( 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 .

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

(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 .

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

(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 % .

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

(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 .

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

(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 ) .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 .

B-MEASURED_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 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 .

B-MEASURED_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 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 B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 ) .

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

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 .

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

(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 .

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

(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 .

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

(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 .

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

(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

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

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 .

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

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 .

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

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 .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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

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

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

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 .

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

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 .

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

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 .

O O O O O O B-MEASURED_VAR O O O B-MEASURED_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

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.


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

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 .

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

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 .

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

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 .

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

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.


E , RT - qPCR of PIF5 expression in 7 - d - old seedlings of same genotypes as shown in D . Fold changes were calculated with respect to expression level in WT . data are means ± s . d . of 3 biological replicates .

E, RT-qPCR analysis of PIF5 expression levels in 7-d-old seedlings of the same genotypes as shown in D. Fold changes were calculated with respect to the expression level in the WT. data are means ± s.d. of 3 biological replicates.


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

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


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

G, Seedlings of WT, arf2-20, pif5-10, and arf2-20 pif5-10 before and after 12 d C-deprivation.


H , Chlorophyll total content reduction in genotypes shown in G after 12 d C - deprivation . data are means ± s . d . of 3 biological replicates .

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

H, Chlorophyll and total cellular protein content reduction in genotypes shown in G after 12 d C-deprivation. data are means ± s.d. of 3 biological replicates.


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

I, Seedlings of WT, abs3-1D, arf2-20 abs3-1D, pif5-10 abs3-1D, and arf2-20 pif5-10 abs3-1D before and after 8 d C-deprivation.


J , Chlorophyll total content reduction in genotypes shown in I after 8 d C - deprivation . data are means ± s . d . of 3 biological replicates .

J, Chlorophyll and total cellular protein content reduction in genotypes shown in I after 8 d C-deprivation. data are means ± s.d. of 3 biological replicates.


C , ChIP - qPCR of ARF2 - GFP binding to promoters of ORE1 SGR1 in WT pif4 - 2 pif5 - 10 backgrounds . In C fold enrichments were calculated with respect to input . PP2A served as a non - binding control . Data are means ± s . d . of 3 technical replicates . two - t , n . s . , not significant , * * * * , p < 0 . 0001 . experiment has been independently repeated with similar results .

O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O O O O O O

C, ChIP-qPCR analysis of ARF2-GFP binding to the promoters of ORE1 and SGR1 in the WT and pif4-2 pif5-10 backgrounds. In C fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


D , RT - qPCR of ORE1 SGR1 expression in seedlings of WT , pARF2 : ARF2 - GFP OEs in WT background , pif4 - 2 pif5 - 10 , pARF2 : ARF2 - GFP OEs in pif4 - 2 pif5 - 10 background before after 4 d C - deprivation . Fold changes were calculated with respect to expression level in WT before C - deprivation . In D data are means ± s . d . of 3 biological replicates . Data are means ± s . d . of 3 technical replicates . two - t , n . s . , not significant , * * * * , p < 0 . 0001 . experiment has been independently repeated with similar results .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O 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 O O O O O 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, RT-qPCR analysis of ORE1 and SGR1 expression levels in 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 4 d C-deprivation. Fold changes were calculated with respect to the expression level in the WT before C-deprivation. In D data are means ± s.d. of 3 biological replicates. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


E , ChIP - qPCR of PIF5 - GFP binding to promoters of ORE1 SGR1 in WT arf2 - 20 backgrounds . In fold enrichments were calculated with respect to input . PP2A served as a non - binding control . Data are means ± s . d . of 3 technical replicates . two - t , n . s . , not significant , * * * * , p < 0 . 0001 . experiment has been independently repeated with similar results .

O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_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 O O O O O O O O O O

E, ChIP-qPCR analysis of PIF5-GFP binding to the promoters of ORE1 and SGR1 in the WT and arf2-20 backgrounds. In fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


F , RT - qPCR of ORE1 SGR1 expression in seedlings of WT , pPIF5 : PIF5 - GFP OEs in WT background , arf2 - 20 , pPIF5 : PIF5 - GFP OEs in arf2 - 20 background before after 4 d C - deprivation . Fold changes were calculated as in D . In F , data are means ± s . d . of 3 biological replicates . Data are means ± s . d . of 3 technical replicates . two - t , n . s . , not significant , * * * * , p < 0 . 0001 . experiment has been independently repeated with similar results .

