( 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 B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-ORGANISM O O O O O B-TISSUE O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O B-ORGANISM O O O O B-ORGANISM O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 .

B-GENEPROD O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

B-GENEPROD O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM 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 B-ORGANISM B-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE B-TISSUE B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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 B-TISSUE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CELL_LINE O O O B-TISSUE O O O O O O B-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CELL_LINE B-EXP_ASSAY O O O O O O O O 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-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O B-GENEPROD O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-ORGANISM 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY B-CELL_TYPE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-ORGANISM O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O B-ORGANISM B-TISSUE B-CELL_TYPE O O O O O B-GENEPROD O O O O O O B-GENEPROD 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD 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 B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O 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 B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD 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 B-ORGANISM O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD 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-SMALL_MOLECULE O O O O 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 B-ORGANISM O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD 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 B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-TISSUE O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-ORGANISM 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O 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 B-ORGANISM O O O O O O O O O O O O O O O O O O B-ORGANISM 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O 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 B-GENEPROD O O O O O B-GENEPROD O O O O B-TISSUE B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O 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-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-TISSUE O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY 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-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O 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 B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-EXP_ASSAY 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 B-TISSUE O O O O B-GENEPROD O O B-TISSUE O O O O B-GENEPROD O O B-GENEPROD O B-TISSUE O B-EXP_ASSAY O O O O O O O O B-ORGANISM 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 B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O B-TISSUE O B-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O 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 B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE 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 B-TISSUE O O B-TISSUE O O O B-GENEPROD O B-TISSUE O B-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE 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 B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O O O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE 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 .

B-TISSUE O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR 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 B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM B-CELL_TYPE 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 B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE 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-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-CELL_TYPE 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-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O 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-CELL_TYPE 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-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-CELL_TYPE 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-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O 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-CELL_TYPE 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-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY 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 B-TISSUE O O O O O O O O O O O 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 B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD 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-GENEPROD I-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O 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-GENEPROD I-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD 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 B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD 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-GENEPROD O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O 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-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O 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-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD 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 B-CELL_LINE B-GENEPROD O B-GENEPROD O O O 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 B-GENEPROD O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_LINE O O O O O O O O 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-CELL_LINE 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 B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD 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.


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

O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O 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) 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 of effects of TAB2 TAB3 depletion on autophagy . HeLa stably expressing GFP - LC3 were transfected with siRNAs specific for TAB2 or TAB3 / or with cDNAs coding for full ‐ HA ‐ tagged TAB2 ( HA - TAB2 ) or TAB3 ( HA - TAB3 ) . Twenty ‐ four hours later , frequency of exhibiting > 5 GFP - LC3 + ( GFP - LC3VAC ) was determined . Results are mean values ± s . d . ( n = 3 ; * P0 . 01 versus siUNR ‐ transfected ) .

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

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) U2OS 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 induction of GFP - LC3 + by TAB2 TAB3 depletion . HeLa stably expressing GFP - LC3 were transfected with a control siRNA ( siUNR ) or with siRNAs targeting TAB2 TAB3 for 24 h . During last 12 h of this period , BafA1 was optionally added . After fixation permeabilization , LAMP2 was detected by immunofluorescence . Representative confocal microphotographs for TAB2 siRNA are shown ( A ) , together with profiles of colocalization of signals ( B ) along indicated direction ( α - ω ) . Columns in ( C ) represent of colocalization of GFP - LC3 LAMP2 ( mean values ± s . d . ; * P0 . 01 versus siUNR ‐ transfected ) , as quantified in at least 50 for each condition . frequency ( mean ± s . d . ) of with > 5 GFP - LC3 + ( GFP - LC3VACcells ) is plotted in ( D ) .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O

(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 indicated genotypes were cultured in complete medium supplemented with BafA1 for 12 h proportion of LC3 ‐ I / LC3 ‐ II was determined by immunoblotting . GAPDH were monitored to ensure equal loading .

O O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O

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

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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 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 indicated siRNAs for 24 h , followed by of GFP - LC3VAC ( mean values ± s . d . , n = 3 , * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected )

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

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(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).


