Toxicity of CY190602 in harboring deletion of chromosome 17p ( including p53 locus ) compared to without del17p . Viability of samples was normalized to background apoptosis for samples . For experiments , viability was determined after 72 h of drug treatment . Data represent mean ± SD .

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

Toxicity of CY190602 in patients harboring deletion of chromosome 17p (including p53 locus) compared to patients without del17p. Viability of samples was normalized to background apoptosis for individual patient samples. For all experiments, cell viability was determined after 72 h of drug treatment. Data represent mean ± SD.


B . Rif1 - ∆ C594 retains function to control replication . Growth of a cdc7 - 1 rif1 - ∆ C594 mutant was compared with growth of cdc7 - 1 RIF1 cdc7 - 1 rif1 ∆ strains at 23ºC ( permissive for cdc7 - 1 allele ) , 26ºC ( mild restrictive ) 30ºC ( strict restrictive )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Rif1-∆C594 retains function to control DNA replication. Growth of a cdc7-1 rif1-∆C594 mutant was compared with growth of cdc7-1 RIF1 and cdc7-1 rif1∆ strains at 23ºC (permissive temperature for cdc7-1 allele), 26ºC (mild restrictive temperature) and 30ºC (strict restrictive temperature)


A . Specimen overview of Chromosome VI - left region showing results of ChIP - Seq of Rif1 Rif1 - ∆ C594 . ChIP reads were normalised against reads from corresponding input samples , relative enrichment is plotted for Chromosome VI coordinates 1 - 80 , 000 . Y - axis shows enrichment values ( linear scale , range is 0 to 3 . 5 ) . Values below 1 are shown in grey , values above 1 ( i . e . sequences enriched in ChIP samples ) are coloured blue ( Rif1 ) red ( Rif1 - ∆ C594 ) . Plots show ChIP results from arrested by α - factor ( G1 ) , released from α - factor at 16ºC for 60 min 90 min , or released from α - factor into 0 . 2 M HU for 60 min at 23ºC

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A. Specimen overview of Chromosome VI-left region showing results of ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for Chromosome VI coordinates 1-80,000. Y-axis shows enrichment values (linear scale, range is 0 to 3.5). Values below 1 are shown in grey, and values above 1 (i.e. sequences enriched in ChIP samples) are coloured blue (Rif1) and red (Rif1-∆C594). Plots show ChIP analysis results from cells arrested by α-factor (G1), released from α-factor at 16ºC for 60 min and 90 min, or released from α-factor into 0.2 M HU for 60 min at 23ºC


B . Rap1 - dependent association of Rif1 with promoter regions of Rap1 - controlled genes . ChIP enrichment around PAU3 ( left ) MAM3 ( right ) , both genes whose transcription is controlled by Rap1 . Values above 1 ( i . e . enriched ) shown by overlaid blue red histograms for Rif1 Rif1 - ∆ C594 , respectively . Values below 1 shown in grey

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Rap1-dependent association of Rif1 with the promoter regions of Rap1-controlled genes. ChIP enrichment around PAU3 (left) and MAM3 (right), both genes whose transcription is controlled by Rap1. Values above 1 (i.e. enriched) shown by overlaid blue and red histograms for Rif1 and Rif1-∆C594, respectively. Values below 1 shown in grey


A . ChIP - Seq of Rif1 Rif1 - ∆ C594 shows tRNA gene origin binding , with widened peaks at early origins ARS606 ARS607 in HU block . Plots show Chromosome VI genome coordinates 160 , 000 - 210 , 000 . Plot colours here in following Figures are as in Fig 2B . Widened peaks are not observed in unperturbed S phase samples , or for Rif1 - ∆ C594

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins shows tRNA gene and origin binding, with widened peaks at early origins ARS606 and ARS607 in HU block. Plots show Chromosome VI genome coordinates 160,000- 210,000. Plot colours here and in following Figures are as in Fig 2B. Widened peaks are not observed in unperturbed S phase samples, or for Rif1-∆C594


B . Association of Rif1 - ∆ C594 at replication origins is enhanced in HU block . ChIP enrichment of Rif1 Rif1 - ∆ C594 around early origin ARS1426 ( left ) late origin ARS1412 ( right )

O O O O O O O O O O O O O O O O O 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. Association of Rif1-∆C594 at replication origins is enhanced in HU block. ChIP enrichment of Rif1 and Rif1-∆C594 around early origin ARS1426 (left) and late origin ARS1412 (right)


C . Differential association of Rif1 Rif1 - ∆ C594 to centromeres . ChIP enrichment of Rif1 Rif1 - ∆ C594 around CEN4 locus

O O O 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. Differential association of Rif1 and Rif1-∆C594 to centromeres. ChIP enrichment of Rif1 and Rif1-∆C594 around the CEN4 locus


A . ChIP - qPCR confirmation of Rif1 Rif1 - ∆ C594 association with replication origins . ChIP was performed using arrested in α - factor ( open bars ) or HU ( grey bars ) , qPCR performed for early origin ARS1426 ( left ) late origin ARS1412 ( right ) . Values shown are ' normalised ChIP efficiencies ' obtained by subtracting value obtained at a control locus ( see Materials Methods ) . Bars indicate averages of two biological replicates , with values from each replicate shown by open circles

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ChIP-qPCR confirmation of Rif1 and Rif1-∆C594 association with replication origins. ChIP was performed using cells arrested in α-factor (open bars) or HU (grey bars), and qPCR analysis performed for early origin ARS1426 (left) and late origin ARS1412 (right). Values shown are 'normalised ChIP efficiencies' obtained by subtracting the value obtained at a control locus (see Materials and Methods). Bars indicate the averages of two biological replicates, with values from each replicate shown by open circles


