver transcriptomic changes induced by acute ERS and/or deletion of Nfil3 (NFIL3 KO) as the ranked gene lists were integrated and corrected for multiple testing using the BubbleGUM tool. For the NFIL3 KO ERS vs WT ERS comparison, the Core Enrichment genes (i.e. the subset of genes that contributes most to the enrichment result) were subjected to functional enrichment analyses using the ToppGene Suite. The top ranked KEGG Pathway with its Bonferroni-corrected p-value is shown.


( E ) Box plots showing mRNA expression for 5 genes from Enrichment from D involved in xenobiotic metabolism issued from transcriptomic analyses . Shown are Log2 FC relative to mean normalized expression in WT Control group ( 5 mice per group ) . Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . Two - way ANOVA with Bonferroni ' s post - hoc was used to assess statistical significance , * P < 0 . 05 .

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(E) Box plots showing mRNA expression for 5 genes from the Core Enrichment from D involved in xenobiotic metabolism issued from the transcriptomic analyses. Shown are Log2 FC relative to the mean normalized expression in the WT Control group (5 mice per group). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance, *P < 0.05.


( F ) Integrated Genome Browser ( IGB ) was used to visualize ChIP - seq profiles for NFIL3 ( green ) several LIVER - ID TFs ( red ) in mouse liver at Gsta3 gene locus . of H3K27ac in MPH from non - parenchymal fraction ( NPC ) are shown in blue . grey bar indicates position of a BRD4 SE .

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(F) The Integrated Genome Browser (IGB) was used to visualize ChIP-seq profiles for NFIL3 (green) and several LIVER-ID TFs (red) in the mouse liver at the Gsta3 gene locus. Levels of H3K27ac in MPH and cells from the non-parenchymal fraction (NPC) are shown in blue. The grey bar indicates the position of a BRD4 SE.


( A ) BRD4 occupancy ( left panels ) H3K27ac ( right panels ) at regulatory regions associated with ERS UP or LIVER - ID genes ( 8 regions at ERS gene loci 10 regions at LIVER - ID gene loci were assessed by ChIP - qPCR in MPH ( 10 independent experiments ) or mouse liver ( 10 mice per group ) to define changes induced by acute ERS . Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . One - sample t - with BH correction for multiple testing was used to determine if mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 .

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(A) BRD4 occupancy (left panels) and H3K27ac levels (right panels) at regulatory regions associated with ERS UP or LIVER-ID genes (8 regions at ERS gene loci and 10 regions at LIVER-ID gene loci were assessed by ChIP-qPCR in MPH (10 independent experiments) or mouse liver (10 mice per group) to define changes induced by acute ERS. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.


( B ) Left panel , BRD4 mRNA ( 4 independent experiments ) or expression ( 5 independent experiments ; densitometric in MPH subjected to acute ERS . bar graphs show means ± SD ( standard deviations ) . Student ' s t - was used to assess statistical significance . Right panel , Total extracts from MPH pre - treated for 3h with 0 . 01µM MZ1 followed by addition of 1µM thapsigargin ( ERS ) for 4h were subjected to Western blot with an antibody against BRD4 . LMNA was used as loading control .

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(B) Left panel, BRD4 mRNA (4 independent experiments) or protein expression levels (5 independent experiments; densitometric quantification in MPH subjected to acute ERS. The bar graphs show means ± SD (standard deviations). Student's t-test was used to assess statistical significance. Right panel, Total protein extracts from MPH pre-treated for 3h with 0.01µM MZ1 followed by addition of 1µM thapsigargin (ERS) for 4h were subjected to Western blot with an antibody against BRD4. LMNA was used as loading control.


( C ) Heatmaps showing Log2 FC ( relative to DMSO / Control condition ) for 5 ERS UP LIVER - ID genes issued from RT - qPCR analyses of MPH pre - treated with 500nM JQ1 ( left ) or 0 . 01µM MZ1 ( right ) followed by addition of 1µM thapsigargin ( ERS ) for 4h ( 3 to 6 independent experiments ) . Two - way ANOVA with Bonferroni ' s post - hoc was used to assess statistical significance .

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(C) Heatmaps showing Log2 FC (relative to the DMSO/Control condition) for 5 ERS UP and LIVER-ID genes issued from RT-qPCR analyses of MPH pre-treated with 500nM JQ1 (left) or 0.01µM MZ1 (right) followed by addition of 1µM thapsigargin (ERS) for 4h (3 to 6 independent experiments). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance.


( D ) Heatmaps showing average H3K27ac ChIP - seq signals in MPH at LIVER - ID domains overlapping ( + ) or not ( - ) with BRD4 SE . arrow indicates position of gene transcriptional start sites .

(D) Heatmaps showing average H3K27ac ChIP-seq signals in MPH at LIVER-ID domains overlapping (+) or not (-) with BRD4 SE. The arrow indicates the position of gene transcriptional start sites.


( E ) Box plots showing Log2 FC ERS / Control in MPH ( 3 independent experiments ) ( left panel ) or mouse liver ( 3 mice per group ) ( right panel ) for genes associated with LIVER - ID + BRD4 SE or LIVER - ID - BRD4 SE Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . Student ' s t - was used to assess statistical significance , * P < 0 . 05 .

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(E) Box plots showing Log2 FC ERS/Control in MPH (3 independent experiments) (left panel) or mouse liver (3 mice per group) (right panel) for genes associated with LIVER-ID + BRD4 SE or LIVER-ID - BRD4 SE Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. Student's t-test was used to assess statistical significance, *P < 0.05.


