( b ) We transfected SBMA NPC lines ( three different NPC lines per patient ) with either the BFP - empty vector or BFP - TFEB expression construct , treated the NPCs with JC - 1 dye and counted the percentage of cells with depolarized mitochondria . Results are presented as Tukey box plots with boxes corresponding to first and third quartiles from the median and whiskers corresponding to 1 . 5 quartiles from the median ; n = 3 independent experiments , * P 0 . 05 , t - test , t ( 6 ) = 3 . 217 .

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

(b) We transfected SBMA NPC lines (three different NPC lines per patient) with either the BFP-empty vector or BFP-TFEB expression construct, treated the NPCs with JC-1 dye and counted the percentage of cells with depolarized mitochondria. Results are presented as Tukey box plots with boxes corresponding to first and third quartiles from the median and whiskers corresponding to 1.5 quartiles from the median; n = 3 independent experiments, *P 0.05, t-test, t(6) = 3.217.


( c ) Representative images from transfection of SBMANPCs with the mCherry - EGFP - LC3 vector and either BFP - empty vector or BFP - TFEB expression construct . BFP - empty vector - expressing SBMANPCs display a preponderance of yellow puncta ( autophagosomes ) , while BFP - TFEB - expressing SBMANPCs exhibit a shift toward almost entirely red puncta ( autolysosomes ) , consistent with rescue of the autophagic flux defect . Scale bars , 20 μm . ( d ) We transfected SBMANPC lines ( three different NPC lines per patient ) with the mCherry - EGFP - LC3 vector and either BFP - empty vector or BFP - TFEB expression construct , counted the number of autophagosomes and autolysosomes in BFP - expressing NPCs and determined the number of autophagic vesicles per cell . TFEB overexpression markedly reduced autophagosome formation in SBMANPCs . * * * P 0 . 001 ; t - test , t ( 30 ) = 4 . 373 . Data in d are presented as mean ± s . e . m . Individual P values are available in the Supplementary Methods Checklist .

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

(c) Representative images from transfection of SBMANPCs with the mCherry-EGFP-LC3 vector and either BFP-empty vector or BFP-TFEB expression construct. BFP-empty vector-expressing SBMANPCs display a preponderance of yellow puncta (autophagosomes), while BFP-TFEB-expressing SBMANPCs exhibit a shift toward almost entirely red puncta (autolysosomes), consistent with rescue of the autophagic flux defect. Scale bars, 20 μm. (d) We transfected SBMANPC lines (three different NPC lines per patient) with the mCherry-EGFP-LC3 vector and either BFP-empty vector or BFP-TFEB expression construct, counted the number of autophagosomes and autolysosomes in BFP-expressing NPCs and determined the number of autophagic vesicles per cell. TFEB overexpression markedly reduced autophagosome formation in SBMANPCs. ***P 0.001; t-test, t(30) = 4.373. Data in d are presented as mean ± s.e.m. Individual P values are available in the Supplementary Methods Checklist.


Detection of NRPB2WT - FLAG , NRPB2P979S - FLAG and NRPB2Y732F - FLAG protein by western blotting in NRPB2WT - FLAG Col - 0 , NRPB2P979S - FLAG Col - 0 and NRPB2Y732F - FLAG Col - 0 plants . Untagged NRPB2 ( Col - 0 ) was used as a negative control . Histone H3 was used as an internal control and total protein level detected by stain - free blot was used as a loading control . Quantification was done by normalizing to the loading control and anti - H3 blot based on 3 independent replicates .

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

Detection of NRPB2WT-FLAG, NRPB2P979S-FLAG and NRPB2Y732F-FLAG protein by western blotting in NRPB2WT-FLAG Col-0, NRPB2P979S-FLAG Col-0 and NRPB2Y732F-FLAG Col-0 plants. Untagged NRPB2 (Col-0) was used as a negative control. Histone H3 was used as an internal control and total protein level detected by stain-free blot was used as a loading control. Quantification was done by normalizing to the loading control and anti-H3 blot based on 3 independent replicates.


Transmission rate of nrpb2 - 2 allele in nrpb2 - 2 + / - line ( n = 197 ) and nrpb2 - + / - lines combined with homozygous NRPB2P979S - FLAG + / + ( n = 280 ) , NRPB2Y732F - FLAG + / + ( n = 240 ) and NRPB2WT - FLAG + / + ( n = 210 ) , respectively . Fisher ´ s exact test was used as a statistic test , three asterisks denote p < 0 . 001 between samples and n . s . stands for not significant .

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

Transmission rate of nrpb2-2 allele in nrpb2-2 +/- line (n=197) and nrpb2- +/- lines combined with homozygous NRPB2P979S-FLAG +/+ (n=280), NRPB2Y732F-FLAG +/+ (n=240) and NRPB2WT-FLAG +/+ (n=210), respectively. Fisher´s exact test was used as a statistic test, three asterisks denote p<0.001 between samples and n.s. stands for not significant.


Image of homozygous mutant nrpb2 - 2 fully complemented by NRPB2WT - FLAG ( top , NRPB2WT + / + nrpb2 - 2 - / - ) and partially complemented by NRPB2Y732F - FLAG ( bottom , NRPB2WT + / + nrpb2 - 2 - / - ) . Plants were grown for 4 weeks in soil . Scale bars represent 1 cm .

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

Image of homozygous mutant nrpb2-2 fully complemented by NRPB2WT-FLAG (top, NRPB2WT +/+ nrpb2-2 -/-) and partially complemented by NRPB2Y732F-FLAG (bottom, NRPB2WT +/+ nrpb2-2 -/-). Plants were grown for 4 weeks in soil. Scale bars represent 1 cm.


Schematic drawing of the experimental design to investigate RNAPII transcription speed in vivo . In brief , Arabidopsis seedlings of NRPB2WT - FLAG Col - 0 and NRPB2Y732F - FLAG Col - 0 were grown on MS media for 12 days and then were transferred to MS liquid media for 2 days . Flagellin peptides ( flagellin 22 ) were added into media and treated samples were collected in a 0 minute ( no treatment ) , 2 minutes and 4 minutes time course . The nascent RNA was isolated and used for reverse transcription and qPCR analyses to reveal RNAPII accumulation at different region in genes . See Methods for technical details .

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

Schematic drawing of the experimental design to investigate RNAPII transcription speed in vivo. In brief, Arabidopsis seedlings of NRPB2WT-FLAG Col-0 and NRPB2Y732F-FLAG Col-0 were grown on MS media for 12 days and then were transferred to MS liquid media for 2 days. Flagellin peptides (flagellin 22) were added into media and treated samples were collected in a 0 minute (no treatment), 2 minutes and 4 minutes time course. The nascent RNA was isolated and used for reverse transcription and qPCR analyses to reveal RNAPII accumulation at different region in genes. See Methods for technical details.


Nascent RNA profile of AT5G41750 . Nascent RNA RT - qPCR assay measuring RNAPII signal at 3 positions ( dark red bars : probe 1 , 2 and 3 ) on the gene upon flagellin 22 treatment in a 0 minute , 2 minutes , 3 minutes and 4 minutes time course . Nascent RNA signal values were normalized to reference gene ACT2 . Error bars represent SEM from 3 independent replicates . The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by the two - sided Student ' s t - test . n . s . denotes not significant ; * denotes p < 0 . 05 and * * denotes p < 0 . 01 . Scale bar ( black ) represent 0 . 5 kb .

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

Nascent RNA profile of AT5G41750. Nascent RNA RT-qPCR assay measuring RNAPII signal at 3 positions (dark red bars: probe 1, 2 and 3) on the gene upon flagellin 22 treatment in a 0 minute, 2 minutes, 3 minutes and 4 minutes time course. Nascent RNA signal values were normalized to reference gene ACT2. Error bars represent SEM from 3 independent replicates. The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by the two-sided Student's t-test. n.s. denotes not significant; * denotes p<0.05 and ** denotes p<0.01. Scale bar (black) represent 0.5 kb.


Nascent RNA profile of AT4G19520 . Nascent RNA RT - qPCR assay measuring RNAPII signal at 3 positions ( dark red bars : probe 1 , 2 and 3 ) on the gene upon flagellin 22 treatment in a 0 minute , 2 minutes , 3 minutes and 4 minutes time course . Nascent RNA signal values were normalized to reference gene ACT2 . Error bars represent SEM from 3 independent replicates . The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by a two - sided Student ' s t - test . n . s . denotes not significant ; * denotes p < 0 . 05 and * * denotes p < 0 . 01 . Scale bar ( black ) represents 0 . 5 kb .