B . Numbers of early late origins associated with Rif1 Rif1 - ∆ C594 peaks . Plot showing numbers of early late origins bound by Rif1 Rif1 - ∆ C594 in G1 HU - blocked , on peak - calling results

O O O O O O O O O O O O O O O O O O O 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. Numbers of early and late origins associated with Rif1 and Rif1-∆C594 peaks. Plot showing numbers of early and late origins bound by Rif1 and Rif1-∆C594 in G1 and HU-blocked cells, based on peak-calling results


C . Replication timing programme is intact in rif1 - ∆ C594 . Replication of selected origins at an HU block analysed by BrdU incorporation . were synchronised by α - factor released into medium containing 0 . 2 M HU 1 . 13 mM BrdU . Plots show of total ARS607 , ARS422 . 5 , or ARS1412 pulled down by IP with anti - BrdU , calculated from two biological replicate samples . Bars indicate average of two biological replicates open circles values from each replicate . Insets in ARS422 , 5 ARS1412 panels show same data with Y - axis scales adjusted to 0 - 0 . 03 %

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C. Replication timing programme is intact in rif1-∆C594 cells. Replication of selected origins at an HU block analysed by BrdU incorporation. Cells were synchronised by α-factor and released into the medium containing 0.2 M HU and 1.13 mM BrdU. Plots show the percentage of total ARS607, ARS422.5, or ARS1412 DNA pulled down by IP with anti-BrdU, calculated from two biological replicate samples. Bars indicate the average of two biological replicates and open circles the values from each replicate. Insets in ARS422,5 and ARS1412 panels show the same data with Y-axis scales adjusted to 0 - 0.03%


Expression of YAP / TAZ a smaller ( asterisk ) in different lines , expression was determined by immunoblotting ( IB ) .

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

Expression of YAP/TAZ and a smaller protein (asterisk) in different cell lines, protein expression was determined by immunoblotting (IB).


shRNA targeting TAZ , but not YAP , knocked down expression of smaller ( asterisk ) .

The shRNA targeting TAZ, but not YAP, knocked down the expression of the smaller protein (asterisk).


Antibodies targeting C - terminus YAP / TAZ , such as TAZ ( CST ) YAP / TAZ , effectively pulled down smaller ( asterisk , dubbed as cTAZ hereafter ) in RKO in an immunoprecipitation ( IP ) . cTAZ was not recognized by TAZ ( SA ) , an antibody targeting N - terminus of TAZ .

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

Antibodies targeting C-terminus YAP/TAZ, such as TAZ (CST) and YAP/TAZ, effectively pulled down the smaller protein (asterisk, dubbed as cTAZ hereafter) in RKO cells in an immunoprecipitation (IP) assay. cTAZ was not recognized by TAZ (SA), an antibody targeting N-terminus of TAZ.


Exogenous TAZ / YAP was not processed proteolytically to cTAZ . RKO were transfected with indicated plasmids expressing C - terminal HA - tagged TAZ or YAP .

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

Exogenous TAZ/YAP was not processed proteolytically to cTAZ. RKO cells were transfected with the indicated plasmids expressing C-terminal HA-tagged TAZ or YAP.


RKO , but not HEK293A , expressed both full - TAZ cTAZ transcripts . RT PCR primers targeting different

RKO, but not HEK293A cells, expressed both full-length TAZ and cTAZ transcripts. RT PCR primers targeting different regions


Alignment of TAZ cTAZ sequences . cTAZ was derived from Sanger sequencing results of short PCR product ( RKO , F2 : R1 )

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

Alignment of TAZ and cTAZ protein sequences. cTAZ protein sequence was derived from the Sanger sequencing results of the short PCR product (RKO, F2:R1)


Knock out of cTAZ in RKO using CRISPR / Cas9 technology . Monoclones ( # 3 # 14 ) were selected .

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

Knock out of cTAZ in RKO cells using CRISPR/Cas9 technology. Monoclones (#3 and #14) were selected.


Cluster of expression of WT , cTAZ - / - , cTAZ - / - / GFP - cTAZ put - back RKO . Genes down - up - regulated more than 2 folds ( P < 0 . 05 ) in cTAZ KO were included in cluster . Green red indicate down - up - regulated genes respectively .

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

Cluster analysis of RNA expression of WT, cTAZ-/-, and cTAZ-/- /GFP-cTAZ put-back RKO cells. Genes down- and up-regulated more than 2 folds (P < 0.05) in cTAZ KO cells were included in cluster analysis. Green and red indicate down- and up-regulated genes respectively.


cTAZ suppressed expression of ISGs . of selected ISGs in WT , cTAZ - / - , cTAZ - / - / GFP - cTAZ RKO were quantified by qPCR . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - .

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

cTAZ suppressed expression of ISGs. RNA levels of selected ISGs in WT, cTAZ-/-, cTAZ-/-/GFP-cTAZ RKO cells were quantified by qPCR. Error bars indicate SD, n =3. **P<0.01; ***P<0.001; Student's t-test.


cTAZ repressed 5 × ISRE - luciferase activity induced by IFN - α . HEK293A were transfected with 5 × ISRE - luciferase reporter along with indicated plasmids , treated with or without IFN - α ( 50 ng / ml ) for 12 hours . expression of ectopic genes was determined by IB . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; one - way ANOVA was used for statistical .

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

cTAZ repressed 5×ISRE-luciferase activity induced by IFN-α. HEK293A cells were transfected with 5×ISRE-luciferase reporter along with the indicated plasmids, treated with or without IFN-α (50 ng/ml) for 12 hours. The expression of ectopic genes was determined by IB. Error bars indicate SD, n =3. ***P<0.001; one-way ANOVA test was used for statistical analysis.