( A ) RT - qPCR analyses of 21 LIVER - ID genes in MPH which were treated with 1µM thapsigargin ( ERS ) for 4h , 8h , 16h or 24h ( 4 independent experiments ) . For each gene , timepoint showing maximal repression by ERS was recorded . depicts of genes maximally repressed by ERS at each timepoint .

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(A) RT-qPCR analyses of 21 LIVER-ID genes in MPH which were treated with 1µM thapsigargin (ERS) for 4h, 8h, 16h or 24h (4 independent experiments). For each gene, the timepoint showing maximal repression by ERS was recorded. The heatmap depicts the percentage of genes maximally repressed by ERS at each timepoint.


( B ) RT - qPCR analyses of Hnf4a , Nr1h4 Foxa2 expression in MPH treated with vehicle ( Control ) or 1µM thapsigargin ( ERS ) for 1h or 4h ( 4 independent experiments ) . bar graph shows means ± SD ( standard deviations ) . One - sample t - with BH correction for multiple testing was used to determine if mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 .

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(B) RT-qPCR analyses of Hnf4a, Nr1h4 and Foxa2 expression in MPH treated with vehicle (Control) or 1µM thapsigargin (ERS) for 1h or 4h (4 independent experiments). The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.


( C ) Densitometric of expression data for 1h timepoint ( average of 9 biological replicates ) for 4h timepoint ( average of 3 biological replicates ) . bar graph shows means ± SD ( standard deviations ) . One - sample t - with BH correction for multiple testing was used to determine if mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 .

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(C) Densitometric quantification of the protein expression data for the 1h timepoint (average of 9 biological replicates) and for the 4h timepoint (average of 3 biological replicates). The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.


( D ) extracts from MPH pre - treated for 30min with 1µM ISRIB followed by addition of 1µM thapsigargin ( ERS ) for 1h were subjected to Western blot with antibodies against HNF4A , NR1H4 , FOXA2 or DDIT3 . LMNA was used as loading control . Results obtained from 3 independent biological replicates are shown . ( E ) Densitometric of expression data shown in panel ( D ) . Repression by ERS in ISRIB condition ( average of 3 biological replicates ) is shown relative to repression by ERS in vehicle condition . bar graph shows means ± SD ( standard deviations ) . One - sample t - with BH correction for multiple testing was used to determine if mean relative repression is statistically different from 100 % , * P < 0 . 05 .

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(D) Nuclear extracts from MPH pre-treated for 30min with 1µM ISRIB followed by addition of 1µM thapsigargin (ERS) for 1h were subjected to Western blot with antibodies against HNF4A, NR1H4, FOXA2 or DDIT3. LMNA was used as loading control. Results obtained from 3 independent biological replicates are shown. (E) Densitometric quantification of the protein expression data shown in panel (D). Repression by ERS in the ISRIB condition (average of 3 biological replicates) is shown relative to repression by ERS in vehicle condition. The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100%, *P < 0.05.


( F ) extracts from MPH pre - treated for 30min with 10µM MG132 or 10µM PP2 followed by addition of 1µM thapsigargin ( ERS ) for 1h were subjected to Western blot or Simple Western immunoassay with antibodies against HNF4A , NR1H4 , FOXA2 or XBP1S . LMNA was used as loading control . Results obtained from 2 independent biological replicates are shown . ( G ) Densitometric of expression data shown in panel ( F ) Repression by ERS in MG132 condition ( average of 4 biological replicates ) is shown relative to repression by ERS in vehicle condition . bar graph shows means ± SD ( standard deviations ) . One - sample t - with BH correction for multiple testing was used to determine if mean relative repression is statistically different from 100 % , * P < 0 . 05 .

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(F) Nuclear extracts from MPH pre-treated for 30min with 10µM MG132 or 10µM PP2 followed by addition of 1µM thapsigargin (ERS) for 1h were subjected to Western blot or Simple Western immunoassay with antibodies against HNF4A, NR1H4, FOXA2 or XBP1S. LMNA was used as loading control. Results obtained from 2 independent biological replicates are shown. (G) Densitometric quantification of the protein expression data shown in panel (F) Repression by ERS in MG132 condition (average of 4 biological replicates) is shown relative to repression by ERS in vehicle condition. The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100%, *P < 0.05.


( H ) ( Left ) extracts issued from MPH ( 2 biological replicates ) were subjected to Western blot with antibodies against p - Y416 - SRC or total SRC . ACTB was used as loading control . ( Right ) Densitometric of expression data . bar graphs show means ± SD ( standard deviations ) . Two - way ANOVA with Bonferroni ' s post - hoc was used to assess statistical significance .

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(H) (Left) Cytoplasmic extracts issued from the MPH (2 biological replicates) were subjected to Western blot with antibodies against p-Y416-SRC or total SRC. ACTB was used as loading control. (Right) Densitometric quantification of the protein expression data. The bar graphs show means ± SD (standard deviations). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance.


( I ) Densitometric of expression data Repression by ERS in PP2 condition ( average of 4 biological replicates ) is shown relative to repression by ERS in vehicle condition . bar graph shows means ± SD ( standard deviations ) . One - sample t - with BH correction for multiple testing was used to determine if mean relative repression is statistically different from 100 % .

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(I) Densitometric quantification of the protein expression data Repression by ERS in the PP2 condition (average of 4 biological replicates) is shown relative to repression by ERS in vehicle condition. The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100%.


( J ) RT - qPCR analyses of selected ERS UP LIVER - ID TF genes monitoring expression changes induced by 4h ERS in MPH pre - treated or not for 30min with 10µM PP2 ( 3 independent experiments ) . Mean Log2 FC ERS / Control are shown . bar graph shows means ± SD ( standard deviations ) . Two - way ANOVA with Bonferroni ' s post - hoc was used to assess statistical significance , * P < 0 . 05 .