Nascent RNA profile of AT4G19520. Nascent RNA RT-qPCR assay measuring RNAPII signal at 3 positions (dark red bars: probe 1, 2 and 3) on the gene upon flagellin 22 treatment in a 0 minute, 2 minutes, 3 minutes and 4 minutes time course. Nascent RNA signal values were normalized to reference gene ACT2. Error bars represent SEM from 3 independent replicates. The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by a two-sided Student's t-test. n.s. denotes not significant; * denotes p<0.05 and ** denotes p<0.01. Scale bar (black) represents 0.5 kb.


plaNET - seq signal of RNAPII in the promoter proximal region of AT1G70600 in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Arrows indicate the RNAPII signal at the region of promoter - proximal stalling .

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

plaNET-seq signal of RNAPII in the promoter proximal region of AT1G70600 in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Arrows indicate the RNAPII signal at the region of promoter-proximal stalling.


Metagene profile of plaNET - seq mean signal of RNAPII in a 1 Kb window centered at the + 1 nucleosome in Arabidopsis genes ( n = 25474 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . The significance of differences of plaNET - seq signal in the region from - 25 bins to + 25 bins around + 1 nucleosome between NRPB2WT and NRPB2Y732F were assessed by a two - sided Mann - Whitney U - test , p = 5 . 20e - 10 .

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

Metagene profile of plaNET-seq mean signal of RNAPII in a 1 Kb window centered at the +1 nucleosome in Arabidopsis genes (n=25474) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). The significance of differences of plaNET-seq signal in the region from -25 bins to +25 bins around +1 nucleosome between NRPB2WT and NRPB2Y732F were assessed by a two-sided Mann-Whitney U-test, p=5.20e-10.


RNAPII stalling index calculated for all the genes with plaNET - Seq FPKM ≥ 10 in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) ( n = 6596 ) . Medians of the stalling index are 1 . 891 and 1 . 222 for NRPB2WT and NRPB2Y732F , respectively . * * * denotes p - value < 0 . 001 by Wilcoxon signed - rank test . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile .

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

RNAPII stalling index calculated for all the genes with plaNET-Seq FPKM ≥ 10 in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red) (n=6596). Medians of the stalling index are 1.891 and 1.222 for NRPB2WT and NRPB2Y732F, respectively. *** denotes p-value <0.001 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.


Metagene profile of plaNET - seq mean signal over whole genes ( length from 0 . 5 Kb to 5 Kb , scaled to 500 bins , n = 27042 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) .

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

Metagene profile of plaNET-seq mean signal over whole genes (length from 0.5 Kb to 5 Kb, scaled to 500 bins, n=27042) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).


Metagene profile of plaNET - seq mean signal of RNAPII in exons ( length from 50 bp to 300 bp , scaled to 100 bins , n = 73925 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Pink dashed line rectangle illustrates the amplitude of differences between the minimum and the maximum of RNAPII signal across the exons . A two - sided Mann - Whitney U - test was used to assess the plaNET - seq signal of NRPB2WT ( blue ) and NRPB2Y732F ( red ) in exons , p < 1e - 16 . Metagene profile of plaNET - seq mean signal of RNAPII in introns ( 50 bp to 300 bp , scaled to 100 bins , n = 102260 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . A two - sided Mann - Whitney U - test was used to assess the plaNET - seq signal of NRPB2WT ( blue ) and NRPB2Y732F ( red ) in introns , p < 1e - 16 .

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

Metagene profile of plaNET-seq mean signal of RNAPII in exons (length from 50 bp to 300 bp, scaled to 100 bins, n=73925) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Pink dashed line rectangle illustrates the amplitude of differences between the minimum and the maximum of RNAPII signal across the exons. A two-sided Mann-Whitney U-test was used to assess the plaNET-seq signal of NRPB2WT (blue) and NRPB2Y732F (red) in exons, p<1e-16. Metagene profile of plaNET-seq mean signal of RNAPII in introns (50 bp to 300 bp, scaled to 100 bins, n=102260) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). A two-sided Mann-Whitney U-test was used to assess the plaNET-seq signal of NRPB2WT (blue) and NRPB2Y732F (red) in introns, p<1e-16.


Bar charts showing the fractions of 3 ' and 5 ' splicing intermediate reads from plaNET - seq in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) .

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

Bar charts showing the fractions of 3' and 5' splicing intermediate reads from plaNET-seq in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).


Genome browser snapshots illustrating enhanced intron splicing in NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) compared to NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) . Scale bars denote 0 . 5 Kb .

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

Genome browser snapshots illustrating enhanced intron splicing in NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F, red) compared to NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT ,blue). Scale bars denote 0.5 Kb.


The fraction of RNA - seq intronic reads calculated for all genes ( n = 24912 ) in NRPB2WT + / + nrpb2 - 2 - / - and NRPB2Y732F + / + nrpb2 - 2 - / - . Two - sided Mann - Whitney U test : * * * * denotes p - value < 2 . 2e - 16 . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile .

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

The fraction of RNA-seq intronic reads calculated for all genes (n=24912) in NRPB2WT +/+ nrpb2-2 -/- and NRPB2Y732F +/+ nrpb2-2 -/-. Two-sided Mann-Whitney U test: **** denotes p-value<2.2e-16. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.


Differentially expressed ( DE ) exons and introns in NRPB2Y732F + / + nrpb2 - 2 - / - compared to NRPB2WT + / + nrpb2 - 2 - / - based on RNA - seq results . Numbers of DE exons and introns were shown in plot . Quantification ( log fold change of FPKM from RNA - seq ) of differentially expressed ( DE ) exons and non - DE exons in NRPB2Y732F + / + nrpb2 - 2 - / - compared to NRPB2WT + / + nrpb2 - 2 - / - . * * denotes p - value < 0 . 01 by Wilcoxon signed - rank test . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile .

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

Differentially expressed (DE) exons and introns in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/- based on RNA-seq results. Numbers of DE exons and introns were shown in plot. Quantification (log fold change of FPKM from RNA-seq) of differentially expressed (DE) exons and non-DE exons in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/-. ** denotes p-value <0.01 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.


Genome browser snapshots illustrating enhanced exon skipping in NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) compared to NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) . Scale bars denote 0 . 5 Kb . Quantification ( log fold change of FPKM from RNA - seq ) of differentially expressed ( DE ) introns and non - DE exons in NRPB2Y732F + / + nrpb2 - 2 - / - compared to NRPB2WT + / + nrpb2 - 2 - / - . * * * * denotes p - value < 0 . 0001 by Wilcoxon signed - rank test . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile .

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

Genome browser snapshots illustrating enhanced exon skipping in NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F, red) compared to NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT ,blue). Scale bars denote 0.5 Kb. Quantification (log fold change of FPKM from RNA-seq) of differentially expressed (DE) introns and non-DE exons in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/-. **** denotes p-value <0.0001 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.


plaNET - seq signal of RNAPII at 3 ' end of AT2G21410 in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Arrows indicate the RNAPII signal peaks at PAS stalling region .

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

plaNET-seq signal of RNAPII at 3' end of AT2G21410 in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Arrows indicate the RNAPII signal peaks at PAS stalling region.


Metagene profile of plaNET - seq mean signal of RNAPII in a 1 kb window centered at PAS ( n = 24448 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . The significance of differences of plaNET - seq signal in the region from PAS to + 100 bins between NRPB2WT and NRPB2Y732F were assessed by Two - sided Mann - Whitney U - test , p = 1 . 53e - 06 .

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

Metagene profile of plaNET-seq mean signal of RNAPII in a 1 kb window centered at PAS (n=24448) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). The significance of differences of plaNET-seq signal in the region from PAS to +100 bins between NRPB2WT and NRPB2Y732F were assessed by Two-sided Mann-Whitney U-test, p = 1.53e-06.


Histogram of transcriptional read - through length ( nt ) from PAS of protein - coding gene ( plaNET - seq FPKM > 5 , n = 9316 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) .

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

Histogram of transcriptional read-through length (nt) from PAS of protein-coding gene (plaNET-seq FPKM>5, n=9316) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).