Knockout of cTAZ promoted ISGs expression in presence of IFN - α . WT cTAZ - / - RKO were treated with or without of IFN - α ( 50 ng / ml ) for indicated time , expression of selected genes was quantified by qPCR . WT or cTAZ - / - group was normalized by basal level , respectively . Error bars indicate SD , n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; Student ' s t - .

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

Knockout of cTAZ promoted ISGs expression in the presence of IFN-α. WT and cTAZ-/- RKO cells were treated with or without of IFN-α (50 ng/ml) for indicated time, the expression of selected genes was quantified by qPCR. WT or cTAZ-/- group was normalized by basal level, respectively. Error bars indicate SD, n =3. *P<0.05; **P<0.01; Student's t-test.


STAT1 interacted with cTAZ , interaction was enhanced by IFN - α . HEK293A were transfected with indicated plasmids , treated with or without IFN - α ( 50 ng / ml ) for 1 hour . IP were performed using Flag antibody .

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

STAT1 interacted with cTAZ, and the interaction was enhanced by IFN-α. HEK293A cells were transfected with indicated plasmids, treated with or without IFN-α (50 ng/ml) for 1 hour. IP were performed using Flag antibody.


cTAZ attenuated heterodimerization between STAT1 STAT2 induced by IFN - α . Following transfection , were treated with or without IFN - α ( 50 ng / ml ) for 1 hour subjected to IP assays using Flag antibody .

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

cTAZ attenuated heterodimerization between STAT1 and STAT2 induced by IFN-α. Following transfection, cells were treated with or without IFN-α (50 ng/ml) for 1 hour and subjected to IP assays using Flag antibody.


Depletion of cTAZ enhanced interaction between STAT1 STAT2 . RKO lines ( WT # 14 ) were treated with IFN - α as in ( B ) . IP assays were performed using STAT1 antibody .

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

Depletion of cTAZ enhanced the interaction between STAT1 and STAT2. RKO cell lines (WT and #14) were treated with IFN-α as in (B). IP assays were performed using STAT1 antibody.


cTAZ attenuated interaction between STAT1 importin α5 ( Im - α5 ) . Following transfection , HEK293A were stimulated using IFN - α IP assays were performed using Flag antibody .

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

cTAZ attenuated interaction between STAT1 and importin α5 (Im-α5). Following transfection, HEK293A cells were stimulated using IFN-α IP assays were performed using Flag antibody.


Deletion of cTAZ promoted accumulation of STAT1 upon IFN - α stimulation . RKO ( WT cTAZ - / - ) treated with or without IFN - α ( 50 ng / ml ) for 1 hour , fixed , subjected to IF using STAT1 antibody ( green ) . was labeled by DAPI ( blue ) . Scale bar , 50 μm . was performed using ImageJ software ( right ) . N > C : ; N = C : plus ; N < C : localization .

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

Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN-α stimulation. RKO (WT and cTAZ-/-) cells treated with or without IFN-α (50 ng/ml) for 1 hour, fixed, and subjected to IF using STAT1 antibody (green). DNA was labeled by DAPI (blue). Scale bar, 50 μm. Quantification was performed using ImageJ software (right). N>C: nuclear; N=C: nuclear plus cytoplasmic; N<C: cytoplasmic localization.


Deletion of cTAZ promoted accumulation of STAT1 upon IFN - α stimulation . were treated subjected to subcellular fractionation .

Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN-α stimulation. Cells were treated and subjected to subcellular fractionation.


cTAZ defective in STAT1 - binding failed to block translocation of STAT1 . HEK293A stably overexpressing STAT1 were transfected with indicated plasmids , treated with or without IFN - α ( 50 ng / ml ) for 1 hour , fixed , subjected to IF Scale bar , 25 μm .

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

cTAZ defective in STAT1-binding failed to block nuclear translocation of STAT1. HEK293A stably overexpressing STAT1 were transfected with the indicated plasmids, treated with or without IFN-α (50 ng/ml) for 1 hour, fixed, and subjected to IF Scale bar, 25 μm.


cTAZ repressed antiviral activity of ectopic STAT1 / 2 . HEK293A were transfected with indicated plasmids , infected with gVSV ( MOI = 0 . 01 ) for 12 hours , fixed , subjected to microscopy ( Left ) . Viral infection replication were marked by green . Scale bar , 100 μm . ( Right ) of GFP positive . About 300 ( from 3 different fields ) were analyzed . Error bars indicate SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - .

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

cTAZ repressed the antiviral activity of ectopic STAT1/2. HEK293A were transfected with indicated plasmids, infected with gVSV (MOI = 0.01) for 12 hours, fixed, and subjected to microscopy (Left). Viral infection and replication were marked by green fluorescence. Scale bar, 100 μm. (Right) Quantification of GFP positive cells. About 300 cells (from 3 different fields) were analyzed. Error bars indicate SD. **P<0.01; ***P<0.001; Student's t-test.


cTAZ and STAT1/2 expression levels of cells


cTAZ effectively repressed antiviral activity of IFN - α . Transfection infection were performed Top : GFP of lysates was measured by a fluorometer . Bottom : viral GFP ectopic cTAZ were detected by IB .

cTAZ effectively repressed antiviral activity of IFN-α. Transfection and infection were performed Top: GFP intensity of cell lysates was measured by a fluorometer. Bottom: viral GFP and ectopic cTAZ protein levels were detected by IB.


Depletion of cTAZ repressed viral replication . RKO lines ( WT two independent cTAZ - / - clones ) were infected with gVSV ( MOI = 0 . 1 ) for 12 hours . Scale bar , 100 μm . of GFP positive were calculated About 300 ( from 3 different fields ) were analyzed . Error bars indicate SD . * * * P < 0 . 001 ; Student ' s t - .