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(J) RT-qPCR analyses of selected ERS UP and LIVER-ID TF genes monitoring expression changes induced by 4h ERS in MPH pre-treated or not for 30min with 10µM PP2 (3 independent experiments). Mean Log2 FC ERS/Control are shown. The bar graph shows means ± SD (standard deviations). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance, *P < 0.05.


( A ) using transcriptomic data from liver of sepsisBIM mice ( 16h after intraperitoneal injection of live E . coli ) .

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(A) analysis using transcriptomic data from the liver of sepsisBIM mice (16h after intraperitoneal injection of live E. coli).


( B - C ) Enrichment plots from GSEA performed using LIVER - ID genes ( B ) or response to ERS gene set ( GO : 0034976 ) ( C ) as gene set transcriptomic differences in sepsisBIM vs Control mouse liver as ranked gene list .

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(B-C) Enrichment plots from GSEA performed using LIVER-ID genes (B) or the response to ERS gene set (GO:0034976) (C) as the gene set and transcriptomic differences in sepsisBIM vs Control mouse liver as the ranked gene list.


( E ) Box plots showing Log2 FC for ERS UP , DOWN or unchanged genes in sepsisBIM vs Control mouse liver ( 6 mice per group ) . Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . One - way ANOVA with Welch ' s correction Dunnett ' s Modified Tukey - Kramer pairwise multiple comparison was used to assess statistical significance , * P < 0 . 05 .

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(E) Box plots showing Log2 FC for ERS UP, DOWN or unchanged genes in sepsisBIM vs Control mouse liver (6 mice per group). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-way ANOVA with Welch's correction and Dunnett's Modified Tukey-Kramer pairwise multiple comparison test was used to assess statistical significance, *P < 0.05.


( F ) RT - qPCR analyses of selected ERS UP genes LIVER - ID TFs monitoring expression changes in liver of sepsisBIM vs Control mice ( 6 mice per group ) . bar graph shows means ± SD ( standard deviations ) . One - sample t - with BH correction for multiple testing was used to determine if mean Log2 FC sepsisBIM / Control is statistically different from 0 , * P < 0 . 05 .

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(F) RT-qPCR analyses of selected ERS UP genes and LIVER-ID TFs monitoring expression changes in the liver of sepsisBIM vs Control mice (6 mice per group). The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC sepsisBIM/Control is statistically different from 0, *P < 0.05.


( G ) extracts from livers of control or sepsisBIM mice were subjected to Western blot or Simple Western immunoassay with antibodies against HNF4A , NR1H4 or FOXA2 . LMNA was used as loading control .

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(G) Nuclear extracts from livers of control or sepsisBIM mice were subjected to Western blot or Simple Western immunoassay with antibodies against HNF4A, NR1H4 or FOXA2. LMNA was used as loading control.


( H ) extracts from livers of sepsisBIM mice pre - treated for 4 consecutive days with vehicle or 500 mpk TUDCA were subjected to Western blot with antibodies against HNF4A , NR1H4 , FOXA2 or XBP1S . TFIIB was used as loading control . ( I ) Densitometric of expression data from 6 mice precondition shown in panel ( H ) ( 3 mice per condition ) bar graph shows means ± SD ( standard deviations ) . One - sample t - with BH correction for multiple testing was used to determine if mean Log2 FC TUDCA / Vehicle is statistically different from 0 , * P < 0 . 05 .

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

(H) Nuclear extracts from livers of sepsisBIM mice pre-treated for 4 consecutive days with vehicle or 500 mpk TUDCA were subjected to Western blot with antibodies against HNF4A, NR1H4, FOXA2 or XBP1S. TFIIB was used as loading control. (I) Densitometric quantification of the protein expression data from 6 mice precondition shown in panel (H) (3 mice per condition) The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC TUDCA/Vehicle is statistically different from 0, *P < 0.05.


( J ) Serum alanine aminotransferase ( ALT ) ( left ) aspartate aminotransferase ( AST ) activities ( right ) from sepsisBIM mice pre - treated for 4 consecutive days with vehicle or 500 mpk TUDCA ( 10 mice per group ) . bar graphs show means ± SD ( standard deviations ) . Student ' s t - was used to assess statistical significance , * P < 0 . 05 .

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

(J) Serum alanine aminotransferase (ALT) (left) and aspartate aminotransferase (AST) activities (right) from sepsisBIM mice pre-treated for 4 consecutive days with vehicle or 500 mpk TUDCA (10 mice per group). The bar graphs show means ± SD (standard deviations). Student's t-test was used to assess statistical significance, *P < 0.05.


( A ) RT - qPCR analyses of selected ERS UP genes LIVER - ID TFs in livers from sepsisCLP mice collected 10h , 30h or 3 days after CLP ( 15 mice per group ) vs livers from healthy pair - fed mice ( Control ) ( 15 mice per group ) . bar graph shows means ± SD ( standard deviations ) . Wilcoxon with BH correction for multiple testing was used to assess statistical significance , * P < 0 . 05 .

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

(A) RT-qPCR analyses of selected ERS UP genes and LIVER-ID TFs in livers from sepsisCLP mice collected 10h, 30h or 3 days after CLP (15 mice per group) vs livers from healthy pair-fed mice (Control) (15 mice per group). The bar graph shows means ± SD (standard deviations). Wilcoxon test with BH correction for multiple testing was used to assess statistical significance, *P < 0.05.