( B ) U2OS stably expressing FYVE - RFP were co ‐ transfected with pcDNA3 . 1 or with vectors encoding TAB2C or TAB3C together with a control siRNA ( siUNR ) or with siRNAs specific for VPS34 , Beclin 1 ( BCN1 ) or TAK1 for 24 h , followed by of exhibiting FYVE - RFP + dots ( FYVE - RFP + ) . Results are mean values ± s . d . ( n = 3 ; * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected ) .

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(B) U2OS cells stably expressing FYVE-RFP were co‐transfected with pcDNA3.1 or with vectors encoding TAB2C or TAB3C together with a control siRNA (siUNR) or with siRNAs specific for VPS34, Beclin 1 (BCN1) or TAK1 for 24 h, followed by the quantification of cells exhibiting FYVE-RFP+ dots (FYVE-RFP+). Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells).


( C ) Inhibition of interaction between endogenous TAB2 , TAB3 BCN1 by C ‐ terminal fragments of TAB2 TAB3 . Forty ‐ eight hours after transfection with pcDNA3 . 1 or plasmids coding for indicated , were lysed , TAB2 or TAB3 was immunoprecipitated BCN1 was immunodetected . GAPDH were monitored to ensure equal loading . This experiment has been done three times , yielding comparable results .

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(C) Inhibition of the interaction between endogenous TAB2, TAB3 and BCN1 by C‐terminal fragments of TAB2 and TAB3. Forty‐eight hours after transfection with pcDNA3.1 or plasmids coding for the indicated proteins, cells were lysed, TAB2 or TAB3 was immunoprecipitated and BCN1 was immunodetected. GAPDH levels were monitored to ensure equal loading. This experiment has been done three times, yielding comparable results.


( D ) Mechanisms of autophagy induction by Beclin ‐ binding domain ( BBD ) ‐ containing C ‐ terminal fragments of TAB2 TAB3 . HeLa were transfected with pcDNA3 . 1 , TAB2C ‐ or TAB3C ‐ encoding plasmids in combination with indicated siRNAs for 24 h , followed by of GFP - LC3VAC ( mean values ± s . d . , n = 3 ; * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected ) .

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(D) Mechanisms of autophagy induction by the Beclin‐binding domain (BBD)‐containing C‐terminal fragments of TAB2 and TAB3. HeLa cells were transfected with pcDNA3.1, TAB2C‐ or TAB3C‐encoding plasmids in combination 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 ) Determination of TAB ‐ binding domain ( TBD ) within Beclin 1 ( BCN1 ) . Yeast two ‐ hybrid technology was used to screen for positive ( + ) or negative interactions ( − ) between BCN1 fragments full ‐ TAB2 .

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(A) Determination of the TAB‐binding domain (TBD) within Beclin 1 (BCN1). Yeast two‐hybrid technology was used to screen for positive (+) or negative interactions (−) between BCN1 fragments and full‐length TAB2.


( C ) Confirmation of TBD by immunoprecipitation . indicated constructs were transfected into HeLa , followed by lysis , immunoprecipitation of HA ‐ tagged full ‐ TAB2 or TAB3 detection of His ‐ tagged constructs .

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(C) Confirmation of the TBD by immunoprecipitation. The indicated constructs were transfected into HeLa cells, followed by lysis, immunoprecipitation of HA‐tagged full‐length TAB2 or TAB3 and detection of His‐tagged constructs.


( D , E ) Inhibition of interaction between endogenous BCN1 , TAB2 TAB3 by a BCN1 fragment corresponding to TBD . HeLa were transfected with pcDNA3 . 1 ( empty vector ) or with plasmids encoding TBD or a His - tagged Beclin 1 variant lacking TBD ( His - BCN1ΔTBD ) , followed by co ‐ immunoprecipitation of endogenous TAB2 ( D ) or TAB3 ( E ) detection of BCN1 .

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(D, E) Inhibition of the interaction between endogenous BCN1, TAB2 and TAB3 by a BCN1 fragment corresponding to the TBD. HeLa cells were transfected with pcDNA3.1 (empty vector) or with plasmids encoding the TBD or a His-tagged Beclin 1 variant lacking the TBD (His-BCN1ΔTBD), followed by co‐immunoprecipitation of endogenous TAB2 (D) or TAB3 (E) and detection of BCN1.