Box plot shows the RNAPII transcriptional read - through length from PAS of protein - coding genes ( plaNET - seq FPKM > 5 n = 9316 ) called based on statistic model ( see Methods ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Median of read - through length in NRPB2WT and NRPB2Y732F mutant are 534 nt and 649 nt . Two - sided Mann - Whitney U - test : * * * denotes p = 9 . 9e - 62 . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile .

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Box plot shows the RNAPII transcriptional read-through length from PAS of protein-coding genes (plaNET-seq FPKM>5 n=9316) called based on statistic model (see Methods) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Median of read-through length in NRPB2WT and NRPB2Y732F mutant are 534 nt and 649 nt. Two-sided Mann-Whitney U-test: *** denotes p = 9.9e-62. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.


Metagene plot of RNAPII signal by plaNET - seq anchored at both PAS of upstream genes and TSS of downstream genes for tandemly oriented genes ( n = 5753 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Red arrow denotes the direction of transcriptional read - through . Pink dashed line rectangle indicates the region corresponding to the second half of PAS - TSS gaps along 5 ' to 3 ' direction . Metagene plot of RNAPII signal by plaNET - seq anchored at PASs of both upstream genes and downstream genes for gene pairs located in " tail to tail " orientation ( n = 1384 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Red arrows denote the directions of transcriptional read - through from both PASs . Pink dashed line rectangles indicate the region corresponding to the second half of PAS - TSS gaps along 5 ' to 3 ' direction .

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

Metagene plot of RNAPII signal by plaNET-seq anchored at both PAS of upstream genes and TSS of downstream genes for tandemly oriented genes (n=5753) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Red arrow denotes the direction of transcriptional read-through. Pink dashed line rectangle indicates the region corresponding to the second half of PAS-TSS gaps along 5' to 3' direction. Metagene plot of RNAPII signal by plaNET-seq anchored at PASs of both upstream genes and downstream genes for gene pairs located in "tail to tail" orientation (n=1384) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Red arrows denote the directions of transcriptional read-through from both PASs. Pink dashed line rectangles indicate the region corresponding to the second half of PAS-TSS gaps along 5' to 3' direction.


A . Binding of ubiquitin chains to immobilized GST - fusion proteins . Indicated proteins were purified from E . coli and incubated with equal amounts of preformed fluorescently labeled ubiquitin chains , which were either K48 - or K63 - linked . The fraction of ubiquitin chains interacting with the bait protein was analyzed by SDS ‑ PAGE and fluorescence scan . Total fluorescence was quantified per lane and normalized to the first lane ( rel . fluo . ) . Asterisk ( * ) indicates lane from the same gel that was cropped and moved .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Binding of ubiquitin chains to immobilized GST-fusion proteins. Indicated proteins were purified from E. coli and incubated with equal amounts of preformed fluorescently labeled ubiquitin chains, which were either K48- or K63-linked. The fraction of ubiquitin chains interacting with the bait protein was analyzed by SDS‑PAGE and fluorescence scan. Total fluorescence was quantified per lane and normalized to the first lane (rel. fluo.). Asterisk (*) indicates lane from the same gel that was cropped and moved.


B . At indicated time points , samples were taken from the reaction mix and analyzed by SDS ‑ PAGE and fluorescence scan . Product fluorescence over total fluorescence per lane was quantified ( rel . fluo . ) . D . Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full ‑ length Ubc1 or Ubc1 - ΔUBA ( aa1 ‑ 150 ) . The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin , while acceptor sites were blocked by introducing the K48R substitution . Reactions were performed in triplicate .

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B. At indicated time points, samples were taken from the reaction mix and analyzed by SDS‑PAGE and fluorescence scan. Product fluorescence over total fluorescence per lane was quantified (rel. fluo.). D. Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full‑length Ubc1 or Ubc1-ΔUBA (aa1‑150). The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin, while acceptor sites were blocked by introducing the K48R substitution. Reactions were performed in triplicate.


b . L143E and / or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC / UBA domain interface . Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63 ‑ Ub2 with Ub ( K48R ) at the distal position or monoubiquitin as acceptor . Experiment and initial rate

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

b. L143E and/or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC/UBA domain interface. Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63‑Ub2 with Ub(K48R) at the distal position or monoubiquitin as acceptor. Experiment and initial rate determination


c . Ubc1 variants were generated with deletions in the linker region ( aa152 ‑ 167 ) , which connects the UBA domain and the UBC domain . Single turnover ubiquitination reaction mixes were incubated for 5 min with K63 - Ub2 or for 20 min with monoubiquitin as acceptor . Final product formation was analyzed by SDS - PAGE and fluorescence scan . Product fluorescence over total fluorescence ( rel . fluo . ) was quantified .

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c. Ubc1 variants were generated with deletions in the linker region (aa152‑167), which connects the UBA domain and the UBC domain. Single turnover ubiquitination reaction mixes were incubated for 5 min with K63-Ub2 or for 20 min with monoubiquitin as acceptor. Final product formation was analyzed by SDS-PAGE and fluorescence scan. Product fluorescence over total fluorescence (rel. fluo.) was quantified.


d . C - terminally blocked M1 - linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full - length Ubc1 or Ubc1 - ΔUBA . Reactions with acceptor K63 - Ub2 were carried out controls . Initial rate

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

d. C-terminally blocked M1-linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full-length Ubc1 or Ubc1-ΔUBA. Reactions with acceptor K63-Ub2 were carried out controls. Initial rate determination


e . CSPs of I44 were plotted over UBA domain concentration . f . KD values for binding of the UBA domain were determined from results in ( E ) assuming two independent but different binding sites .

O O O O O O O O O O O O O O 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. CSPs of I44 were plotted over UBA domain concentration. f. KD values for binding of the UBA domain were determined from results in (E) assuming two independent but different binding sites.


a . The activity of Ubc1 was compared with the K48 - linked chain building enzyme Ubc7 / Cue1 in single turnover ubiquitination experiments in presence of either monoubiquitin , K48 ‑ Ub2 or K63 - Ub2 . Initial rates were

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a. The activity of Ubc1 was compared with the K48-linked chain building enzyme Ubc7/Cue1 in single turnover ubiquitination experiments in presence of either monoubiquitin, K48‑Ub2 or K63-Ub2. Initial rates were determined


b . The K63 - linked chain building enzyme Ubc13 with its cofactor Uev1a or the Ubc1 homologue Ube2K ( h . sapiens ) were employed in single turnover ubiquitination experiments in presence of K63 - Ub2 with Ub ( K48R ) at the proximal or distal position or in presence of K48 ‑ Ub2 . Initial rate determination c . Experiment as in ( B ) but with the Ubc1 homologue ubc - 20 ( c . elegans ) .

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b. The K63-linked chain building enzyme Ubc13 with its cofactor Uev1a or the Ubc1 homologue Ube2K (h. sapiens) were employed in single turnover ubiquitination experiments in presence of K63-Ub2 with Ub(K48R) at the proximal or distal position or in presence of K48‑Ub2. Initial rate determination c. Experiment as in (B) but with the Ubc1 homologue ubc-20 (c. elegans).


D . Alexa488 - labeled monoubiquitin and C - terminally capped K63 - Ub4 were used in a 4 : 1 molar ratio for in vitro ubiquitination reactions with full ‑ length Ubc1 or Ubc1 - ΔUBA . Where indicated , Alexa488 - labeled monoubiquitin was methylated prior to the reaction ( met - Ub ) to block de novo chain assembly . Reactions were incubated overnight , quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH ( lanes 4 - 6 ) . Samples were analyzed by SDS - PAGE and fluorescence scan .

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D. Alexa488-labeled monoubiquitin and C-terminally capped K63-Ub4 were used in a 4:1 molar ratio for in vitro ubiquitination reactions with full‑length Ubc1 or Ubc1-ΔUBA. Where indicated, Alexa488-labeled monoubiquitin was methylated prior to the reaction (met-Ub) to block de novo chain assembly. Reactions were incubated overnight, quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH (lanes 4-6). Samples were analyzed by SDS-PAGE and fluorescence scan.


F . Ubc1 - EA was genomically integrated into various yeast strains . Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10 ‑ fold dilutions . Where noted cells were incubated at elevated temperature ( 37 ° C ) or in presence of hydroxyurea ( HU ) to induce DNA replication stress .