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

Depletion of cTAZ repressed viral replication. RKO cell lines (WT and two independent cTAZ -/- clones) were infected with gVSV (MOI = 0.1) for 12 hours. Scale bar, 100 μm. The ratio of GFP positive cells were calculated About 300 cells (from 3 different fields) were analyzed. Error bars indicate SD. ***P<0.001; Student's t-test.


Depletion of cTAZ repressed viral replication . were treated GFP of lysates was measured . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - .

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

Depletion of cTAZ repressed viral replication. Cells were treated GFP intensity of cell lysates was measured. Error bars indicate SD, n = 3. **P<0.01; ***P<0.001; Student's t-test.


Depletion of cTAZ repressed viral replication . were treated viral genomic was quantified by qPCR ( normalized to human GAPDH ) . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; Student ' s t - .

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

Depletion of cTAZ repressed viral replication. Cells were treated the viral genomic RNA was quantified by qPCR (normalized to human GAPDH). Error bars indicate SD, n = 3. ***P<0.001; Student's t-test.


cTAZ deficiency enhanced ISGs expression . were treated expression of IFIH1 DDX58 were assessed by qPCR . Error bars indicate SD , n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - .

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

cTAZ deficiency enhanced ISGs expression. Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR. Error bars indicate SD, n = 3. *P<0.05; **P<0.01; ***P<0.001; Student's t-test.


Overexpression of cTAZ promotes infection . DOX - inducible cTAZ - overexpressing RKO lines were pre - treated with DOX for 48 hours ( 1 μg / ml ) . gVSV infection GFP were performed Error bars indicate SD , n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - .

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

Overexpression of cTAZ promotes virus infection. DOX-inducible cTAZ-overexpressing RKO cell lines were pre-treated with DOX for 48 hours (1 μg/ml). gVSV infection and GFP Fluorescence intensity quantification were performed Error bars indicate SD, n = 3. **P<0.01; ***P<0.001; Student's t-test.


Overexpression of cTAZ repressed ISGs expression . were treated expression of IFIH1 DDX58 were assessed by qPCR . Error bars indicate SD , n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; Student ' s t - .

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

Overexpression of cTAZ repressed ISGs expression. Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR. Error bars indicate SD, n = 3. *P<0.05; **P<0.01; Student's t-test.


gVSV induced expression of cTAZ . RKO were infected with gVSV ( MOI = 0 , 0 . 001 , 0 . 01 , 0 . 01 , 1 ) for 8 hours . expression was determined by IB ( Left ) quantified ( Right ) . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; two - way ANOVA was used for statistical .

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

gVSV induced expression of cTAZ. RKO cells were infected with gVSV (MOI = 0, 0.001, 0.01, 0.01, 1) for 8 hours. Protein expression was determined by IB (Left) and quantified (Right). Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.


IFN - α induced expression of cTAZ . RKO were treated with different doses of IFN - α for 8 hours . expression was determined Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; two - way ANOVA was used for statistical .

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

IFN-α induced expression of cTAZ. RKO cells were treated with different doses of IFN-α for 8 hours. Protein expression was determined Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.


IFN - α induced mRNA level of cTAZ . RKO were treated with different doses of IFN - α for 8 hours . were measured by qPCR . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; two - way ANOVA was used for statistical .

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

IFN-α induced mRNA level of cTAZ. RKO cells were treated with different doses of IFN-α for 8 hours. RNA levels were measured by qPCR. Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.


STAT1 / 2 synergistically activated a cTAZ promoter . HEK293A were transfected with cTAZ reporter with or without STAT . Promoter activity was determined using luciferase , expression was determined by IB . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; Student ' s t - .

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

STAT1/2 synergistically activated a cTAZ promoter. HEK293A cells were transfected with cTAZ reporter with or without STAT proteins. Promoter activity was determined using luciferase assay, and protein expression was determined by IB. Error bars indicate SD, n = 3. ***P<0.001; Student's t-test.


STAT1 / 2 binding sites on cTAZ promoter . HEK293A were transfected with cTAZ promoters ( wild type or mutants ) . Luciferase activity responses to STAT1 / 2 expression were measured . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; Student ' s t - .

STAT1/2 binding sites on cTAZ promoter. HEK293A were transfected with cTAZ promoters (wild type or mutants). Luciferase activity and responses to STAT1/2 expression were measured. Error bars indicate SD, n = 3. **P<0.01; Student's t-test.


STAT1 occupied cTAZ promoter in an IFN - α - sensitive manner . RKO were treated with or without IFN - α ( 50 ng / ml ) for 1 hour subjected to ChIP assays . cTAZ PCR primers were designed to target STAT1 / 2 binding site . ISG15 promoter was included as a positive control .

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STAT1 occupied cTAZ promoter in an IFN-α-sensitive manner. RKO cells were treated with or without IFN-α (50 ng/ml) for 1 hour and subjected to ChIP assays. cTAZ PCR primers were designed to target STAT1/2 binding site. ISG15 promoter was included as a positive control.


of - Seq of differentially expressed lncRNAs ( ≥ 2 - fold change FDR < 0 . 05 ) in mesenchymal ( MES ) 1123 proneural ( PN ) 528 GSCs . GSCs were pre - cultured for 16 h in DMEM / F12 medium with EGF ( 2 ng / ml ) bFGF ( 2 ng / ml ) then followed by co - culturing with or without 20 - μg / ml TGF - β1 for 3 h .

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Heatmap of RNA-Seq analysis of differentially expressed lncRNAs (≥ 2-fold change and FDR< 0.05) in mesenchymal (MES) 1123 and proneural (PN) 528 GSCs. GSCs were pre-cultured for 16 h in DMEM/F12 medium with EGF (2 ng/ml) and bFGF (2 ng/ml) and then followed by co-culturing with or without 20-μg/ml TGF-β1 for 3 h.