( B ) Box plots showing Log2 FC ERS / Control in mouse liver for ERS UP genes 8h 34h after tunicamycin injection in WT or ATF6 KO mice ( 3 mice per experimental condition ) . Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . One - sample t - with BH correction for multiple testing was used to determine if mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 . NS , not significant . ( C ) Similar analyses to panel ( B ) for ERS DOWN LIVER - ID genes ( 3 mice per experimental condition ) . Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . One - sample t - with BH correction for multiple testing was used to determine if mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Box plots showing Log2 FC ERS/Control in mouse liver for ERS UP genes 8h and 34h after tunicamycin injection in WT or ATF6 KO mice (3 mice per experimental condition). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05. NS, not significant. (C) Similar analyses to panel (B) for ERS DOWN and LIVER-ID genes (3 mice per experimental condition). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.


( D ) Main observations from ( Huck et al . , 2019 ) ( left ) box plots showing Log2 FC HNF4A KO / Control in mouse liver ( 3 mice per group ) for LIVER - ID genes TFs 5 days after PHx ( right ) . Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . One - sample t - with BH correction for multiple testing was used to determine if mean Log2 FC HNF4A KO / Control is statistically different from 0 , * P < 0 . 05 .

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

(D) Main observations from (Huck et al., 2019) (left) and box plots showing Log2 FC HNF4A KO/Control in mouse liver (3 mice per group) for LIVER-ID genes and TFs 5 days after PHx (right). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC HNF4A KO/Control is statistically different from 0, *P < 0.05.


( F ) Expression of LIVER - ID TF encoding genes was analyzed in livers of deceased critically - ill with sepsis displaying agonal bilirubin below ( Bil < 2 ; n = 34 ) or above ( Bil > 2 ; n = 28 ) 2mg / dL . Median fold - change expression level of each group for different genes has been used to generate box plots . Expression in critically - ill groups are expressed relative to those in control group ( n = 18 ) . Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . One - t - was used to assess whether expression of LIVER - ID TF encoding genes in Bil > 2 group is significantly greater than in Bil < 2 group , * P < 0 . 05 .

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

(F) Expression of LIVER-ID TF encoding genes was analyzed in the livers of deceased critically-ill patients with sepsis displaying agonal bilirubin levels below (Bil<2; n=34) or above (Bil>2; n=28) 2mg/dL. Median fold-change expression level of each group for the different genes has been used to generate the box plots. Expression levels in the critically-ill groups are expressed relative to those in the control group (n=18). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-tailed t-test was used to assess whether expression of LIVER-ID TF encoding genes in the Bil>2 group is significantly greater than in the Bil<2 group, *P < 0.05.


( G ) RT - qPCR analyses of indicated LIVER - ID TF encoding genes monitoring expression in livers of Bil < 2 ( n = 34 ) or Bil > 2 ( n = 28 ) groups of deceased critically - ill with sepsis vs control donors ( n = 18 ) . Data are shown as box plots , with mRNA of critically - ill groups expressed relative to those of control group . Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . Wilcoxon was used to assess statistically significant differences with Bil > 2 group , * P < 0 . 05 .

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

(G) RT-qPCR analyses of indicated LIVER-ID TF encoding genes monitoring expression in the livers of Bil<2 (n=34) or Bil>2 (n=28) groups of deceased critically-ill patients with sepsis vs control donors (n=18). Data are shown as box plots, with mRNA levels of the critically-ill groups expressed relative to those of the control group. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. Wilcoxon test was used to assess statistically significant differences with the Bil>2 group, *P < 0.05.


( H ) RT - qPCR analyses of indicated ERS UP genes performed Box plots are composed of a box from 25th to 75th percentile with median as a min to max as whiskers . Wilcoxon was used to assess statistically significant differences with Bil > 2 group , * P < 0 . 05 .

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

(H) RT-qPCR analyses of indicated ERS UP genes performed Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. Wilcoxon test was used to assess statistically significant differences with the Bil>2 group, *P < 0.05.


( A - B ) Human fibroblasts ( BJ ) were treated with vehicle ( water for 8 times 24 hours ) or abemaciclib ( 1 μM for 8 times 24 hours ) . population doubling over 8 days treatment is plotted ( A ) . 1 or 8 dpt , were incubated with EdU for 20 hours stained quantified ( n = 9 samples from 3 independent experiments ; scale bar , 150 μm ) ( B ) . Data information : Data are means ± SD . Unpaired student t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . dpt , days post treatment .

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

(A-B) Human fibroblasts (BJ) were treated with vehicle (water for 8 times 24 hours) or abemaciclib (1 μM for 8 times 24 hours). The population doubling over the 8 days treatment is plotted (A). 1 or 8 dpt, cells were incubated with EdU for 20 hours and stained and quantified (n=9 samples from 3 independent experiments; scale bar, 150 μm) (B). Data information: Data are means ±SD. Unpaired student t-test *p<0.05, **p<0.01, ***p<0.001. dpt, days post treatment.