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

F, RT-qPCR analysis of ORE1 and SGR1 expression levels in 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 4 d C-deprivation. Fold changes were calculated as in D. In F, data are means ± s.d. of 3 biological replicates. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


G , Hypocotyl elongation in seedlings of WT , pPIF5 : PIF5 - GFP OEs in WT background , arf2 - 20 , pPIF5 : PIF5 - GFP OEs in arf2 - 20 background . Seedlings were grown under continuous for one week .

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

G, Hypocotyl elongation in seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, arf2-20, and pPIF5:PIF5-GFP OEs in the arf2-20 background. Seedlings were grown under continuous light for one week.


H , RT - qPCR of PIL1 YUC8 expression in seedlings shown in G . Fold changes were calculated with respect to expression level in WT . Color legends in H are same as in F . In H , data are means ± s . d . of 3 biological replicates . Data are means ± s . d . of 3 technical replicates . two - t , n . s . , not significant , * * * * , p < 0 . 0001 . experiment has been independently repeated with similar results .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H, RT-qPCR analysis of PIL1 and YUC8 expression levels in seedlings shown in G. Fold changes were calculated with respect to the expression level in the WT. Color legends in H are the same as in F. In H, data are means ± s.d. of 3 biological replicates. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


J , ChIP - qPCR of PIF5 binding to promoters of PIL1 YUC8 in WT arf2 - 20 backgrounds . In J , fold enrichments were calculated with respect to input . PP2A served as a non - binding control . Data are means ± s . d . of 3 technical replicates . two - t , n . s . , not significant , * * * * , p < 0 . 0001 . experiment has been independently repeated with similar results .

O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_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 O O O O O O O O O O O O

J, ChIP-qPCR analysis of PIF5 binding to the promoters of PIL1 and YUC8 in the WT and arf2-20 backgrounds. In J, fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 3 technical replicates. two-tailed t test, n.s., not significant, ****, p<0.0001. The experiment has been independently repeated with similar results.


A , RT - qPCR of ARF2 expression in 7 - d - old seedlings of WT , pARF2 : ARF2 - GFP OEs in WT background , mateq , pARF2 : ARF2 - GFP OEs in mateq background . Fold changes were calculated with respect to expression level in WT . B , RT - qPCR of PIF5 expression in 7 - d - old seedlings of WT , pPIF5 : PIF5 - GFP OEs in WT background , mateq , pPIF5 : PIF5 - GFP OEs in mateq background . Fold changes were calculated as in A . In A , B , data are means ± s . d . of 3 biological replicates .

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

A, RT-qPCR analysis of ARF2 expression levels in 7-d-old seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, mateq, and pARF2:ARF2-GFP OEs in the mateq background. Fold changes were calculated with respect to the expression level in the WT. B, RT-qPCR analysis of PIF5 expression levels in 7-d-old seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, mateq, and pPIF5:PIF5-GFP OEs in the mateq background. Fold changes were calculated as in A. In A, B, data are means ± s.d. of 3 biological replicates.


C , Seedlings of same genotypes as in A before after 6 d 9 d C - deprivation . D , Seedlings of same genotypes as in B before after 6 d 11 d C - deprivation .

O O O O O O O O O O O O O O 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, Seedlings of the same genotypes as in A before and after 6 d and 9 d C-deprivation. D, Seedlings of the same genotypes as in B before and after 6 d and 11 d C-deprivation.


E F , Chlorophyll total content reduction in genotypes shown in A ( E ) after 6 d 9 d C - deprivation in genotypes shown in B ( F ) after 6 d 11 d C - deprivation . Color legends in E are same as in A . Color legends in F are same as in B . data are means ± s . d . of 3 biological replicates .

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

E and F, Chlorophyll and total cellular protein content reduction in genotypes shown in A (E) after 6 d and 9 d C-deprivation and in genotypes shown in B (F) after 6 d and 11 d C-deprivation. Color legends in E are the same as in A. Color legends in F are the same as in B. data are means ± 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 . Color legends in G are same as in A . Color legends in are same as in B . data are means ± s . d . of 3 biological replicates .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. Color legends in G are the same as in A. Color legends in are the same as in B. data are means ± s.d. of 3 biological replicates.