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F. Ubc1-EA was genomically integrated into various yeast strains. Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10‑fold dilutions. Where noted cells were incubated at elevated temperature (37 °C) or in presence of hydroxyurea (HU) to induce DNA replication stress.


CSF extract was treated with DMSO , CsA , buffer or His - CnA420 - 508 at the indicated concentrations . Meiotic exit was induced by calcium addition and samples were taken at the indicated time points . Samples were immunoblotted for cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin .

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CSF extract was treated with DMSO, CsA, buffer or His-CnA420-508 at the indicated concentrations. Meiotic exit was induced by calcium addition and samples were taken at the indicated time points. Samples were immunoblotted for cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin.


CSF extract was treated with DMSO or CsA . Both reactions were divided and supplemented with the indicated amounts of calcium or H2O . After 20min , samples were taken and immunoblotted for cyclin B2 for which a low and high exposure is shown . The cyclin B2 membrane was stripped and reprobed for α - tubulin .

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CSF extract was treated with DMSO or CsA. Both reactions were divided and supplemented with the indicated amounts of calcium or H2O. After 20min, samples were taken and immunoblotted for cyclin B2 for which a low and high exposure is shown. The cyclin B2 membrane was stripped and reprobed for α-tubulin.


CSF extract was supplemented with Myc - XErp1 wt IVT at the indicated dilutions . An empty IVT reaction not expressing Myc - XErp1 served as control . All reactions were divided and treated with DMSO or CsA . Calcium was added to all reactions and samples were taken at the indicated time points , treated with λ - phosphatase and immunoblotted for XErp1 , the Myc - tag , cyclin B2 and α - tubulin . Asterisks indicate unspecific bands .

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CSF extract was supplemented with Myc-XErp1 wt IVT at the indicated dilutions. An empty IVT reaction not expressing Myc-XErp1 served as control. All reactions were divided and treated with DMSO or CsA. Calcium was added to all reactions and samples were taken at the indicated time points, treated with λ-phosphatase and immunoblotted for XErp1, the Myc-tag, cyclin B2 and α-tubulin. Asterisks indicate unspecific bands.


CSF extract was supplemented with Myc - XErp1 wt IVT . The reaction was divided and treated with DMSO or CsA . Both reactions were treated with calcium , samples were taken at the indicated time points and as indicated incubated with λ - phosphatase . Samples were immunoblotted for XErp1 and cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin . Asterisk indicates unspecific bands .

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CSF extract was supplemented with Myc-XErp1 wt IVT. The reaction was divided and treated with DMSO or CsA. Both reactions were treated with calcium, samples were taken at the indicated time points and as indicated incubated with λ-phosphatase. Samples were immunoblotted for XErp1 and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.


CSF extract was supplemented with Myc - XErp1 DSG - DSA - ZBR - ( S33N S38N S284N S288N C583A ) IVT and as indicated treated with DMSO or CsA . Both reactions were treated with calcium and samples were taken at the indicated time points . Samples were immunoblotted for the Myc - tag , cyclin B2 and α - tubulin . A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A . Arrow marks the meiotic phosphorylation state of Myc - XErp1 . Asterisk indicates unspecific bands .

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

CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT and as indicated treated with DMSO or CsA. Both reactions were treated with calcium and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag, cyclin B2 and α-tubulin. A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A. Arrow marks the meiotic phosphorylation state of Myc-XErp1. Asterisk indicates unspecific bands.


CSF extract was supplemented with Myc - XErp1 DSG - DSA - ZBR - ( S33N S38N S284N S288N C583A ) IVT that was either wild - type or mutated to alanine at the p90RSK - target sites S335 , T336 and S342 ( Rsk3A ) . Both reactions were divided and treated with DMSO or CsA . Calcium was added and samples were taken at the indicated time points . Samples were immunoblotted for the Myc - tag and cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin . Asterisk indicates unspecific bands .

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

CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT that was either wild-type or mutated to alanine at the p90RSK-target sites S335, T336 and S342 (Rsk3A). Both reactions were divided and treated with DMSO or CsA. Calcium was added and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.


CSF extract was treated with Myc - XErp1 CaMKII - ZBR - ( T195A C583A ) IVT . An empty IVT reaction not expressing XErp1 was used as control . The extracts were treated with DMSO or CsA as indicated . Calcium was added , samples were taken at the indicated time points and immunoblotted for cyclin B2 , pSer335 XErp1 and α - tubulin . The pSer335 XErp1 membrane was stripped and reprobed for the Myc - tag . Asterisks indicate unspecific bands .

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

CSF extract was treated with Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT. An empty IVT reaction not expressing XErp1 was used as control. The extracts were treated with DMSO or CsA as indicated. Calcium was added, samples were taken at the indicated time points and immunoblotted for cyclin B2, pSer335 XErp1 and α-tubulin. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisks indicate unspecific bands.


Myc - XErp1 CaMKII - ZBR - ( T195A C583A ) IVT was in vitro phosphorylated by recombinant PKA and isolated by α - Myc immunoprecipitation . The immunoprecipitate was supplemented with calcium and treated with recombinant hs _ CaN and calmodulin as indicated . Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1 . The pSer335 XErp1 membrane was stripped and reprobed for the Myc - tag . Asterisk indicates the IgG heavy chain .

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

Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT was in vitro phosphorylated by recombinant PKA and isolated by α-Myc immunoprecipitation. The immunoprecipitate was supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisk indicates the IgG heavy chain.


CSF extract was supplemented with mRNA encoding Flag - Cdc20 that was either wild - type or mutated to serine at Thr64 , Thr68 and Thr79 ( 3TS ) . After expression , calcium was added , samples were taken at the indicated time points and resolved by either conventional or Phos - tagTM SDS - PAGE . Samples were immunoblotted for the Flag - tag , Cdc27 and cyclin B2 .

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CSF extract was supplemented with mRNA encoding Flag-Cdc20 that was either wild-type or mutated to serine at Thr64, Thr68 and Thr79 (3TS). After expression, calcium was added, samples were taken at the indicated time points and resolved by either conventional or Phos-tagTM SDS-PAGE. Samples were immunoblotted for the Flag-tag, Cdc27 and cyclin B2.


CSF extract was supplemented with Myc - XErp1 wt IVT and treated with DMSO or CsA . Calcium was added , samples were taken at the indicated time points , resolved by either conventional or Phos - tagTM SDS - PAGE and immunoblotted for Cdc20 , Cdc27 and cyclin B2 . A control sample was treated with λ - phosphatase . A pseudocoloured representation of the Cdc20 and the Cdc27 immunoblots is shown in Fig . EV4A . The cyclin B2 membrane was stripped and reprobed for α - tubulin .

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

CSF extract was supplemented with Myc-XErp1 wt IVT and treated with DMSO or CsA. Calcium was added, samples were taken at the indicated time points, resolved by either conventional or Phos-tagTM SDS-PAGE and immunoblotted for Cdc20, Cdc27 and cyclin B2. A control sample was treated with λ-phosphatase. A pseudocoloured representation of the Cdc20 and the Cdc27 immunoblots is shown in Fig. EV4A. The cyclin B2 membrane was stripped and reprobed for α-tubulin.


CSF extract was supplemented with Flag - Cdc20 wt IVT coupled to α - Flag beads . Calcium was added and Flag - Cdc20 was isolated by α - Flag immunoprecipitation at the indicated time points . Input and pellet ( IP : Flag ) samples were immunoblotted for the Flag - tag , pThr68 Cdc20 and p150 ( Glued ) . The pThr68 Cdc20 membrane was stripped and reprobed for cyclin B2 . Asterisk in the cyclin B2 immunoblot indicates the IgG heavy chain . Asterisks in other immunoblots indicate unspecific bands .

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

CSF extract was supplemented with Flag-Cdc20 wt IVT coupled to α-Flag beads. Calcium was added and Flag-Cdc20 was isolated by α-Flag immunoprecipitation at the indicated time points. Input and pellet (IP: Flag) samples were immunoblotted for the Flag-tag, pThr68 Cdc20 and p150(Glued). The pThr68 Cdc20 membrane was stripped and reprobed for cyclin B2. Asterisk in the cyclin B2 immunoblot indicates the IgG heavy chain. Asterisks in other immunoblots indicate unspecific bands.