( C ) was extracted from BJ treated with vehicle or abemaciclib ( 1 μM for 8 times 24 hours ) or nutlin - 3a ( 10 μM for 8 times 24 hours ) or doxorubicin ( 250 nM for 24 hours ) ChIP assays using an antibody against p53 were performed . qRT - PCR was performed using primers amplifying promoter regions of CDKN1A , GADD45A MDM2 genes containing p53 binding sites . Values indicate fold enrichment relative to vehicle group ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(C) Chromatin was extracted from BJ cells treated with vehicle or abemaciclib (1 μM for 8 times 24 hours) or nutlin-3a (10 μM for 8 times 24 hours) or doxorubicin (250 nM for 24 hours) and ChIP assays using an antibody against p53 were performed. qRT-PCR was performed using primers amplifying promoter regions of CDKN1A, GADD45A and MDM2 genes containing p53 binding sites. Values indicate fold enrichment relative to the vehicle group (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


( E ) 3 * 103 BJ of indicated genotypes were re - plated after vehicle or abemaciclib treatment ( 1 μM ) stained with 0 . 2 % crystal violet 8 dpt ( n = 3 independent experiments ) . ( F ) 8 dpt , treated BJ were incubated with EdU for 20 hours stained ( n = 9 samples from 3 independent experiments ) . ( Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(E) 3*103 BJ cells of the indicated genotypes were re-plated after vehicle or abemaciclib treatment (1 μM) and stained with 0.2% crystal violet 8 dpt (n=3 independent experiments). (F) 8 dpt, treated BJ cells were incubated with EdU for 20 hours and stained (n=9 samples from 3 independent experiments). ( Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


( G ) 3 * 103 mouse dermal fibroblasts ( MDFs ) of indicated genotypes were re - plated after vehicle or abemaciclib treatment ( 4 μM for 8 times 24 hours ) stained with 0 . 2 % crystal violet 8 dpt ( n = 3 independent experiments ) . ( H ) 8 dpt , treated MDFs were incubated with EdU for 20 hours stained ( n = 9 samples from 3 independent experiments ) . ( Data information : Data are means ± SD . Unpaired student t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(G) 3*103 mouse dermal fibroblasts (MDFs) of the indicated genotypes were re-plated after vehicle or abemaciclib treatment (4 μM for 8 times 24 hours) and stained with 0.2% crystal violet 8 dpt (n=3 independent experiments). (H) 8 dpt, treated MDFs were incubated with EdU for 20 hours and stained (n=9 samples from 3 independent experiments). ( Data information: Data are means ±SD. Unpaired student t-test *p<0.05, **p<0.01, ***p<0.001.


( I ) was isolated from vehicle or abemaciclib ( 1 μM ) treated scramble / shp53 BJ mRNA for indicated genes quantified by qRT - PCR relative to tubulin ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(I) RNA was isolated from vehicle or abemaciclib (1 μM) treated scramble/shp53 BJ cells and mRNA for the indicated genes quantified by qRT-PCR relative to tubulin (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


p16 - 3MR mice were treated with vehicle ( PBS , 7 consecutive days ) , doxorubicin ( 5 mg / kg , 3 consecutive days ) or abemaciclib ( 50 mg / kg , 7 consecutive days ) . n = 6 mice / group . 14 dpt , bioluminescence was visualized quantified by IVIS spectrum in vivo imaging system , as shown by representative bioluminescence ( A ) ( B ) . Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant .

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

p16-3MR mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days) or abemaciclib (50 mg/kg, 7 consecutive days). n=6 mice/group. 14 dpt, bioluminescence was visualized and quantified by the IVIS spectrum in vivo imaging system, as shown by representative bioluminescence images (A) and quantification (B). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.


p16 - 3MR mice were treated with vehicle ( PBS , 7 consecutive days ) , doxorubicin ( 5 mg / kg , 3 consecutive days ) or abemaciclib ( 50 mg / kg , 7 consecutive days ) . n = 6 mice / group . isolated from kidneys mRNA encoding p16 quantified by qRT - PCR ( C ) . SA - β - gal activities in vehicle - , doxorubicin - or abemaciclib - treated mouse kidney sections at 15 dpt quantified ( E ) . Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant .

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

p16-3MR mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days) or abemaciclib (50 mg/kg, 7 consecutive days). n=6 mice/group. RNA isolated from kidneys and mRNA encoding p16 quantified by qRT-PCR (C). SA-β-gal activities in vehicle-, doxorubicin- or abemaciclib-treated mouse kidney sections at 15 dpt quantified (E). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.


( G ) was isolated from abemaciclib ± pifithrin - α treated kidneys mRNA encoding p16 p21 genes was quantified by qRT - PCR normalized on tubulin ( n = 5 mice / group ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant .

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

(G) RNA was isolated from abemaciclib ± pifithrin-α treated kidneys and mRNA encoding p16 and p21 genes was quantified by qRT-PCR and normalized on tubulin (n=5 mice/group). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.


For doxorubicin or abemaciclib treated mice ( n = 6 mice / group ) , of T , B , granulocytes macrophages were determined by flow cytometry ( N ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant .

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

For the doxorubicin or abemaciclib treated mice (n=6 mice/group), Percentage of T cells, B cells, granulocytes and macrophages were determined by flow cytometry analysis (N). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.


( A ) 8 dpt , were stained for γ - H2AX ( scale bar , 60 μm ) quantified ( vehicle = 288 , doxorubicin = 280 , palbociclib = 276 , abemaciclib = 266 from 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = Not significant . Doxo , doxorubicin . Abe , abemaciclib . dpt , days post treatment .

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

(A) 8 dpt, cells were stained for γ-H2AX (scale bar, 60 μm) and quantified (vehicle=288 cells, doxorubicin=280 cells, palbociclib=276 cells, abemaciclib=266 cells from 3 independent experiments). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.= Not significant. Doxo, doxorubicin. Abe, abemaciclib. dpt, days post treatment.


( B ) BJ transduced with a NF - κB reporter were treated with vehicle , doxorubicin , paclitaxel ( 50 nM for 24 hours ) , palbociclib ( 1 μM for 8 times 24 hours ) or abemaciclib luciferase activity measured 8 dpt . TNFα treatment was used as positive control ( n = 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = Not significant .