I , RT - qPCR of PIL1 YUC8 expression in 7 - d - old seedlings of same genotypes as shown in B . Fold changes were calculated as in A . Color legends in I are same as in B . data are means ± s . d . of 3 biological replicates .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I, RT-qPCR analysis of PIL1 and YUC8 expression levels in 7-d-old seedlings of the same genotypes as shown in B. Fold changes were calculated as in A. Color legends in I are the same as in B. data are means ± s.d. of 3 biological replicates.


B , ChIP - qPCR of ARF2 - GFP binding to promoters of ABS3 . Fold enrichments were calculated with respect to input . PP2A served as a non - binding control . Data are means ± s . d . of 4 technical replicates . experiment has been independently repeated 3 times with similar results .

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

B, ChIP-qPCR analysis of ARF2-GFP binding to the promoters of ABS3. Fold enrichments were calculated with respect to the input. PP2A served as a non-binding control. Data are means ± s.d. of 4 technical replicates. The experiment has been independently repeated 3 times with similar results.


C , pABS3 : GFP or pABS3 ∆ S2 : GFP was expressed in WT mesophyll protoplasts with or without p35S : ARF2 - HA . p35S : mCherry was co - transfected served as a transfection control . Protoplasts were examined with microscopy . Experiments were independently repeated 3 times with similar results . Bars , 100 μm . D , Dot plots of GFP relative in protoplasts expressing WT mutant forms of pABS3 : GFP with or without p35S : ARF2 - HA . Data are median with interquartile range , n is indicated in parentheses , two - Mann - Whitney , n . s . , not significant , * * * * , 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 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 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

C, pABS3:GFP or pABS3∆S2:GFP was expressed in WT mesophyll protoplasts with or without p35S:ARF2-HA. p35S:mCherry was co-transfected and served as a transfection control. Protoplasts were examined with fluorescence microscopy. Experiments were independently repeated 3 times with similar results. Bars, 100 μm. D, Dot plots of GFP relative fluorescence intensity in protoplasts expressing WT and mutant forms of pABS3:GFP with or without p35S:ARF2-HA. Data are median with interquartile range, n is indicated in parentheses, two-tailed Mann-Whitney test, n.s., not significant, ****, p<0.0001.


E , RT - qPCR of ABS3 expression in 7 - d - old seedlings of WT , pARF2 : ARF2 - GFP OEs in WT background , pif4 - 2 pif5 - 10 , pARF2 : ARF2 - GFP OEs in pif4 - 2 pif5 - 10 background . Fold changes were calculated with respect to expression level in WT . Data are means ± s . d . of 3 biological replicates .

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

E, RT-qPCR analysis of ABS3 expression levels in 7-d-old 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. Fold changes were calculated with respect to the expression level in the WT. Data are means ± s.d. of 3 biological replicates.


( A ) of wild type ( Col ) , kup9 mutants ( kup9 - 1 kup9 - 2 ) kup9 - 1 / ProKUP9 : KUP9 complementation lines ( COM1 COM2 ) . Seeds were germinated grown on low - K + ( LK , 50 μM ) or high - K + ( HK , 5 mM ) medium for 7 d . Scale bar , 1 cm .

O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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) Phenotypes of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on low-K+ (LK, 50 μM) or high-K+ (HK, 5 mM) medium for 7 d. Scale bar, 1 cm.


( B ) root of tested Data are means ± SE ( n = 25 , ) . Student ' s t - ( * * P < 0 . 01 ) was used to analyze statistical significance , " # " represents control .

O O O O O O O O O O O O O O O O O O O O O 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) Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents the control.


( D ) RT - qPCR of KUP9 transcript in various materials . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 120 ) .

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

(D) RT-qPCR analysis of KUP9 transcript levels in various materials. Data are means ± SE (n = 3, biological replicates; each replicate contains 120 individual plants).


( E ) ( F ) K + content of various materials tested in ( A ) . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 120 - 150 ) .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) and (F) K+ content of the various plant materials tested in (A). Data are means ± SE (n = 3, biological replicates; each replicate contains 120-150 individual plants).