CSF extract was supplemented with Flag - Cdc20 wt IVT . Flag - Cdc20 was re - isolated by α - Flag immunoprecipitation , supplemented with calcium and treated with recombinant hs _ CaN and calmodulin as indicated . Samples were taken at the indicated time points and immunoblotted for the Flag - tag and pThr68 Cdc20 . The pThr68 Cdc20 membrane was stripped and reprobed for the catalytic calcineurin subunit CnA . Asterisk indicates the IgG heavy chain .

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

CSF extract was supplemented with Flag-Cdc20 wt IVT. Flag-Cdc20 was re-isolated by α-Flag immunoprecipitation, supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the Flag-tag and pThr68 Cdc20. The pThr68 Cdc20 membrane was stripped and reprobed for the catalytic calcineurin subunit CnA. Asterisk indicates the IgG heavy chain.


( a ) Left , cells were fixed and stained for anti - pTyr - 416 - Src ( green ) and with DAPI ( blue ) . Solid arrows indicate cells with active Src in adhesions ; dashed arrows show cells with active Src in puncta . Scale bars , 20 μm . Right , percentage of cells with active Src localizing to intracellular structures was quantified . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) .

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

(a) Left, cells were fixed and stained for anti-pTyr-416-Src (green) and with DAPI (blue). Solid arrows indicate cells with active Src in adhesions; dashed arrows show cells with active Src in puncta. Scale bars, 20 μm. Right, percentage of cells with active Src localizing to intracellular structures was quantified. Data are presented as mean±s.d. and significance is P0.001 (n=3).


( b ) Cells were fixed and stained for anti - FAK ( red ) and anti - pTyr - 416 - Src ( green ) . Merged and higher - magnification images of the outlined areas are also shown . Solid and dashed arrows show Src localization to adhesions or to puncta , respectively . Scale bars , 20 μm .

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

(b) Cells were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Merged and higher-magnification images of the outlined areas are also shown. Solid and dashed arrows show Src localization to adhesions or to puncta, respectively. Scale bars, 20 μm.


( c ) Lysates from cells were immunoblotted with anti - FAK , anti - Src , anti - Yes , anti - Fyn , anti - pTyr - 416 - Src and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(c) Lysates from cells were immunoblotted with anti-FAK, anti-Src, anti-Yes, anti-Fyn, anti-pTyr-416-Src and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Cells were fixed and stained for anti - pTyr - 416 - Src ( red ) and co - stained for anti - LC3B , anti - Atg7 or anti - Atg12 ( all green ) . Scale bars , 20 μm . ( b ) Higher - magnification images of the areas outlined in a . Solid arrows indicate co - localization in focal adhesions and dashed arrows show puncta in the cytoplasm .

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

(a) Cells were fixed and stained for anti-pTyr-416-Src (red) and co-stained for anti-LC3B, anti-Atg7 or anti-Atg12 (all green). Scale bars, 20 μm. (b) Higher-magnification images of the areas outlined in a. Solid arrows indicate co-localization in focal adhesions and dashed arrows show puncta in the cytoplasm.


( c ) LC3B was immunoprecipitated ( IP ) from FAK + / + and FAK − / − cells and then immunoblotted with anti - Src , anti - pTyr - 416 - Src and anti - LC3B antibodies .

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

(c) LC3B was immunoprecipitated (IP) from FAK+/+ and FAK−/− cells and then immunoblotted with anti-Src, anti-pTyr-416-Src and anti-LC3B antibodies.


( d ) Cells were transfected with 40 nM scrambled , Atg5 or Atg12 siRNA for 72 h and immunoblotted with anti - Atg5 , anti - Atg12 and anti - actin antibodies ( lower panels ) or fixed and stained for anti - pTyr - 416 - Src ( green ) , anti - paxillin ( red ) and with DAPI ( blue ) . Dashed arrows point to intracellular phospho - Src - containing puncta ; solid arrows point to peripheral phospho - Src . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(d) Cells were transfected with 40 nM scrambled, Atg5 or Atg12 siRNA for 72 h and immunoblotted with anti-Atg5, anti-Atg12 and anti-actin antibodies (lower panels) or fixed and stained for anti-pTyr-416-Src (green), anti-paxillin (red) and with DAPI (blue). Dashed arrows point to intracellular phospho-Src-containing puncta; solid arrows point to peripheral phospho-Src. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Top , cells were transiently transfected with an RFP - GFP tandem fluorescent - tagged LC3 ( RFP - GFP - LC3 ) . Scale bars , 20 μm . Bottom , the number of yellow puncta and the number of RFP LC3 - positive puncta in the merged images were counted and the total number of puncta per cell was calculated . Data are presented as mean ± s . d . ( n = 3 ) .

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

(a) Top, cells were transiently transfected with an RFP-GFP tandem fluorescent-tagged LC3 (RFP-GFP-LC3). Scale bars, 20 μm. Bottom, the number of yellow puncta and the number of RFP LC3-positive puncta in the merged images were counted and the total number of puncta per cell was calculated. Data are presented as mean±s.d. (n=3).


b ) Cells were treated with 10 μM chloroquine for 24 h and then immunoblotted using anti - pTyr - 416 - Src , anti - Src , anti - LC3B and anti - actin antibodies . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each cell type following treatment with chloroquine and is presented as a percentage increase for the immunoblot shown .

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

b) Cells were treated with 10 μM chloroquine for 24 h and then immunoblotted using anti-pTyr-416-Src, anti-Src, anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type following treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.


( c ) Cells were treated with 200 nM epoxomicin for 4 h and then immunoblotted and probed with anti - pTyr - 416 - Src , anti - Src , anti - p53 and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(c) Cells were treated with 200 nM epoxomicin for 4 h and then immunoblotted and probed with anti-pTyr-416-Src, anti-Src, anti-p53 and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Top , FAK − / − cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti - Src antibody ( green ) . Solid arrows indicate Src at the cell periphery ; dashed arrows indicate Src in autophagosomes . Scale bars , 20 μm . Bottom , quantification of dasatinib - treated cells with Src in puncta . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) . Middle , lysates from cells treated with dasatinib were immunoblotted with anti - Src and anti - pTyr - 416 - Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti - LC3B and anti - actin antibodies . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown .

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

(a) Top, FAK−/− cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti-Src antibody (green). Solid arrows indicate Src at the cell periphery; dashed arrows indicate Src in autophagosomes. Scale bars, 20 μm. Bottom, quantification of dasatinib-treated cells with Src in puncta. Data are presented as mean±s.d. and significance is P0.001 (n=3). Middle, lysates from cells treated with dasatinib were immunoblotted with anti-Src and anti-pTyr-416-Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.


( b ) Top , FAK − / − cells were transiently transfected with SrcY527F - GFP or with Src - 251 - GFP ( green ) and stained for anti - LC3B ( red ) . Scale bars , 20 μm . Bottom , quantification of cells with co - localization between Src and LC3B . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) .

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

(b) Top, FAK−/− cells were transiently transfected with SrcY527F-GFP or with Src-251-GFP (green) and stained for anti-LC3B (red). Scale bars, 20 μm. Bottom, quantification of cells with co-localization between Src and LC3B. Data are presented as mean±s.d. and significance is P0.001 (n=3).


( c ) Left , FAK − / − cells stably re - expressing wild - type FAK , FAKY397F or FAKY4F - Y9F were fixed and stained for anti - FAK ( red ) and anti - pTyr - 416 - Src ( green ) . Solid arrows indicate co - localization at adhesions and dashed arrows indicate active Src in autophagosomes . Scale bars , 20 μm . Right , lysates from these cells were also immunoblotted with anti - FAK and anti - actin antibodies .

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

(c) Left, FAK−/− cells stably re-expressing wild-type FAK, FAKY397F or FAKY4F-Y9F were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate active Src in autophagosomes. Scale bars, 20 μm. Right, lysates from these cells were also immunoblotted with anti-FAK and anti-actin antibodies.


( d ) Left , wild - type FAK , FAKY397F or FAKY4F - Y9F cells were transfected with RFP - GFP - LC3 . Solid arrows indicate co - localization and dotted arrows indicate its absence . Scale bars , 20 μm .

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

(d) Left, wild-type FAK, FAKY397F or FAKY4F-Y9F cells were transfected with RFP-GFP-LC3. Solid arrows indicate co-localization and dotted arrows indicate its absence. Scale bars, 20 μm.