Representative of LINC00115 expression in clinical GBM tumors adjacent tissue using RNAscope . are representative of two independent experiments . Scale bars : 50 μm .

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Representative images of LINC00115 expression in clinical GBM tumors and adjacent tissue using RNAscope analysis. Images are representative of two independent experiments. Scale bars: 50 μm.


Kaplan - Meier of with high versus low LINC00115 - expressing GBM tumors from B . Median survival ( in months ) : low , 13 . 9 ; high , 8 . 1 . Black bars , censored data . Data information : , * , p < 0 . 05 , by log - rank .

Kaplan-Meier analysis of patients with high versus low LINC00115-expressing GBM tumors from B. Median survival (in months): low, 13.9; high, 8.1. Black bars, censored data. Data information: , *, p < 0.05, by log-rank test.


Expression of LINC00115 are higher in LGG ( low grade gliomas ) versus normal brain tissues GBM versus LGG . Expression data of LINC00115 were downloaded from REMBRANDT dataset . Data are presented as mean ± . * * , p < 0 . 01 , * * * , p < 0 . 001 , by two - t .

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Expression levels of LINC00115 are higher in LGG (low grade gliomas) versus normal brain tissues and GBM versus LGG. Expression data of LINC00115 were downloaded from the REMBRANDT dataset. Data are presented as mean ± SEM. **, p < 0.01, ***, p < 0.001, by two-tailed t test.


E to G . Kaplan - Meier of with high versus low LINC00115 - expressing Grade III GBM tumors from REMBRANDT ( E ) or TCGA ( G ) 26 [ ] , GBM from GSE83300 50 [ ] ( F ) datasets . Median survival ( in months ) in E : low , 20 . 2 ; high , 15 . 9 ; in F : low , 19 . 4 ; high , 13 . 0 ; in G : low , 33 . 2 ; high , 25 . 4 . Black bars , censored data . Data information : , * , p < 0 . 05 , by log - rank .

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E to G. Kaplan-Meier analysis of patients with high versus low LINC00115-expressing Grade III and GBM tumors from the REMBRANDT (E) or the TCGA (G) 26[], and GBM from GSE83300 50[] (F) datasets. Median survival (in months) in E: low, 20.2; high, 15.9; in F: low, 19.4; high, 13.0; in G: low, 33.2; high, 25.4. Black bars, censored data. Data information: , *, p < 0.05, by log-rank test.


qRT - PCR of LINC00115 knockdown using two different shRNAs , shL115 - 1 shL115 - 2 in 1123 528 GSCs . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , by One - way

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qRT-PCR analysis of LINC00115 knockdown using two different shRNAs, shL115-1 and shL115-2 in 1123 and 528 GSCs. Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, by One-way ANOVA


Effects of LINC00115 knockdown on GSC proliferation ( B ) data are representative of three independent experiments . Error bars , ± SD . * , by two - t - in B .

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Effects of LINC00115 knockdown on GSC proliferation (B) data are representative of three independent experiments. Error bars, ± SD. *, by two-tailed t-test in B.


C . Effects of LINC00115 knockdown on neuro - like sphere formation ( C ) . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , by One - way ANOVA

C. Effects of LINC00115 knockdown on neuro-like sphere formation (C). Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, by One-way ANOVA in


D . Representative bioluminescence of brains with indicated 528 GSC xenografts expressing shC , shL115 - 1 , or shL115 - 2 at 45 days after implantation . represent results of 5 mice per group of 2 independent experiments . E . of bioluminescence activity in D .

D. Representative bioluminescence images of brains with indicated 528 GSC tumor xenografts expressing shC, shL115-1, or shL115-2 at 45 days after implantation. Images represent results of 5 mice per group of 2 independent experiments. E. Quantification of the bioluminescence activity in D.


F . Representative hematoxylin eosin ( H & E ) - stained of mouse brain sections with GSC1123 xenografts expressing shC , shL115 - 1 , or shL115 - 2 . represent results of five mice per group of two independent experiments . Scale bars : 1 mm . G . of volume in F .

F. Representative hematoxylin and eosin (H&E)-stained images of mouse brain sections with GSC1123 xenografts expressing shC, shL115-1, or shL115-2. Images represent results of five mice per group of two independent experiments. Scale bars: 1 mm. G. Quantification of tumor volume in F.


H . Immunofluorescence ( IF ) of Nestin expression in 1123 GSC control LINC00115 knockdown xenograft tumors from F . are representative of two independent experiments . Scale bars : 50 μm .

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H. Immunofluorescence (IF) analysis of Nestin expression in 1123 GSC control and LINC00115 knockdown xenograft tumors from F. Images are representative of two independent experiments. Scale bars: 50 μm.


I . Kaplan - Meier of implantation with 1123 / shC , 1123 / shL115 - 1 or 1123 / shL115 - 2 GSCs . n = 10 . Data information : * * , p < 0 . 01 , by log - rank .

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I. Kaplan-Meier analysis of animal implantation with 1123/shC, 1123/shL115-1 or 1123/shL115-2 GSCs. n = 10. Data information: **, p < 0.01, by log-rank test.


B C . MS2 - RIP followed by qRT - PCR of endogenous microRNAs association with LINC00115 . 12XMS2 empty vector , LINC00115 - 12XMS2 , or LINC00115 - mut - 12XMS2 with Flag - tagged MS2 were co - transfected into 1123 GSCs . Data information data are representative of three independent experiments . Error bars , ± SD . , * * * , p < 0 . 001 , by two - t - .