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

(B) BJ cells transduced with a NF-κB reporter were treated with vehicle, doxorubicin, paclitaxel (50 nM for 24 hours), palbociclib (1 μM for 8 times 24 hours) or abemaciclib and luciferase activity measured 8 dpt. TNFα treatment was used as positive control (n=3 independent experiments). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.= Not significant.


( E ) 8 dpt , qRT - PCR of indicated NF - κB associated SASP genes was performed using indicated treated BJ 8 dpt ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = Not significant .

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

(E) 8 dpt, qRT-PCR of indicated NF-κB associated SASP genes was performed using indicated treated BJ cells 8 dpt (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.= Not significant.


( G ) in serum - free conditioned media ( CM ) collected from doxorubicin - or abemaciclib - treated BJ fibroblasts ( 8 dpt ) were measured by cytokine array . Purple boxes indicated internal positive controls from each ; Red boxes highlighted most typical SASP factors .

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

(G) Proteins in the serum-free conditioned media (CM) collected from doxorubicin- or abemaciclib-treated BJ fibroblasts (8 dpt) were measured by cytokine array. Purple boxes indicated internal positive controls from each membrane; Red boxes highlighted the most typical SASP factors.


p16 - 3MR mice were treated with vehicle ( PBS , 7 consecutive days ) , doxorubicin ( 5 mg / kg , 3 consecutive days ) or abemaciclib ( 50 mg / kg , 7 consecutive days ) . isolated from kidneys mRNA encoding indicated NF - κB associated genes ( J ) quantified by qRT - PCR . n = 6 mice / group . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = Not significant .

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

p16-3MR mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days) or abemaciclib (50 mg/kg, 7 consecutive days). RNA isolated from kidneys and mRNA encoding indicated NF-κB associated genes (J) quantified by qRT-PCR. n=6 mice/group. Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.= Not significant.


p16 - 3MR mice were treated with vehicle ( PBS , 7 consecutive days ) , doxorubicin ( 5 mg / kg , 3 consecutive days ) or abemaciclib ( 50 mg / kg , 7 consecutive days ) . From a subset of mice , RFP + were sorted from renal cortex using FACS mRNA encoding indicated NF - κB associated pro - inflammatory genes quantified by qRT - PCR . values of fold change over vehicle were plotted in . n = 4 mice / group ( K ) .

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p16-3MR mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days) or abemaciclib (50 mg/kg, 7 consecutive days). From a subset of the mice, RFP+ cells were sorted from the renal cortex using FACS and mRNA encoding indicated NF-κB associated pro-inflammatory genes quantified by qRT-PCR. The values of fold change over vehicle were plotted in the heatmap. n=4 mice/group (K).


( B ) Human BJ fibroblasts , hTERT - RPE1 epithelial lung mesenchymal were treated with vehicle ( DMSO for 8 times 24 hours ) or doxorubicin ( 250 nM for 24 hours ) or paclitaxel ( 50 nM for 24 hours ) or palbociclib ( 1 μM for 8 times 24 hours ) or abemaciclib ( 1 μM for 8 times 24 hours ) . At 8 dpt , was isolated mRNA for indicated p53 - associated SASP genes quantified by qRT - PCR relative to tubulin ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(B) Human BJ fibroblasts, hTERT-RPE1 epithelial cells and lung mesenchymal stem cells were treated with vehicle (DMSO for 8 times 24 hours) or doxorubicin (250 nM for 24 hours) or paclitaxel (50 nM for 24 hours) or palbociclib (1 μM for 8 times 24 hours) or abemaciclib (1 μM for 8 times 24 hours). At 8 dpt, RNA was isolated and mRNA for the indicated p53-associated SASP genes quantified by qRT-PCR relative to tubulin (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


( D E ) was isolated from vehicle , abemaciclib ( 1 μM ) ( D ) or palbociclib ( 1 μM ) ( E ) treated scramble / shp53 BJ quantified by qRT - PCR for IGFBP3 or LIF genes ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(D and E) RNA was isolated from vehicle, abemaciclib (1 μM) (D) or palbociclib (1 μM) (E) treated scramble/shp53 BJ cells and quantified by qRT-PCR for IGFBP3 or LIF genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


( F ) isolated from kidneys treated with doxorubicin or abemaciclib mRNA encoding indicated p53 - associated SASP genes quantified by qRT - PCR ( n = 6 mice / group ) . ( G ) was isolated from kidneys of p16 - 3MR mice treated with abemaciclib ( 50 mg / kg , 7 consecutive days ) ± pifithrin - α ( 2 mg / kg , 7 consecutive days ) , mRNA encoding p53 associated SASP genes was quantified by qRT - PCR normalized on tubulin ( n = 5 mice / group ) . ( Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(F) RNA isolated from kidneys treated with doxorubicin or abemaciclib and mRNA encoding indicated p53-associated SASP genes quantified by qRT-PCR (n=6 mice/group). (G) RNA was isolated from kidneys of p16-3MR mice treated with abemaciclib (50 mg/kg, 7 consecutive days) ± pifithrin-α (2 mg/kg, 7 consecutive days), and mRNA encoding p53 associated SASP genes was quantified by qRT-PCR and normalized on tubulin (n=5 mice/group). ( Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


( I ) was isolated from vehicle or nutlin - 3a ( 1 μM for 8 times 24 hours ) treated BJ at 8 dpt quantified by qRT - PCR for p53 target cycle genes , SASP genes ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(I) RNA was isolated from vehicle or nutlin-3a (1 μM for 8 times 24 hours) treated BJ cells at 8 dpt and quantified by qRT-PCR for p53 target cell cycle genes, SASP genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.