( e ) Lysates were immunoblotted with anti - LC3B and actin antibodies in the absence and presence of chloroquine . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each cell type after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(e) Lysates were immunoblotted with anti-LC3B and actin antibodies in the absence and presence of chloroquine. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Wild - type FAK cells were suspended in 1 . 4 % methylcellulose solution in growth media and plated on agarose for 3 days . Top and bottom right , cells were recovered and cytospins prepared that were stained for anti - pTyr - 416 - Src ( red ) , LC3B ( green ) and with DAPI ( blue ) or for paxillin ( red ) , LC3B ( green ) and with DAPI ( blue ) . Higher - magnification images of the outlined areas are also shown . Solid arrows indicate localization of pTyr - 416 - Src in LC3B - positive puncta . Scale bars , 20 μm . Bottom left , immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti - pTyr - 397 FAK , anti - FAK , anti - pTyr - 416 and anti - Src antibodies .

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

(a) Wild-type FAK cells were suspended in 1.4% methylcellulose solution in growth media and plated on agarose for 3 days. Top and bottom right, cells were recovered and cytospins prepared that were stained for anti-pTyr-416-Src (red), LC3B (green) and with DAPI (blue) or for paxillin (red), LC3B (green) and with DAPI (blue). Higher-magnification images of the outlined areas are also shown. Solid arrows indicate localization of pTyr-416-Src in LC3B-positive puncta. Scale bars, 20 μm. Bottom left, immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti-pTyr-397 FAK, anti-FAK, anti-pTyr-416 and anti-Src antibodies.


( b ) FAK + / + cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti - pTyr - 416 - Src ( green ) and with DAPI ( blue ) . A higher - magnification image of the area outlined is also shown . Scale bar , 20 μm .

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

(b) FAK+/+ cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti-pTyr-416-Src (green) and with DAPI (blue). A higher-magnification image of the area outlined is also shown. Scale bar, 20 μm.


( c ) LC3B was immunoprecipitated ( IP ) from adherent or suspended FAK + / + and then immunoblotted with anti - Src and anti - LC3B antibodies . Lysates were also immunoblotted with anti - pTyr - 397 - FAK and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(c) LC3B was immunoprecipitated (IP) from adherent or suspended FAK+/+ and then immunoblotted with anti-Src and anti-LC3B antibodies. Lysates were also immunoblotted with anti-pTyr-397-FAK and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Top , FAK − / − cells were sparsely plated and then treated with 3 - MA ( for 48 h ) , chloroquine or dasatinib ( both for 24 h ) . Cells were then left at 37 ° C for 1 week to allow colony formation , fixed and then stained with crystal violet . Bottom , the number of colonies formed was then quantified and normalized against untreated controls . Data are presented as mean ± s . d . and significance is P0 . 001 for 3 - MA , P0 . 01 for chloroquine and P > 0 . 5 for dasatinib ( n = 3 ) .

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

(a) Top, FAK−/− cells were sparsely plated and then treated with 3-MA (for 48 h), chloroquine or dasatinib (both for 24 h). Cells were then left at 37 °C for 1 week to allow colony formation, fixed and then stained with crystal violet. Bottom, the number of colonies formed was then quantified and normalized against untreated controls. Data are presented as mean±s.d. and significance is P0.001 for 3-MA, P0.01 for chloroquine and P>0.5 for dasatinib (n=3).


( b ) Top , wild - type FAK and FAK − / − cells were transiently transfected with two individual Atg5 shRNAs ( 1 and 2 ) , selected in puromycin and then the clonogenic assay was carried out as described above . Bottom , quantification ; data are presented as mean ± s . d . and significance is P > 0 . 1 for Atg5 A and B in wild - type FAK cells and P0 . 01 for Atg5 A and P0 . 05 for Atg5 B in FAK − / − cells ( n = 3 ) .

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

(b) Top, wild-type FAK and FAK−/− cells were transiently transfected with two individual Atg5 shRNAs (1 and 2), selected in puromycin and then the clonogenic assay was carried out as described above. Bottom, quantification; data are presented as mean±s.d. and significance is P>0.1 for Atg5 A and B in wild-type FAK cells and P0.01 for Atg5 A and P0.05 for Atg5 B in FAK−/− cells (n=3).


( c ) Cells were also stained with TUNEL . The positive control was DNase1 - treated cells and the negative control lacked TUNEL reaction mix . Quantification is shown and data are presented as mean ± s . d . and significance is P0 . 005 for 3 - MA and P0 . 001 for Atg5 siRNA ( n = 3 ) .

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

(c) Cells were also stained with TUNEL. The positive control was DNase1-treated cells and the negative control lacked TUNEL reaction mix. Quantification is shown and data are presented as mean±s.d. and significance is P0.005 for 3-MA and P0.001 for Atg5 siRNA (n=3).


( d ) FAK − / − cells were untreated or treated with dasatinib , 3 - MA , chloroquine or bafilomycin A ( BFA ) for 24 h , or were transfected with scrambled or Atg5 siRNA for 72 h and then immunoblotted with an anti - PARP antibody . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(d) FAK−/− cells were untreated or treated with dasatinib, 3-MA, chloroquine or bafilomycin A (BFA) for 24 h, or were transfected with scrambled or Atg5 siRNA for 72 h and then immunoblotted with an anti-PARP antibody. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Cells untreated , or treated with chloroquine for 24 h , were fixed and stained for anti - pTyr - 416 - Src ( red ) and anti - c - Cbl ( green ) . Solid arrows indicate co - localization at adhesions and dashed arrows indicate co - localization in autophagosomes . Scale bars , 20 μm .

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

(a) Cells untreated, or treated with chloroquine for 24 h, were fixed and stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate co-localization in autophagosomes. Scale bars, 20 μm.


( b ) c - Cbl was immunoprecipitated ( IP ) from FAK + / + and FAK − / − cells and then immunoblotted with anti - Src , anti - pTyr - 416 - Src and anti - c - Cbl antibodies .

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

(b) c-Cbl was immunoprecipitated (IP) from FAK+/+ and FAK−/− cells and then immunoblotted with anti-Src, anti-pTyr-416-Src and anti-c-Cbl antibodies.


( c , d ) Cells were transfected with scrambled siRNA or with c - Cbl siRNA for 72 h and then immunoblotted with anti - c - Cbl and actin antibodies ( c ) or stained for anti - pTyr - 416 - Src ( red ) and anti - c - Cbl ( green ; d , left ) . Dashed arrows indicate co - localization and solid arrows indicate Src at focal adhesions . Scale bars , 20 μm . Quantification is shown ( d , right ) . Data are presented as mean ± s . d . and significance is P > 0 . 5 for FAK + / + cells and P0 . 001 for FAK − / − cells ( n = 3 ) .

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(c,d) Cells were transfected with scrambled siRNA or with c-Cbl siRNA for 72 h and then immunoblotted with anti-c-Cbl and actin antibodies (c) or stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green; d, left). Dashed arrows indicate co-localization and solid arrows indicate Src at focal adhesions. Scale bars, 20 μm. Quantification is shown (d, right). Data are presented as mean±s.d. and significance is P>0.5 for FAK+/+ cells and P0.001 for FAK−/− cells (n=3).


( e ) LC3B was immunoprecipitated from FAK − / − cells expressing either scrambled or c - Cbl siRNA and then immunoblotted with anti - Src and anti - LC3B antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(e) LC3B was immunoprecipitated from FAK−/− cells expressing either scrambled or c-Cbl siRNA and then immunoblotted with anti-Src and anti-LC3B antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) c - Cbl siRNA was transfected into FAK − / − cells expressing siRNA resistant , HA - tagged wild - type c - Cbl or c - Cbl mutants with defective E3 ligase activity ( c - CblC381A or c - Cbl - 70Z ) . Left , cells were stained for anti - p - Tyr - 416 - Src ( green ) , anti - HA ( red ) and with DAPI ( blue ) . Solid arrows show co - localization in intracellular puncta . Scale bars , 20 μm . Right , quantification of percentage of cells that contained active Src in intracellular puncta . Data are presented as mean ± s . d . and significance is P > 0 . 5 ( n = 3 ) .