( A ) Root meristem zones of wild type ( Col ) , kup9 mutants ( kup9 - 1 kup9 - 2 ) kup9 - 1 / ProKUP9 : KUP9 complementation lines ( COM1 COM2 ) . Seeds were germinated grown on LK or HK medium for 7 d . meristem zone lengths are marked with red lines . Scale bars , 50 μm .

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

(A) Root meristem zones of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on LK or HK medium for 7 d. The meristem zone lengths are marked with red lines. Scale bars, 50 μm.


( B ) , ( C ) ( D ) represent different zone lengths , different zone numbers , of indicated seedlings germinated grown on LK or HK medium for 7 d . MEZ , meristem zone ; EZ , elongation zone ; MAZ , maturation zone . Data are means ± SE ( n = 10 - 15 , ) . Student ' s t - ( * * P < 0 . 01 ) was used to analyze statistical significance , " # " represents control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) and (D) represent different zone lengths, different zone cell numbers, and cell length of the indicated seedlings germinated and grown on LK or HK medium for 7 d. MEZ, meristem zone; EZ, elongation zone; MAZ, maturation zone. Data are means ± SE (n = 10-15, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents the control.


( A ) to ( F ) Expression of KUP9 as determined in ProKUP9 : GUS . GUS of 5 - day - old seedling ( A ) , root maturation zone ( B ) , transverse section of maturation zone ( C ) ( D ) , root tip ( E ) , root apex ( F ) . Ep , epidermis ; Co , cortex ; En , endodermis ; Pe , pericyle ; Xy , xylem ; Ph , phloem . Scale bars , 5 mm in ( A ) , 1 mm in ( B ) , 10 μm in ( C ) , 5 μm in ( D ) , 1 mm in ( E ) , 20 μm in ( F ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) to (F) Expression of KUP9 as determined in the ProKUP9:GUS line. GUS staining of 5-day-old seedling (A), root maturation zone (B), transverse section of maturation zone (C) and (D), primary root tip (E), and root apex (F). Ep, epidermis; Co, cortex; En, endodermis; Pe, pericyle; Xy, xylem; Ph, phloem. Scale bars, 5 mm in (A), 1 mm in (B), 10 μm in (C), 5 μm in (D), 1 mm in (E), and 20 μm in (F).


( G ) ( H ) Expression of KUP9 as determined in kup9 - 1 / ProKUP9 : KUP9 - GFP transgenic . Green shows subcellular localization of KUP9 - GFP . PI ( propidium iodide ) ER tracker were used to indicate positions of plasma ( PM ) endoplasmic reticulum ( ER ) are shown as red cyan , respectively . Scale bars for root tip , 10 μm in ( G ) 5 μm in ( H ) . Scale bars for EZ MAZ , 20 μm .

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

(G) and (H) Expression of KUP9 protein as determined in the kup9-1/ProKUP9:KUP9-GFP transgenic plants. Green fluorescence shows the subcellular localization of KUP9-GFP. PI (propidium iodide) and ER tracker were used to indicate the positions of the plasma membrane (PM) and endoplasmic reticulum (ER) and are shown as red and cyan fluorescence, respectively. Scale bars for root tip, 10 μm in (G) and 5 μm in (H). Scale bars for EZ and MAZ, 20 μm.


( I ) Co - localization analyses of KUP9 - GFP with ER marker NIP1 ; 1 - mCherry in their crossing . Scale bars , 20 μm .

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

(I) Co-localization analyses of KUP9-GFP with ER marker NIP1;1-mCherry in their crossing line. Scale bars, 20 μm.


( J ) Co - localization analyses of KUP9 - GFP with ER marker BIP2 using immunological . Scale bars , 5 μm .

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

(J) Co-localization analyses of KUP9-GFP with the ER marker BIP2 using immunological staining. Scale bars, 5 μm.


( A ) QC observation in wild type kup9 mutants using QC marker lines ProWOX5 : GFP ProQC25 : GUS . Seeds were germinated on HK medium for 4 d , then seedlings were transferred to LK or HK medium for indicated times . Scale bars , 10 μm .