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B and C. MS2-RIP followed by qRT-PCR analysis of endogenous microRNAs association with LINC00115. 12XMS2 empty vector, LINC00115-12XMS2, or LINC00115-mut- 12XMS2 with Flag-tagged MS2 were co-transfected into 1123 GSCs. Data information data are representative of three independent experiments. Error bars, ± SD. , ***, p < 0.001, by two-tailed t-test.


E . pull - down qRT - PCR assays of LINC00115 binding with miR - 200s . GSC1123 lysates were incubated with biotin - labeled LINC00115 . Data information data are representative of three independent experiments . Error bars , ± SD . p < 0 . 05 , * * , p < 0 . 01 , by One - way ANOVA .

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E. RNA pull-down and qRT-PCR assays of LINC00115 binding with miR-200s. GSC1123 cell lysates were incubated with biotin-labeled LINC00115. Data information data are representative of three independent experiments. Error bars, ± SD. p < 0.05, **, p < 0.01, by One-way ANOVA.


F . Luciferase activity of 1123 GSCs co - transfected with miR - 200s luciferase reporters containing LINC00115 WT , mutant , or empty vector ( EV ) . Data information : data are representative of three independent experiments . Error bars , ± SD . p < 0 . 05 , * * , p < 0 . 01 , by One - way ANOVA .

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F. Luciferase activity analysis of 1123 GSCs co-transfected with miR-200s and luciferase reporters containing LINC00115 WT, mutant, or empty vector (EV). Data information: data are representative of three independent experiments. Error bars, ± SD. p < 0.05, **, p < 0.01, by One-way ANOVA.


A B . Correlation of expression between LINC00115 ZEB1 from TCGA GBM - Seq dataset ( A ) REMBRANDT GBM array dataset ( B ) . * * * , p < 0 . 001 , by two - t - .

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A and B. Correlation of expression between LINC00115 and ZEB1 from TCGA GBM RNA-Seq dataset (A) and REMBRANDT GBM array dataset (B). ***, p < 0.001, by two-tailed t-test.


C . LINC00115 knockdown reduced ZEB1 mRNA expression in 1123 528 GSCs . Data information : , data are representative of three independent experiments . Error bars , ± SD . * * , p < 0 . 01 , by One - way

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C. LINC00115 knockdown reduced ZEB1 mRNA expression in 1123 and 528 GSCs. Data information: , data are representative of three independent experiments. Error bars, ± SD. **, p < 0.01, by One-way ANOVA


D . Effects of LINC00115 knockdown on ZEB1 , vimentin , E - cadherin expression . Data information : data are representative of three independent experiments . Error bars , ± SD . * * ,

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D. Effects of LINC00115 knockdown on ZEB1, vimentin, and E-cadherin protein expression. Data information: data are representative of three independent experiments. Error bars, ± SD. **,


E . Effects of re - expression of shRNA - resistant LINC00115 WT ( L115 - WT ) , miR - 200 binding mutant ( L115 - mut ) with or without miR - 200b on ZEB1 signaling activation . Data information data are representative of three independent experiments . Error bars , ± SD .

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E. Effects of re-expression of shRNA-resistant LINC00115 WT (L115-WT), miR-200 binding mutant (L115-mut) with or without miR-200b on ZEB1 signaling activation. Data information data are representative of three independent experiments. Error bars, ± SD.


F . Neuro - like sphere formation of effects of re - expression of shRNA - resistant LINC00115 WT mutant with or without miR - 200b . Data information : data are representative of three independent experiments . Error bars , ± SD . * * , p < 0 . 01 , by two - t - in F .

F. Neuro-like sphere formation assay of effects of re-expression of shRNA-resistant LINC00115 WT and mutant with or without miR-200b. Data information: data are representative of three independent experiments. Error bars, ± SD. **, p < 0.01, by two-tailed t-test in F.


G . Kaplan - Meier of implantation with 1123 GSCs with indicated modifications . n = 10 . Statistical was performed by log - rank .

G. Kaplan-Meier analysis of animal implantation with 1123 GSCs with indicated modifications. n = 10. Statistical analysis was performed by log-rank test.


H I . GSEA of EMT signature genes using ranked gene expression changes in TGF - β1 - treated 1123 / shLINC00115 GSCs versus control ( H ) or in 1123 GSCs with ectopic expression of LINC00115 or an empty vector ( EV ) ( I ) . NES , normalized enrichment score .

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H and I. GSEA of EMT signature genes using ranked gene expression changes in TGF-β1-treated 1123/shLINC00115 GSCs versus the control (H) or in 1123 GSCs with ectopic expression of LINC00115 or an empty vector (EV) (I). NES, normalized enrichment score.


LINC00115 knockdown reduced ZNF596 mRNA expression . Data information : In data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , by One - way

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LINC00115 knockdown reduced ZNF596 mRNA expression. Data information: In data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, by One-way ANOVA


LINC00115 knockdown reduced ZNF596 ( D ) expression . Data information : data are representative of three independent experiments . Error bars , ± SD .

LINC00115 knockdown reduced ZNF596 protein (D) expression. Data information: data are representative of three independent experiments. Error bars, ± SD.


E . Effects of exogenous ZNF596 expression on LINC00115 knockdown - inhibited ZNF596 expression . Data information : data are representative of three independent experiments . Error bars , ± SD .

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E. Effects of exogenous ZNF596 expression on LINC00115 knockdown-inhibited ZNF596 protein expression. Data information: data are representative of three independent experiments. Error bars, ± SD.


F . ZNF596 overexpression had no effect on LINC00115 expression . Data information data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , by One - way

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F. ZNF596 overexpression had no effect on LINC00115 expression. Data information data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, by One-way ANOVA


G . Expression of exogenous ZNF596 rescued LINC00115 knockdown - inhibited proliferation . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , by two - t - in G .

