( 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 .

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

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

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

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

(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 ) 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 .

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

(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 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 .

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

(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 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 .

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

(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 ) 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 .

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

(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 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 .

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 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 . 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 .

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 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 ) 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 .

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

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 O O O O O O O O O O O O O O O O O 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 , 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 .

O 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 O O B-CONTROLLED_VAR 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 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 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 .

O 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 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 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 .

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

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

(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 ) . 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 .

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

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 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 ) .

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

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 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 .

O O O O O O 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 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 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 .

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 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 ) 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 .

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 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 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 ) 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 .

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

(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 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

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 O O B-CONTROLLED_VAR 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 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 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 .

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , 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 .

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


C 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 .

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

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 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 .

O O O O B-MEASURED_VAR 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 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 .

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

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


F , 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

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

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


B , Interaction between ARF2 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 .

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


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

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


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

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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.


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 .

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


B , RT - qPCR 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 .

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

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 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 .

O O B-MEASURED_VAR 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

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

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


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 .

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 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 .

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.


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


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 B-MEASURED_VAR 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

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.


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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 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 .

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 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 .

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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 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 .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_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 O O O O O O O O O O O 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 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 .

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

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 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 O O O O O O O O O O O O O O O O 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 the WT background , arf2 - 20 , and pPIF5 : PIF5 - GFP OEs in the arf2 - 20 background . Seedlings were grown under continuous light for one week .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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G and H, RT-qPCR analysis of ORE1 and SGR1 expression levels in genotypes shown in A (G) and B (H) before and after 4 d C-deprivation. Fold changes were calculated with respect to the expression level in the WT before C-deprivation. 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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(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 ) 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 .

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

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(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 ) 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 ) .

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(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 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 .

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(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 ) 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 .

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(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 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 ) .

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(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 ) 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 .

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(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 line . Scale bars , 20 μm .

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(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 the ER marker BIP2 using immunological staining . Scale bars , 5 μm .

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


( 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 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 .

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(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 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 .

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(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.


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 .

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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.


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 .

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


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 .

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 cells . HDEL - mCherry was used as an ER marker . Scale bar , 10 μm .

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(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 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 .

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(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 and OST4 - GFP - KUP9 in Xenopus oocytes . Scale bar , 100 μm .

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


( 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 .

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(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 + 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 .

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(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 ) 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 .

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

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(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 ) 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 .

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(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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . Proteins binding to BCN1 are listed and their interacting domains are indicated by black bars . Numbers refer to amino ‐ acid positions .

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(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 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 ) .

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(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 and TAB3 used in this study .

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


( 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 ) 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 ) .

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(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 + 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 ) .

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(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 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 .

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(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.


( D , E ) Quantification of autophagosomes and autophagolysosomes by transmission electron microscopy . Representative images of HeLa cells transfected with siUNR or with TAB2 ‐ or TAB3 ‐ targeting siRNAs are shown in ( D ) , and quantitative results are depicted in ( E ) ( mean values ± s . d . , n = 3 ; * P0 . 01 versus siUNR ‐ transfected cells ) .

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(D, E) Quantification of autophagosomes and autophagolysosomes by transmission electron microscopy. Representative images of HeLa cells transfected with siUNR or with TAB2‐ or TAB3‐targeting siRNAs are shown in (D), and quantitative results are depicted in (E) (mean values±s.d., n=3; *P0.01 versus siUNR‐transfected cells).


( F ) Epistatic analysis of the effects of TAB2 and TAB3 depletion on autophagy . HeLa cells stably expressing GFP - LC3 were transfected with siRNAs specific for TAB2 or TAB3 and / or with cDNAs coding for full ‐ length HA ‐ tagged TAB2 ( HA - TAB2 ) or TAB3 ( HA - TAB3 ) . Twenty ‐ four hours later , the frequency of cells exhibiting > 5 GFP - LC3 + cytosolic puncta ( GFP - LC3VAC cells ) was determined . Results are mean values ± s . d . ( n = 3 ; * P0 . 01 versus siUNR ‐ transfected cells ) .

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(F) Epistatic analysis of the effects of TAB2 and TAB3 depletion on autophagy. HeLa cells stably expressing GFP-LC3 were transfected with siRNAs specific for TAB2 or TAB3 and/or with cDNAs coding for full‐length HA‐tagged TAB2 (HA-TAB2) or TAB3 (HA-TAB3). Twenty‐four hours later, the frequency of cells exhibiting >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was determined. Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐transfected cells).


( G ) U2OS cells stably expressing FYVE - RFP were transfected with siUNR , or with siRNAs specific for TAB2 , TAB3 , VPS34 , Beclin 1 ( BCN1 ) and TAK1 , in the indicated combinations . Forty ‐ eight hours later , the percentage of cells with RFP - FYVE + puncta cells was determined . Results are mean values ± s . d . ( n = 3 ; * P0 . 01 versus siUNR ‐ transfected cells ) .

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(G) U2OS cells stably expressing FYVE-RFP were transfected with siUNR, or with siRNAs specific for TAB2, TAB3, VPS34, Beclin 1 (BCN1) and TAK1, in the indicated combinations. Forty‐eight hours later, the percentage of cells with RFP-FYVE+ puncta cells was determined. Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐transfected cells).