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

(A) QC cell observation in wild type and the kup9 mutants using the QC marker lines ProWOX5:GFP and ProQC25:GUS. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium for the indicated times. Scale bars, 10 μm.


( B ) QC division rates of wild type kup9 mutants after LK treatment for 96 h . statistical method is described in Materials Methods . Data are means ± SE ( n = 150 , ) . Student ' s t - ( * * P < 0 . 01 ) was used to analyze statistical significance , " # " represents control .

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

(B) QC division rates of wild type and the kup9 mutants after LK treatment for 96 h. The statistical method is described in Materials and Methods. Data are means ± SE (n = 150, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


( A ) Auxin in root tips , as indicated by ProDR5 : GFP reporter . Seeds were germinated on HK medium for 4 d , then seedlings were transferred to LK or HK medium for indicated times . Scale bar , 10 μm . ( B ) of GFP in QC shown in ( A ) . Data are means ± SE ( n = 20 , ) . Student ' s t - ( * * P < 0 . 01 ) was used to analyze statistical significance .

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

(A) Auxin levels in root tips, as indicated by the ProDR5:GFP reporter line. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium for the indicated times. Scale bar, 10 μm. (B) Quantification of GFP fluorescence in the QC cells shown in (A). Data are means ± SE (n = 20, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance.


( A ) of various . Seeds were germinated grown on LK or HK medium with or without NAA for 7 d . Scale bar , 1 cm .

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

(A) Phenotypes of various plants. Seeds were germinated and grown on LK or HK medium with or without NAA for 7 d. Scale bar, 1 cm.


( C ) QC observation using QC marker ProWOX5 : GFP . Seeds were germinated on HK medium for 4 d , then seedlings were transferred to LK or HK medium with or without NAA ( 100 nM ) for 96 h . Scale bar , 10 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O

(C) QC cell observation using the QC marker line ProWOX5:GFP. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium with or without NAA (100 nM) for 96 h. Scale bar, 10 μm.


( D ) QC division rates of tested statistical method is described in Materials Methods . Data are means ± SE ( n = 50 - 60 , ) . Student ' s t - ( * * P < 0 . 01 ) was used to analyze statistical significance , " # " represents control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) QC division rates of the plants tested The statistical method is described in Materials and Methods. Data are means ± SE (n = 50-60, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


of kup9 - 1 / ProWOX5 : AMI1 ( A ) transgenic lines . Seeds were germinated grown on LK or HK medium with or without IAM for 7 d . Scale bars , 1 cm .

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

Phenotypes of the kup9-1/ProWOX5:AMI1 (A) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm.


root of tested Data are means ± SE ( n = 25 , ) . Student ' s t - ( * * P < 0 . 01 ) was used to analyze statistical significance , " # " represents control .

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

Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


of kup9 - 1 / ProKUP9 : AMI1 ( C ) transgenic lines . Seeds were germinated grown on LK or HK medium with or without IAM for 7 d . Scale bars , 1 cm .

Phenotypes of the kup9-1/ProKUP9:AMI1 (C) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm.


( A ) Expression of KUP9 - GFP in BY - 2 tobacco . HDEL - mCherry was used as an ER marker . Scale bar , 10 μm .

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

(A) Expression of KUP9-GFP in BY-2 tobacco cells. HDEL-mCherry was used as an ER marker. Scale bar, 10 μm.


( B ) 3H - IAA accumulation in BY - 2 expressing indicated . PIN4 - GFP KUP10 - GFP were used as positive control negative control , respectively . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 3 - 4 mL suspension ) . Student ' s t - ( * P < 0 . 05 , * * P < 0 . 01 ) was used to analyze statistical significance , " # " represent control .

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

(B) 3H-IAA accumulation in the BY-2 cells expressing the indicated proteins. PIN4-GFP and KUP10-GFP were used as positive control and negative control, respectively. Data are means ± SE (n = 3, biological replicates; each replicate contains 3-4 mL suspension cells). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control.


( C ) Expression of KUP9 - GFP OST4 - GFP - KUP9 in Xenopus oocytes . Scale bar , 100 μm .

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

(C) Expression of KUP9-GFP and OST4-GFP-KUP9 in Xenopus oocytes. Scale bar, 100 μm.