G. Expression of exogenous ZNF596 rescued LINC00115 knockdown-inhibited cell proliferation. Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05 , by two-tailed t-test in G.


H . Representative H & E of mouse brain sections with 1123 / shC 1123 / shL115 GSC xenografts with empty vector ( EV ) or ectopic ZNF596 expression . represent results of five mice per group of two independent experiments . Scale bars : 1 mm . I . of in H .

H. Representative H&E staining images of mouse brain sections with 1123/shC and 1123/shL115 GSC tumor xenografts with empty vector (EV) or ectopic ZNF596 expression. Images represent results of five mice per group of two independent experiments. Scale bars: 1 mm. I. Quantification of tumor size in H.


J K . Correlation of expression between LINC00115 ZEB1 from TCGA GBM - Seq dataset ( J ) REMBRANDT GBM array dataset ( K ) . * * , p < 0 . 01 , by two - t - .

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J and K. Correlation of expression between LINC00115 and ZEB1 from TCGA GBM RNA-Seq dataset (J) and REMBRANDT GBM array dataset (K). **, p < 0.01, by two-tailed t-test.


Expression of shRNA - resistant LINC00115 WT but not mutant or empty vector ( EV ) rescued LINC00115 shRNA - inhibited ZNF596 mRNA ( A ) expression in 1123 GSCs .

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Expression of shRNA-resistant LINC00115 WT but not mutant or empty vector (EV) rescued LINC00115 shRNA-inhibited ZNF596 mRNA (A) expression in 1123 GSCs.


Expression of shRNA - resistant LINC00115 WT but not mutant or empty vector ( EV ) rescued LINC00115 shRNA - inhibited ZNF596 ( B ) expression in 1123 GSCs .

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Expression of shRNA-resistant LINC00115 WT but not mutant or empty vector (EV) rescued LINC00115 shRNA-inhibited ZNF596 protein (B) expression in 1123 GSCs.


D . MS2 - RIP followed by qRT - PCR for endogenous microRNAs association with ZNF596 . 12XMS2 empty vector , ZNF596 - 5 ' - UTR - WT - 12XMS2 , or ZNF596 - 5 ' - UTR - mut - 12XMS2 with Flag - tagged MS2 were co - transfected into 1123 GSCs .

D. MS2-RIP followed by qRT-PCR for endogenous microRNAs association with ZNF596. 12XMS2 empty vector, ZNF596-5'-UTR-WT-12XMS2, or ZNF596-5'-UTR-mut -12XMS2 with Flag-tagged MS2 were co-transfected into 1123 GSCs.


F . Luciferase activity in 1123 GSCs co - transfected with miR - 200s luciferase reporters containing ZNF596 5 ' - UTR WT , mutant , or empty vector ( EV ) .

F. Luciferase activity in 1123 GSCs co-transfected with miR-200s and luciferase reporters containing ZNF596 5'-UTR WT, mutant, or empty vector (EV).


GSEA of EZH2 - targeted signature genes using ranked gene expression changes in 1123 GSCs with ZNF596 sgRNA or a control sgRNA . NES , normalized enrichment score .

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GSEA of EZH2-targeted signature genes using ranked gene expression changes in 1123 GSCs with ZNF596 sgRNA or a control sgRNA. NES, normalized enrichment score.


qRT - PCR of effect of ZNF596 knockout on EZH2 expression . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 , by One - way

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qRT-PCR analysis of effect of ZNF596 knockout on EZH2 expression. Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001, by One-way ANOVA


ZNF596 knockout impaired EZH2 expression , its downstream H3K27me3 , STAT3 phosphorylation in 1123 528 GSCs . Data information , data are representative of three independent experiments . Error bars , ± SD .

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ZNF596 knockout impaired EZH2 expression, its downstream H3K27me3, and STAT3 phosphorylation in 1123 and 528 GSCs. Data information , data are representative of three independent experiments. Error bars, ± SD.


LINC00115 regulated ZNF596 / EZH2 / STAT3 signaling . 1123 528 GSCs were treated with or without EZH2 inhibitor GSK343 ( 3 μM ) for 6 Data information : data are representative of three independent experiments . Error bars , ± SD .

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LINC00115 regulated ZNF596/EZH2/STAT3 signaling. 1123 and 528 GSCs were treated with or without EZH2 inhibitor GSK343 (3 μM) for 6 Data information: data are representative of three independent experiments. Error bars, ± SD.


LINC00115 ZNF596 regulate EZH2 promoter activity . Data information : data are representative of three independent experiments . Error bars , ± SD . , by two - t -

LINC00115 and ZNF596 regulate EZH2 promoter activity. Data information: data are representative of three independent experiments. Error bars, ± SD. , by two-tailed t-test


ChIP - qPCR of ZNF596 binding with EZH2 promoter . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 , by One - way

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ChIP-qPCR analysis of ZNF596 binding with the EZH2 promoter. Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001, by One-way ANOVA


EZH2 inhibitor GSK343 impaired ZNF596 overexpression - reversed LINC00115 knockdown - inhibited neuro - like sphere formation . 1123 528 GSCs with an inducible ZNF596 shRNA were treated with or without Dox ( 2 μg / ml ) / or EZH2 inhibitor GSK343 ( 1 μM ) for 7 days . Data information : data are representative of three independent experiments . Error bars , ± SD . by two - t -

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EZH2 inhibitor GSK343 impaired ZNF596 overexpression-reversed and LINC00115 knockdown-inhibited neuro-like sphere formation. 1123 and 528 GSCs with an inducible ZNF596 shRNA were treated with or without Dox (2 μg/ml) and/or EZH2 inhibitor GSK343 (1 μM) for 7 days. Data information: data are representative of three independent experiments. Error bars, ± SD. by two-tailed t-test