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

(a) c-Cbl siRNA was transfected into FAK−/− cells expressing siRNA resistant, HA-tagged wild-type c-Cbl or c-Cbl mutants with defective E3 ligase activity (c-CblC381A or c-Cbl-70Z). Left, cells were stained for anti-p-Tyr-416-Src (green), anti-HA (red) and with DAPI (blue). Solid arrows show co-localization in intracellular puncta. Scale bars, 20 μm. Right, quantification of percentage of cells that contained active Src in intracellular puncta. Data are presented as mean±s.d. and significance is P>0.5 (n=3).


( c ) GST - pulldown assay of HA - tagged wild - type c - Cbl , c - CblAA ( W802A , L805A ) and c - CblAAAA ( W802A , L803A , S804A , L805A ) expressed in HEK293T cells was carried out using GST - LC3 .

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

(c) GST-pulldown assay of HA-tagged wild-type c-Cbl, c-CblAA (W802A, L805A) and c-CblAAAA (W802A, L803A, S804A, L805A) expressed in HEK293T cells was carried out using GST-LC3.


( d ) c - Cbl siRNA was transfected into FAK − / − cells expressing siRNA - resistant HA - tagged wild - type c - Cbl or c - CblAAAA . Left , cells were stained for anti - pTyr - 416 - Src ( green ) , anti - HA ( red ) and with DAPI ( blue ) . Solid arrows indicate pTyr - 416 localization to adhesions and dashed arrows indicate localization to intracellular puncta . Scale bars , 20 μm . Right , quantification of percentage of cells that contained active Src in intracellular puncta . Data are presented as mean ± s . d . and significance is P0 . 01 ( n = 3 ) . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(d) c-Cbl siRNA was transfected into FAK−/− cells expressing siRNA-resistant HA-tagged wild-type c-Cbl or c-CblAAAA. Left, cells were stained for anti-pTyr-416-Src (green), anti-HA (red) and with DAPI (blue). Solid arrows indicate pTyr-416 localization to adhesions and dashed arrows indicate localization to intracellular puncta. Scale bars, 20 μm. Right, quantification of percentage of cells that contained active Src in intracellular puncta. Data are presented as mean±s.d. and significance is P0.01 (n=3). Uncropped images of blots are shown in Supplementary Fig. S9.


( A ) The mRNA levels of QKI relative to those of 36B4 in BAT and ingWAT from HFD mice ( n = 8 ) and lean controls ( n = 8 ) .

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(A)The mRNA levels of QKI relative to those of 36B4 in BAT and ingWAT from HFD mice (n = 8) and lean controls (n = 8).


WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed a NCD or HFD for 20 weeks . The arrow indicates the time when the high fat diet （ HFD ） starts , and body weight was monitored weekly . ( n = 10 , per genotype )

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WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed a NCD or HFD for 20 weeks. The arrow indicates the time when the high fat diet（HFD）starts, and body weight was monitored weekly. (n = 10, per genotype)


Representative pictures of adipocyte - specific QKI - deficient mice under NCD or HFD conditions .

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Representative pictures of adipocyte-specific QKI-deficient mice under NCD or HFD conditions.


Gross appearance of tissues from BAT , eWAT and ingWAT fat pads from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice after 20 weeks on the NCD or HFD .

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Gross appearance of tissues from BAT, eWAT and ingWAT fat pads from WT fl/fl and QKI KO fl/fl:AP2-Cre mice after 20 weeks on the NCD or HFD.


H & E staining of BAT , eWAT and ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( Scale bar represents 50 μm )

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H&E staining of BAT, eWAT and ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm)


The gross appearance of BAT , eWAT and ingWAT fat pads after administration of AAV - ShQKI or AAV - NC vectors in local adipose ( ingWAT and BAT ) .

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The gross appearance of BAT, eWAT and ingWAT fat pads after administration of AAV-ShQKI or AAV-NC vectors in local adipose (ingWAT and BAT).


The increase in relative body weights following HFD feeding . ( n = 8 , per group )

The increase in relative body weights following HFD feeding. (n = 8, per group)


H & E staining of the liver , BAT and ingWAT from AAV - NC or AAV - ShQKI mice . ( Scale bar represents 50 μm ) .

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H&E staining of the liver, BAT and ingWAT from AAV-NC or AAV-ShQKI mice. (Scale bar represents 50 μm).


Representative CT images of WT fl / fl and QKI KO fl / fl : AP2 - Cre mice on an NCD or HFD , showing the whole - body fat composition .

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Representative CT images of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on an NCD or HFD, showing the whole-body fat composition.


Glucose tolerance tests ( GTT ) and Insulin tolerance tests ( ITT ) were performed on WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . Blood glucose concentrations during GTT and ITT were monitored . ( n = 10 , per genotype )

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Glucose tolerance tests (GTT) and Insulin tolerance tests (ITT) were performed on WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. Blood glucose concentrations during GTT and ITT were monitored. (n= 10, per genotype)


Blood glucose and plasma insulin levels in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( n = 8 , per genotype )

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Blood glucose and plasma insulin levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype)


Plasma Free Fatty Acid ( FFA ) and Glycerol levels in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( n = 8 , per genotype )

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Plasma Free Fatty Acid (FFA) and Glycerol levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype)


H & E and Oil Red O staining , and liver triglyceride ( TG ) content in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( Scale bar represents 50 μm )

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H&E and Oil Red O staining, and liver triglyceride (TG) content in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm)


O2 consumption rates were measured via indirect calorimetry using CLAMS under chow - fed conditions ( n = 8 , per genotype ) . And average light and dark O2 consumption rates are shown on the right .

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O2 consumption rates were measured via indirect calorimetry using CLAMS under chow-fed conditions (n = 8, per genotype). And average light and dark O2 consumption rates are shown on the right.


Heat production rates in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice ( n = 8 , per genotype ) . And average light and dark heat production rates are shown on the right .

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Heat production rates in WT fl/fl and QKI KO fl/fl:AP2-Cre mice (n = 8, per genotype). And average light and dark heat production rates are shown on the right.


Heatmaps of log2 ( fold change QKI KO fl / fl : AP2 - Cre BAT vs . WT fl / fl BAT ) for genes involved in thermogenesis and oxidation . ( 3 samples , per group )

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Heatmaps of log2 (fold change QKI KO fl/fl:AP2-Cre BAT vs. WT fl/fl BAT) for genes involved in thermogenesis and oxidation. (3 samples, per group)


Photograph of WT fl / fl and QKI KO fl / fl : AP2 - Cre classical BAT in 4 % paraformaldehyde solution , representative transmission electron micrographs , and UCP1 staining of BAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice .

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Photograph of WT fl/fl and QKI KO fl/fl:AP2-Cre classical BAT in 4% paraformaldehyde solution, representative transmission electron micrographs, and UCP1 staining of BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice.


The average diameter of lipid droplets and the number of mitochondria per field from WT fl / fl and QKI KO fl / fl : AP2 - Cre BAT . ( n = 3 biological replicates )

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The average diameter of lipid droplets and the number of mitochondria per field from WT fl/fl and QKI KO fl/fl:AP2-Cre BAT. (n = 3 biological replicates)


The relative mRNA levels of genes related to thermogenesis in BAT analyzed by RT - PCR . ( n = 4 biological replicates )

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The relative mRNA levels of genes related to thermogenesis in BAT analyzed by RT-PCR. (n = 4 biological replicates)


Total oxygen consumption rates ( OCR ) per gram in BAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice under basal conditions were measured using a Clark electrode . ( n = 3 biological replicates )

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Total oxygen consumption rates (OCR) per gram in BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice under basal conditions were measured using a Clark electrode. (n = 3 biological replicates)


The relative mRNA levels of genes related to thermogenesis in ingWAT analyzed by RT - PCR . ( n = 4 biological replicates )

The relative mRNA levels of genes related to thermogenesis in ingWAT analyzed by RT-PCR. (n = 4 biological replicates)


Total oxygen consumption rates ( OCR ) per gram in ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice under basal conditions . ( n = 3 biological replicates )

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Total oxygen consumption rates (OCR) per gram in ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice under basal conditions. (n = 3 biological replicates)


Representative recording of Continuous measurement of oxygen consumption rate ( OCR ) in isolated mitochondria from BAT of WT fl / fl and QKI KO fl / fl : AP2 - Cre mice on NCD ; Oxygen consumption was performed under basal conditions , following the addition of 0 . 25 mg mitochondria ( Mit ) , 5 mM substrate glycerol - 3 - phosphate ( G3P ) , 2 mM GDP , and 450 mM ADP , 2 μg / ml oligomycin , and 1 . 5 μM FCCP . Quantification of experimental data . ( n = 3 biological replicates , per group )