( D ) 3H - IAA accumulation in oocytes expressing KUP9 - GFP PIN4 at indicated times . PIN4 was used as a positive control . oocytes were incubated in bath solution containing 3H - IAA for indicated times . Data are means ± SE ( n = 6 , biological replicates ; each replicate contains 6 oocytes ) .

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

(D) 3H-IAA accumulation in the oocytes expressing KUP9-GFP and PIN4 at the indicated times. PIN4 was used as a positive control. The oocytes were incubated in the bath solution containing 3H-IAA for the indicated times. Data are means ± SE (n = 6, biological replicates; each replicate contains 6 oocytes).


( E ) IAA content in oocytes expressing indicated . oocytes were injected with same amount of IAA incubated in IAA - free bath solution for 6 h . IAA content was measured using UPLC - MS . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) .

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

(E) IAA content in the oocytes expressing the indicated proteins. The oocytes were injected with the same amount of IAA and incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).


( F ) K + efflux activity of oocytes expressing KUP9 NRT1 . 5 . oocytes were not injected with IAA . oocytes were incubated in K + - free bath solution for 6 h . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-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

(F) K+ efflux activity of the oocytes expressing KUP9 and NRT1.5. The oocytes were not injected with IAA. The oocytes were incubated in K+-free bath solution for 6 h. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).


( G ) K + efflux activity of oocytes expressing indicated . oocytes were injected with H2O , IAA , or BA . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-MEASURED_VAR I-MEASURED_VAR 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

(G) K+ efflux activity of the oocytes expressing the indicated proteins. The oocytes were injected with H2O, IAA, or BA. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).


( H ) K + efflux activity of oocytes expressing indicated . IAA was injected into oocytes . Then oocytes were incubated in K + - free bath solutions at different pH for 6 h . Data are means ± SE ( n = 3 - 6 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) K+ efflux activity of the oocytes expressing the indicated proteins. IAA was injected into the oocytes. Then the oocytes were incubated in K+-free bath solutions at different pH for 6 h. Data are means ± SE (n = 3-6, biological replicates; each replicate contains 6 oocytes).


( I ) IAA efflux activity of oocytes expressing indicated under different external K + conditions ( - K , 0 mM ; + K , 50 mM ) . oocytes were injected with same amount of IAA , then incubated in IAA - free bath solution for 6 h . IAA content was measured using UPLC - MS . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) . Student ' s t - ( * P < 0 . 05 , * * P < 0 . 01 ) was used to analyze statistical significance , " # " represent control .

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

(I) IAA efflux activity of oocytes expressing the indicated proteins under different external K+ conditions (-K, 0 mM; +K, 50 mM). The oocytes were injected with the same amount of IAA, and then incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control.


( A ) K + of various materials using dye Asante Potassium Green - 2 ( AM ) . Scale bar , 50 μm . ( B ) of K + under LK HK conditions . Data are means ± SE ( n = 25 , ) . Student ' s t - ( * * P < 0 . 01 ) was used to analyze statistical significance , " # " represents control .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) K+ levels of various materials using the fluorescent dye Asante Potassium Green-2 (AM). Scale bar, 50 μm. (B) Quantification of K+ concentration under LK and HK conditions. Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


( C ) to ( E ) contents of free IAA ( C ) , IA - Asp ( D ) , IA - Glu ( E ) in root tips of wild type kup9 - 1 mutant . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 1000 - 1200 ) . Student ' s t - ( * P < 0 . 05 , * * P < 0 . 01 ) was used to analyze statistical significance , " # " represents control .

O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

(C) to (E) The contents of free IAA (C), IA-Asp (D), and IA-Glu (E) in the root tips of wild type and the kup9-1 mutant. Data are means ± SE (n = 3, biological replicates; each replicate contains 1000-1200 individual plants). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represents control.


( F ) Phenotype of kup9 - 1 / ProKUP9 : PIN8 transgenic lines . Seeds were germinated grown on LK or HK medium for 7 d . Scale bar , 1 cm .

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

(F) Phenotype of the kup9-1/ProKUP9:PIN8 transgenic lines. Seeds were germinated and grown on LK or HK medium for 7 d. Scale bar, 1 cm.