( A - D ) Impact of bafilomycin A1 ( BafA1 ) on the induction of GFP - LC3 + puncta by TAB2 and TAB3 depletion . HeLa cells stably expressing GFP - LC3 were transfected with a control siRNA ( siUNR ) or with siRNAs targeting TAB2 and TAB3 for 24 h . During the last 12 h of this period , BafA1 was optionally added . After fixation and permeabilization , LAMP2 was detected by immunofluorescence . Representative confocal microphotographs for the TAB2 siRNA are shown ( A ) , together with the profiles of colocalization of fluorescent signals ( B ) along the indicated direction ( α - ω ) . Columns in ( C ) represent the percentage of colocalization of GFP - LC3 and LAMP2 ( mean values ± s . d . ; * P0 . 01 versus siUNR ‐ transfected cells ) , as quantified in at least 50 cells for each condition . The frequency ( mean ± s . d . ) of cells with > 5 GFP - LC3 + cytosolic puncta ( GFP - LC3VACcells ) is plotted in ( D ) .

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(A-D) Impact of bafilomycin A1 (BafA1) on the induction of GFP-LC3+ puncta by TAB2 and TAB3 depletion. HeLa cells stably expressing GFP-LC3 were transfected with a control siRNA (siUNR) or with siRNAs targeting TAB2 and TAB3 for 24 h. During the last 12 h of this period, BafA1 was optionally added. After fixation and permeabilization, LAMP2 was detected by immunofluorescence. Representative confocal microphotographs for the TAB2 siRNA are shown (A), together with the profiles of colocalization of fluorescent signals (B) along the indicated direction (α-ω). Columns in (C) represent the percentage of colocalization of GFP-LC3 and LAMP2 (mean values±s.d.; *P0.01 versus siUNR‐transfected cells), as quantified in at least 50 cells for each condition. The frequency (mean±s.d.) of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VACcells) is plotted in (D).


( E ) Impact of BafA1 on LC3 lipidation . MEFs with the indicated genotypes were cultured in complete medium supplemented with BafA1 for 12 h and the proportion of LC3 ‐ I / LC3 ‐ II was determined by immunoblotting . GAPDH levels were monitored to ensure equal loading .

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(E) Impact of BafA1 on LC3 lipidation. MEFs with the indicated genotypes were cultured in complete medium supplemented with BafA1 for 12 h and the proportion of LC3‐I/LC3‐II was determined by immunoblotting. GAPDH levels were monitored to ensure equal loading.


( F , G ) Impact of autophagy ‐ relevant proteins and of the TAK1 ‐ IKK signalling axis on GFP - LC3 aggregation induced by the depletion of TAB2 or TAB3 . HeLa cells stably expressing GFP - LC3 were transfected with siUNR or with siRNAs targeting the indicated proteins , alone or in combination , and 48 h later GFP - LC3VAC cells were quantified ( mean values ± s . d . , n = 4 ; * P0 . 01 versus siUNR ‐ transfected cells ) .

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(F, G) Impact of autophagy‐relevant proteins and of the TAK1‐IKK signalling axis on GFP-LC3 aggregation induced by the depletion of TAB2 or TAB3. HeLa cells stably expressing GFP-LC3 were transfected with siUNR or with siRNAs targeting the indicated proteins, alone or in combination, and 48 h later GFP-LC3VAC cells were quantified (mean values±s.d., n=4; *P0.01 versus siUNR‐transfected cells).


( H ) Inhibition of autophagy by dominant ‐ negative ( DN ) TAK1 . HeLa cells stably expressing GFP - LC3 were co ‐ transfected with pcDNA3 . 1 ( empty vector ) or with plasmids encoding WT ( TAK1WT ) or a DN TAK1 variant ( TAK1K63W ) together with the indicated siRNAs for 24 h , followed by the quantification of GFP - LC3VAC cells ( mean values ± s . d . , n = 3 , * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected cells )

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 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 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

(H) Inhibition of autophagy by dominant‐negative (DN) TAK1. HeLa cells stably expressing GFP-LC3 were co‐transfected with pcDNA3.1 (empty vector) or with plasmids encoding WT (TAK1WT) or a DN TAK1 variant (TAK1K63W) together with the indicated siRNAs for 24 h, followed by the quantification of GFP-LC3VAC cells (mean values±s.d., n=3, *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells)


( A ) Effects of full ‐ length TAB2 and TAB3 or their deletion mutants ( as in Figure 1C ) on autophagy . HeLa cells stably expressing GFP - LC3 were transfected with pcDNA3 . 1 ( empty vector ) or with plasmids encoding the indicated TAB2 and TAB3 variants for 24 h , then driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α ( PFTα ) for 4 h . Finally , the frequency ( mean ± s . d . , n = 3 ) of cells with > 5 GFP - LC3 + cytosolic puncta ( GFP - LC3VAC cells ) was assessed ( * P0 . 01 versus control cells transfected with the same plasmid ; # P0 . 01 versus pcDNA3 . 1 ‐ transfected cells treated with the same pro ‐ autophagic trigger ) .

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 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 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 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

(A) Effects of full‐length TAB2 and TAB3 or their deletion mutants (as in Figure 1C) on autophagy. HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 (empty vector) or with plasmids encoding the indicated TAB2 and TAB3 variants for 24 h, then driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα) for 4 h. Finally, the frequency (mean±s.d., n=3) of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was assessed (*P0.01 versus control cells transfected with the same plasmid; #P0.01 versus pcDNA3.1‐transfected cells treated with the same pro‐autophagic trigger).