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Representative recording of Continuous measurement of oxygen consumption rate (OCR) in isolated mitochondria from BAT of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on NCD; Oxygen consumption was performed under basal conditions, following the addition of 0. 25 mg mitochondria (Mit), 5 mM substrate glycerol-3-phosphate (G3P), 2 mM GDP, and 450 mM ADP, 2 μg/ml oligomycin, and 1.5 μM FCCP. Quantification of experimental data. (n=3 biological replicates, per group)


UCP1 - dependent oxygen consumption rates estimated as GDP - inhabitable rates , based on the data shown in ( K ) . ( n = 3 biological replicates )

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UCP1-dependent oxygen consumption rates estimated as GDP-inhabitable rates, based on the data shown in (K). (n=3 biological replicates)


The protein levels of PGC1α and QKI in siRNA - mediated QKI knockdown differentiated cell , and the quantification graph was shown on the right . ( n = 3 biological replicates )

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The protein levels of PGC1α and QKI in siRNA-mediated QKI knockdown differentiated cell, and the quantification graph was shown on the right. (n=3 biological replicates)


Immunostaining of PGC1α and UCP1 in NC , Si - QKI 1 , and Si - QKI 2 adipocytes . ( Scale bar represents 20 μm ) .

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Immunostaining of PGC1α and UCP1 in NC, Si-QKI 1, and Si-QKI 2 adipocytes. (Scale bar represents 20 μm).


The relative mRNA levels of genes related to thermogenesis in adipocytes treated with NC , QKI siRNA or both QKI and PGC1α siRNA . ( n = 3 biological replicates )

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The relative mRNA levels of genes related to thermogenesis in adipocytes treated with NC, QKI siRNA or both QKI and PGC1α siRNA. (n=3 biological replicates)


The oxygen consumption rates in adipocytes treated with NC , QKI siRNA or both QKI and PGC1α siRNA . ( n = 3 biological replicates )

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The oxygen consumption rates in adipocytes treated with NC, QKI siRNA or both QKI and PGC1α siRNA. (n=3 biological replicates)


Representative pictures of oil red O staining in three adipocyte types : ShScramble + ovCon cells , QKI knockdown cells ( ShQKI + ovCon ) and QKI knockdown with QKI - 5 re - expression cells ( ShQKI + ovQKI ) .

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Representative pictures of oil red O staining in three adipocyte types: ShScramble+ovCon cells, QKI knockdown cells (ShQKI+ovCon) and QKI knockdown with QKI-5 re-expression cells (ShQKI+ovQKI).


The protein levels of PGC1α in these adipocytes , and the quantification graph was shown on the right . ( n = 3 biological replicates )

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The protein levels of PGC1α in these adipocytes, and the quantification graph was shown on the right. (n=3 biological replicates)


The relative mRNA levels of genes associated with energy dissipation in ShScramble + ovCon , ShQKI + ovCon and ShQKI + ovQKI differentiated cells . The results represent 3 independent experiments .

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The relative mRNA levels of genes associated with energy dissipation in ShScramble+ovCon, ShQKI+ovCon and ShQKI+ovQKI differentiated cells. The results represent 3 independent experiments.


Construction of a series of luciferase reporter vectors ( PGC1α full - length 3 ' UTR T , A , B , C fragments and PGC1α 3 ' UTR QRE motif sites mutant B M1 , B M2 , C M1 , C M2 fragments ) . Luciferase activity assays to examine the functional QRE motif sites in the PGC1α 3 ' UTR affected by QKI . ( n = 4 biological replicates )

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Construction of a series of luciferase reporter vectors (PGC1α full-length 3'UTR T, A, B, C fragments and PGC1α 3'UTR QRE motif sites mutant B M1, B M2, C M1, C M2 fragments). Luciferase activity assays to examine the functional QRE motif sites in the PGC1α 3'UTR affected by QKI. (n=4 biological replicates)


Construction of a series of luciferase reporter vectors ( UCP1 full - length 3 ' UTR T fragment and UCP1 3 ' UTR QRE motif sites mutant T M1 , T M2 , T M3 fragments ) . Luciferase activity assays to examine the functional QRE motif sites in the UCP1 3 ' UTR affected by QKI . ( n = 4 biological replicates )

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Construction of a series of luciferase reporter vectors (UCP1 full-length 3'UTR T fragment and UCP1 3'UTR QRE motif sites mutant T M1, T M2, T M3 fragments). Luciferase activity assays to examine the functional QRE motif sites in the UCP1 3'UTR affected by QKI. (n=4 biological replicates)


Based on RIP - sequencing data , the distribution of QKI - binding peaks within UCP1 and PGC1α transcript regions .

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

Based on RIP-sequencing data, the distribution of QKI-binding peaks within UCP1 and PGC1α transcript regions.


RNA immunoprecipitation followed by real - time PCR . Specific primer for PGC1α 3 ' UTR region 1 , region 2 and UCP1 3 ' UTR to detect these QRE motif sites region bound to endogenous QKI in brown fat tissue , and data showing the PGC1α and UCP1 transcripts abundance in QKI IP compared with IgG IP . ( n = 4 biological replicates )

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RNA immunoprecipitation followed by real-time PCR. Specific primer for PGC1α 3'UTR region 1, region 2 and UCP1 3'UTR to detect these QRE motif sites region bound to endogenous QKI in brown fat tissue, and data showing the PGC1α and UCP1 transcripts abundance in QKI IP compared with IgG IP. (n=4 biological replicates)


293T cells were transfected with PGC1α wild type or QRE sites mutant 3 ' UTR fragments . RNA immunoprecipitation ( RIP ) to identify these QRE motif sites region bound to endogenous QKI by using anti - QKI antibodies . The results showed that endogenous QKI interacted with full - length wild type QRE motif regions within 3 ' UTRs of

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

293T cells were transfected with PGC1α wild type or QRE sites mutant 3'UTR fragments. RNA immunoprecipitation (RIP) to identify these QRE motif sites region bound to endogenous QKI by using anti-QKI antibodies. The results showed that endogenous QKI interacted with full-length wild type QRE motif regions within 3'UTRs of PGC1α


293T cells were transfected with UCP1 wild type or QRE sites mutant 3 ' UTR fragments . RNA immunoprecipitation ( RIP ) to identify these QRE motif sites region bound to endogenous QKI by using anti - QKI antibodies . The results showed that endogenous QKI interacted with full - length wild type QRE motif regions within 3 ' UTRs of UCP1 .

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

293T cells were transfected with UCP1 wild type or QRE sites mutant 3'UTR fragments. RNA immunoprecipitation (RIP) to identify these QRE motif sites region bound to endogenous QKI by using anti-QKI antibodies. The results showed that endogenous QKI interacted with full-length wild type QRE motif regions within 3'UTRs of UCP1.


The mRNA stability in adipocytes was measured by incubating cells with actinomycin D ( 5 μg / ml ) . Actinomycin D was added to stop transcription , and RNAs were harvested at the indicated times ( x - axis ) after transcription inhibition . Real - time PCR was used to determine remaining UCP1 , PGC1α and Actin mRNA levels compared with the starting time . Increasing PGC1α mRNA half - life by depletion of QKI ( ShScramble T1 / 2 = 4 . 73 h , ShQKI T1 / 2 = 8 . 04 h ) . ( n = 4 biological replicates )

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

The mRNA stability in adipocytes was measured by incubating cells with actinomycin D (5 μg/ml). Actinomycin D was added to stop transcription, and RNAs were harvested at the indicated times (x-axis) after transcription inhibition. Real-time PCR was used to determine remaining UCP1, PGC1α and Actin mRNA levels compared with the starting time. Increasing PGC1α mRNA half-life by depletion of QKI (ShScramble T1/2 = 4.73 h, ShQKI T1/2 = 8.04 h). (n=4 biological replicates)


The nuclear and cytoplasmic fractionation was valid by immunoblotting using cytoplasmic and nuclear protein markers ( GAPDH and Lamin B1 ) to show the purity of fractions .

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

The nuclear and cytoplasmic fractionation was valid by immunoblotting using cytoplasmic and nuclear protein markers (GAPDH and Lamin B1) to show the purity of fractions.