( J ) Mouse dermal fibroblasts ( MDF ) or MDF - p53 - / - fibroblasts were transfected with empty vector ( EV ) or p53 - overexpressing vector ( p53OE ) , then were treated with vehicle ( water ) or doxorubicin ( 250 nM for 24 hours ) qRT - PCR was performed for Igfbp3 Lif genes ( n = 3 independent experiments ) . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . EV , empty

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(J) Mouse dermal fibroblasts (MDF) or MDF-p53-/- fibroblasts were transfected with empty vector (EV) or p53-overexpressing vector (p53OE), then the cells were treated with vehicle (water) or doxorubicin (250 nM for 24 hours) and qRT-PCR was performed for Igfbp3 and Lif genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001. EV, empty vector


( A ) MCF7 were incubated with serum - free CM collected from treated BJ fibroblasts ( 8 dpt ) containing EdU for 45 hours EdU + quantified ( n = 9 samples from 3 independent experiments ) . Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant . CM , conditioned media .

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(A) MCF7 cells were incubated with serum-free CM collected from treated BJ fibroblasts (8 dpt) containing EdU for 45 hours and EdU+ cells quantified (n=9 samples from 3 independent experiments). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant. CM, conditioned media.


( B ) MCF7 migrated through pores of trans - well after 24 hours incubation with CM ( 8 dpt ; scale bar , 1 mm ; n = 9 samples from 3 independent experiments ) were stained with 0 . 2 % crystal violet quantified . Data information : Data are means ± SD . One - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant . CM , conditioned media .

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(B) MCF7 cells migrated through the pores of trans-well after 24 hours incubation with CM (8 dpt; scale bar, 1 mm; n=9 samples from 3 independent experiments) were stained with 0.2% crystal violet and quantified. Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant. CM, conditioned media.


( H I ) 2 * 105 of vehicle - treated ( water for 8 times 24 hours ) mouse dermal fibroblasts ( MDF ) were injected into left flank of p16 - 3MR mice . Doxorubicin - induced ( 250 nM for 24 hours ) ( H ) or abemaciclib - induced ( 4 μM for 8 times 24 hours ) ( I ) MDFs were injected into right flank of same . 7 dpt , above - mentioned mice were injected with coelenterazine bioluminescence from p16 - 3MR mouse was visualized / quantified by IVIS spectrum in vivo imaging system quantified . Lower panel , scheme of experimental design ( n = 4 mice / group ) . Data information : Data are means ± SD . Paired student t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant .

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(H and I) 2*105 cells of vehicle-treated (water for 8 times 24 hours) mouse dermal fibroblasts (MDF) were injected into the left flank of the p16-3MR mice. Doxorubicin-induced (250 nM for 24 hours) (H) or abemaciclib-induced (4 μM for 8 times 24 hours) (I) MDFs were injected into the right flank of the same animal. 7 dpt, the above-mentioned mice were injected with coelenterazine and bioluminescence from the p16-3MR mouse was visualized/quantified by the IVIS spectrum in vivo imaging system and quantified. Lower panel, scheme of experimental design (n=4 mice/group). Data information: Data are means ±SD. Paired student t-test *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.


( J ) Equal amount ( 2 * 105 ) of doxorubicin - induced ( 250 nM for 24 hours , right flank ) abemaciclib - induced ( 4 μM for 8 times 24 hours , left flank ) senescent MDF - 3MR were subcutaneously injected into same wild - type mice . At 1 dpt , 7 dpt 15 dpt , above - mentioned mice were injected with coelenterazine bioluminescence from injected MDF - 3MR was visualized / quantified by IVIS spectrum in vivo imaging system quantified ( n = 4 mice / group ) . Upper panel , scheme of experimental design . Data information : Data are means ± SD . Two - way ANOVA * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , N . S . = not significant .

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(J) Equal amount (2*105 cells) of doxorubicin-induced (250 nM for 24 hours, right flank) and abemaciclib-induced (4 μM for 8 times 24 hours, left flank) senescent MDF-3MR cells were subcutaneously injected into the same wild-type mice. At 1 dpt, 7 dpt and 15 dpt, the above-mentioned mice were injected with coelenterazine and bioluminescence from the injected MDF-3MR cells was visualized/quantified by the IVIS spectrum in vivo imaging system and quantified (n=4 mice/group). Upper panel, scheme of experimental design. Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.


A , Seedlings of WT , abs3 - 1D , FD11 - 55 , arf2 - 20 before after 6 d C - deprivation . B , Seedlings of WT , abs3 - 1D , FD18 - 70 , pif5 - 10 before after 6 d C - deprivation .

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A, Seedlings of WT, abs3-1D, FD11-55, and arf2-20 before and after 6 d C-deprivation. B, Seedlings of WT, abs3-1D, FD18-70, and pif5-10 before and after 6 d C-deprivation.


C D , RT - qPCR of ABS3 expression in 7 - d - old seedlings of same genotypes as shown in A ( C ) B ( D ) . Fold changes were calculated with respect to expression level in WT . two - t , * * * * , p < 0 . 0001 , n . s . , not significant . data are means ± standard deviation ( s . d . ) of 3 biological replicates .

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C and D, RT-qPCR analysis of ABS3 expression levels in 7-d-old seedlings of the same genotypes as shown in A (C) and B (D). Fold changes were calculated with respect to the expression level in the WT. two-tailed t test, ****, p<0.0001, n.s., not significant. data are means ± standard deviation (s.d.) of 3 biological replicates.