( G ) root of tested Data are means ± SE ( n = 25 , ) . Student ' s t - ( * * P < 0 . 01 ) was used to analyze statistical significance .

O O O O O O O O O O O O O O O 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) Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance.


( H ) QC division rates of indicated . statistical method is described in Materials Methods . Data are means ± SE ( n = 50 - 60 , ) . Student ' s t - ( * * P < 0 . 01 ) was used to analyze statistical

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) QC division rates of the indicated plants. The statistical method is described in Materials and Methods. Data are means ± SE (n = 50-60, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance


( A ) Beclin 1 ( BCN1 ) interactors identified by yeast two ‐ hybrid technology . binding to BCN1 are listed their interacting domains are indicated by black bars . Numbers refer to ‐ acid positions .

O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(A) Beclin 1 (BCN1) interactors identified by yeast two‐hybrid technology. Proteins binding to BCN1 are listed and their interacting domains are indicated by black bars. Numbers refer to amino‐acid positions.


( B ) Identification of TAB2 TAB3 fragments interacting with BCN1 in yeast two ‐ hybrid system . Blue lines depict fragments of TAB2 or TAB3 that were found to interact with BCN1 ( numbers on right refer to amount of yeast clones identified for each fragment ) . minimal domain required for interaction is referred to as Beclin ‐ binding domain ( BBD ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Identification of TAB2 and TAB3 fragments interacting with BCN1 in the yeast two‐hybrid system. Blue lines depict the fragments of TAB2 or TAB3 that were found to interact with BCN1 (numbers on the right refer to the amount of yeast clones identified for each fragment). The minimal domain required for the interaction is referred to as Beclin‐binding domain (BBD).


( C ) Constructs derived from TAB2 TAB3 used in this study .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O

(C) Constructs derived from TAB2 and TAB3 used in this study.


( D , E ) Co ‐ immunoprecipitation of TAB2 or TAB3 with BCN1 . indicated constructs , namely HA ‐ tagged T7 ‐ tagged TAB2 , TAB3 constructs in ( D ) ( E ) , respectively , Flag ‐ tagged Beclin 1 ( Flag - BCN1 ) were transfected into HeLa alone or in combination . Twenty ‐ four hours later , TAB2 TAB3 were immunoprecipitated with antibodies specific for HA ( D ) or T7 ( E ) precipitate was separated by SDS - PAGE revealed with an antibody specific for Flag .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Co‐immunoprecipitation of TAB2 or TAB3 with BCN1. The indicated constructs, namely HA‐tagged and T7‐tagged TAB2, and TAB3 constructs in (D) and (E), respectively, and Flag‐tagged Beclin 1 (Flag-BCN1) were transfected into HeLa cells alone or in combination. Twenty‐four hours later, TAB2 and TAB3 were immunoprecipitated with antibodies specific for HA (D) or T7 (E) and the precipitate was separated by SDS-PAGE and revealed with an antibody specific for Flag.


( F ) Immunoprecipitation of endogenous BCN1 with endogenous TAB2 or TAB3 . HeLa were subjected to autophagy induction with starvation conditions , 1 μM rapamycin or 30 μM pifithrin α ( PFTα ) for indicated time then processed for TAB2 or TAB3 immunoprecipitation followed by immunodetection of BCN1 , TAK1 , TAB2 TAB3 . Results in ( E ) ( F ) are representative for three independent experiments .

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

(F) Immunoprecipitation of endogenous BCN1 with endogenous TAB2 or TAB3. HeLa cells were subjected to autophagy induction with starvation conditions, 1 μM rapamycin or 30 μM pifithrin α (PFTα) for the indicated time and then processed for TAB2 or TAB3 immunoprecipitation followed by the immunodetection of BCN1, TAK1, TAB2 and TAB3. Results in (E) and (F) are representative for three independent experiments.


( A , B ) Inhibition of autophagy by dominant ‐ negative ( DN ) TAK1 . HeLa were co ‐ transfected with a GFP - LC3 ‐ encoding construct plus pcDNA3 . 1 ( empty vector ) , or plasmids for expression of WT TAK1 ( TAK1WT ) or DN TAK1K63W mutant . One day later , were either left untreated ( control ) or driven into autophagy by starvation or by administration of 1 μM rapamycin or 30 μM pifithrin α ( PFTα ) , followed by immunoblotting for detection of TAK1 endogenous LC3 ( A ) or immunofluorescence microscopy for of with GFP - LC3 ( GFP - LC3VAC ) ( B ) ( mean values ± s . d . , n = 3 ; * P0 . 01 versus control ) . GAPDH were monitored to ensure equal loading .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Inhibition of autophagy by dominant‐negative (DN) TAK1. HeLa cells were co‐transfected with a GFP-LC3‐encoding construct plus pcDNA3.1 (empty vector), or plasmids for the expression of WT TAK1 (TAK1WT) or the DN TAK1K63W mutant. One day later, cells were either left untreated (control) or driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα), followed by immunoblotting for the detection of TAK1 and endogenous LC3 (A) or immunofluorescence microscopy for the quantification of cells with cytosolic GFP-LC3 puncta (GFP-LC3VAC cells) (B) (mean values±s.d., n=3; *P0.01 versus control cells). GAPDH levels were monitored to ensure equal loading.


( C , D ) Inhibition of autophagy by knockdown of VPS34 , Beclin 1 ( BCN1 ) TAK1 . siRNAs that effectively deplete VPS34 , BCN1 TAK1 were co ‐ transfected with a GFP - LC3 ‐ encoding plasmid in HeLa . Autophagy was then induced as in ( A ) frequency of GFP - LC3VAC ( mean values ± s . d . , n = 3 ; * P0 . 01 versus control ) was determined .

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

(C, D) Inhibition of autophagy by knockdown of VPS34, Beclin 1 (BCN1) and TAK1. siRNAs that effectively deplete VPS34, BCN1 and TAK1 were co‐transfected with a GFP-LC3‐encoding plasmid in HeLa cells. Autophagy was then induced as in (A) and the frequency of GFP-LC3VAC cells (mean values±s.d., n=3; *P0.01 versus control cells) was determined.


( E , F ) same setting shown in ( C , D ) was performed with U2OS FYVE - RFP ( mean values ± s . d . , n = 3 ; * P0 . 01 , * * P0 . 001 versus control ) .

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

(E, F) The same setting shown in (C, D) was performed with U2OS cells and FYVE-RFP (mean values±s.d., n=3; *P0.01, **P0.001 versus control cells).


( A , B ) Detection of autophagic GFP - LC3 + . HeLa or U2OS stably expressing GFP - LC3 were transfected with siRNAs targeting TAK1 , TAB1 , TAB2 or TAB3 or with a control siRNA ( siUNR ) . One day later , subcellular localization abundance of GFP - LC3 or immunostained TAB2 or TAB3 was determined by epifluorescence microscopy . Representative are shown in ( A ) ( HeLa ) quantitative results ( mean values ± s . d . , n = 3 ; * P0 . 01 versus siUNR ‐ transfected ) are depicted in ( B ) ( U2OS ) .

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

(A, B) Detection of autophagic GFP-LC3+ puncta. HeLa or U2OS cells stably expressing GFP-LC3 were transfected with siRNAs targeting TAK1, TAB1, TAB2 or TAB3 or with a control siRNA (siUNR). One day later, the subcellular localization and abundance of GFP-LC3 or immunostained TAB2 or TAB3 was determined by epifluorescence microscopy. Representative images are shown in (A) (HeLa cells) and quantitative results (mean values±s.d., n=3; *P0.01 versus siUNR‐transfected cells) are depicted in (B) (U2OS cells).


( C ) Lipidation of LC3 induced by TAB2 or TAB3 knockdown . Representative immunoblots showing conversion of non ‐ lipidated LC3 ( LC3 ‐ I ) to its lipidated variant ( LC3 ‐ II ) as well as SQSTM1 / p62 are shown . GAPDH were monitored to ensure equal loading .

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

(C) Lipidation of LC3 induced by TAB2 or TAB3 knockdown. Representative immunoblots showing the conversion of non‐lipidated LC3 (LC3‐I) to its lipidated variant (LC3‐II) as well as SQSTM1/p62 protein levels are shown. GAPDH levels were monitored to ensure equal loading.