E F , Chlorophyll total content reduction in genotypes shown in A ( E ) B ( F ) after 6 d C - deprivation . data are means ± standard deviation ( s . d . ) of 3 biological replicates .

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E and F, Chlorophyll and total cellular protein content reduction in genotypes shown in A (E) and B (F) after 6 d C-deprivation. data are means ± standard deviation (s.d.) of 3 biological replicates.


G H , RT - qPCR of ORE1 SGR1 expression in genotypes shown in A ( G ) B ( H ) before after 4 d C - deprivation . Fold changes were calculated with respect to expression level in WT before C - deprivation . data are means ± standard deviation ( s . d . ) of 3 biological replicates .

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G and H, RT-qPCR analysis of ORE1 and SGR1 expression levels in genotypes shown in A (G) and B (H) before and after 4 d C-deprivation. Fold changes were calculated with respect to the expression level in the WT before C-deprivation. data are means ± standard deviation (s.d.) of 3 biological replicates.


C , of log2FC values of 2 , 587 ABS3 - regulated genes ( abs3 - 1D vs . WT ) under C - deprivation in indicated comparisons .

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C, Heatmap of log2FC values of 2,587 ABS3-regulated genes (abs3-1D vs. WT) under C-deprivation in indicated comparisons.


F , of log2FC values of 1 , 326 ARF2 , PIF5 , C - deprivation co - regulated genes in indicated comparisons . C - deprivation : abs3 - 1D 4 d C - deprivation vs . abs3 - 1D 0 d C - deprivation ; ARF2 : abs3 - 1D vs . arf2 - 20 abs3 - 1D after 4 d C - deprivation ; PIF5 : abs3 - 1D vs . abs3 - 1D pif5 - 10 after 4 d C - deprivation . Enlarged were expression changes of senescence genes identified in DAVID GO

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F, Heatmap of log2FC values of 1,326 ARF2, PIF5, and C-deprivation co-regulated genes in indicated comparisons. C-deprivation: abs3-1D 4 d C-deprivation vs. abs3-1D 0 d C-deprivation; ARF2: abs3-1D vs. arf2-20 abs3-1D after 4 d C-deprivation; PIF5: abs3-1D vs. abs3-1D pif5-10 after 4 d C-deprivation. Enlarged were expression changes of senescence genes identified in DAVID GO analysis


A , Interaction between ARF2 PIF5 in yeast two - hybrid . Growth of yeast co - transformed with indicated BD AD vectors was monitored on quadruple dropout medium ( QDO , SD - Ade / - His / - Leu / - Trp ) QDO medium containing X - α - gal .

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A, Interaction between ARF2 and PIF5 in the yeast two-hybrid assay. Growth of yeast cells co-transformed with indicated BD and AD vectors was monitored on quadruple dropout medium (QDO, SD-Ade/-His/-Leu/-Trp) and QDO medium containing X-α-gal.


B , Interaction between ARF2 PIF5 in protoplast BiFC assays . YN - ARF2 PIF5 - YC were coexpressed with marker NLS - mCherry in Arabidopsis mesophyll protoplasts . Coexpression of YN - ARF2 YC alone or YN alone with PIF5 - YC served as negative controls . Bars , 10 μm .

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B, Interaction between ARF2 and PIF5 in protoplast BiFC assays. YN-ARF2 and PIF5-YC were coexpressed with the nuclear marker NLS-mCherry in Arabidopsis mesophyll protoplasts. Coexpression of YN-ARF2 and YC alone or YN alone with PIF5-YC served as negative controls. Bars, 10 μm.


C , Direct interaction between GST - ARF2 MBP - PIF5 - His in in vitro GST pull - down assays .

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C, Direct interaction between GST-ARF2 and MBP-PIF5-His in in vitro GST pull-down assays.


D , Co - immunoprecipitation of ARF2 - HA with PIF5 - GFP in vivo . ARF2 - HA was coexpressed with PIF5 - GFP or GFP alone in protoplasts . co - immunoprecipitated by GFP - Trap beads were immunoblotted with anti - GFP anti - HA antibodies .

D, Co-immunoprecipitation of ARF2-HA with PIF5-GFP in vivo. ARF2-HA was coexpressed with PIF5-GFP or GFP alone in protoplasts. Proteins co-immunoprecipitated by GFP-Trap beads were immunoblotted with anti-GFP and anti-HA antibodies.


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A, Seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, pif4-2 pif5-10, and pARF2:ARF2-GFP OEs in the pif4-2 pif5-10 background before and after 6 d C-deprivation.


B , RT - qPCR of ARF2 expression in 7 - d - old seedlings of same genotypes as shown in A . Fold changes were calculated with respect to expression level in WT . data are means ± s . d . of 3 biological replicates .

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B, RT-qPCR analysis of ARF2 expression levels in 7-d-old seedlings of the same genotypes as shown in A. Fold changes were calculated with respect to the expression level in the WT. data are means ± s.d. of 3 biological replicates.


C , Chlorophyll total content reduction in genotypes shown in A after 6 d C - deprivation . Color legends in C are same as in B . data are means ± s . d . of 3 biological replicates .

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C, Chlorophyll and total cellular protein content reduction in genotypes shown in A after 6 d C-deprivation. Color legends in C are the same as in B. data are means ± s.d. of 3 biological replicates.


D , Seedlings of WT , pPIF5 : PIF5 - GFP OEs in WT background , arf2 - 20 , pPIF5 : PIF5 - GFP OEs in arf2 - 20 background before after 6 d C - deprivation .

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D, Seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, arf2-20, and pPIF5:PIF5-GFP OEs in the arf2-20 background before and after 6 d C-deprivation.