Growth kinetics of subcutaneous xenograft with indicated genetic modifications after treatment with or without Dox / or GSK343 . - bearing mice ( five mice per group ) were fed with 10 % sucrose water with or without 2 mg / ml Dox combined with treatment with or without GSK343 ( 5 mg / kg from Monday to Friday via intraperitoneal injection ) for three beginning five days post - xenograft transplantation . Data were from three independent experiments with five mice per group . Data information : * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 , by two - t -

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Growth kinetics of subcutaneous xenograft tumor with indicated genetic modifications after treatment with or without Dox and/or GSK343. Tumor-bearing mice (five mice per group) were fed with 10% sucrose water with or without 2 mg/ml Dox and combined with treatment with or without GSK343 (5 mg/kg body weight from Monday to Friday via intraperitoneal injection) for three weeks beginning five days post-xenograft transplantation. Data were from three independent experiments with five mice per group. Data information: *, p < 0.05, **, p < 0.01, ***, p < 0.001 , by two-tailed t-test


Kaplan - Meier of implantation with GSC1123 with indicated modifications . n = 10 . Statistical was performed by log - rank .

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Kaplan-Meier analysis of animal implantation with GSC1123 with indicated modifications. n = 10. Statistical analysis was performed by log-rank test.


Representative of RNAscope for LINC00115 for ZEB1 ZNF596 expression in two GBM specimens , Sample # 1 ( positive ) Sample # 2 ( negative ) . Scale bar , 50 μm . are representative of two independent experiments .

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Representative images of RNAscope for LINC00115 and IHC for ZEB1 and ZNF596 expression in two GBM specimens, Sample #1 (positive) and Sample #2 (negative). Scale bar, 50 μm. Images are representative of two independent experiments.


Correlation of detected expression between ZEB1 or ZNF596 , LINC00115 from a separate cohort of a total of 75 paraffin - embedded clinical GBM samples . Statistical was performed by Chi - square .

Correlation of detected expression between ZEB1 or ZNF596, and LINC00115 from a separate cohort of a total of 75 paraffin-embedded clinical GBM samples. Statistical analysis was performed by Chi-square test.


Kaplan - Meier survival of with GBM tumors expressing high or low of LINC00115 with high or low of ZEB1 or ZNF596 expression . Median survival ( in months ) : LINC00115hi / ZEB1hi , 5 . 4 ; LINC00115lo / ZEB1lo , 27 . 9 ; LINC00115hi / ZNF596hi , 4 . 2 ; LINC00115lo / ZNF596lo , 23 . 8 . Statistical was performed by log - rank .

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Kaplan-Meier survival analysis of patients with GBM tumors expressing high or low levels of LINC00115 with high or low levels of ZEB1 or ZNF596 expression. Median survival (in months): LINC00115hi/ZEB1hi, 5.4; LINC00115lo/ZEB1lo, 27.9; LINC00115hi/ZNF596hi, 4.2; LINC00115lo/ZNF596lo, 23.8. Statistical analysis was performed by log-rank test.


( left ) Luciferase expression in HEK293 driven by deletion constructs of POLG proximal promoter . ( right ) Luciferase expression with mutated CAAT - boxes in POLG promoter . error bars indicate standard deviation in 3 biological replicates . AU ; arbitrary units .

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(left) Luciferase expression in HEK293 cells driven by deletion constructs of POLG proximal promoter. (right) Luciferase expression with mutated CAAT-boxes in POLG promoter. The error bars indicate standard deviation in 3 biological replicates. AU; arbitrary units.


Expression pattern driven by 500 bp Polg proximal promoter in E12 . 5 mouse . LacZ - positive populations in developing midbrain ( black arrows ) , dorsal root ganglia ( grey arrows ) motoneuron progenitors ( arrowhead ) of neural tube . Somites show some expression ( arrow ) . Sectioning planes indicated by red lines . Scale bars 100 µm .

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Expression pattern driven by 500 bp Polg proximal promoter in E12.5 mouse embryo. LacZ-positive cell populations in the developing midbrain (black arrows), dorsal root ganglia (grey arrows) and motoneuron progenitors (arrowhead) of the neural tube. Somites show some expression (white arrow). Sectioning planes indicated by red lines. Scale bars 100 µm.


- ( G ) POLG enhancer elements are functional in vivo drive expression in E12 . 5 transgenic mice . Sectioning planes indicated by red lines . Black in ( E ) marks expression in dorsal neural tube , whereas rostral caudal regions lack dorsal expression ( black arrows ) .

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- (G) POLG enhancer elements are functional in vivo and drive expression in E12.5 transgenic mice. Sectioning planes indicated by red lines. Black line in (E) marks the expression in the dorsal neural tube, whereas rostral and caudal regions lack dorsal expression (black arrows).


( H ) - ( J ) Neural tube lacZ expression driven by ( H ) EE1 : immature neuronal precursors ( grey arrow ) , ( I ) EE2 : dorsal neural tube ( grey arrow ) dorsal root ganglia ( black arrow ) , ( J ) EE3 : dorsal neural tube ( grey arrow ) . Scale bars 100 µm .

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(H)- (J) Neural tube lacZ expression driven by (H) EE1: immature neuronal precursors (grey arrow), (I) EE2: dorsal neural tube (grey arrow) and dorsal root ganglia (black arrow), and (J) EE3: dorsal neural tube (grey arrow). Scale bars 100 µm.


K ) - ( M ) Midbrain lacZ expression , driven by ( K ) EE1 , ( L ) EE2 ( M ) EE3 . Black arrow indicates neuronal population from EE1 stained in ( N ) . Scale bars 100 µm .

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K)- (M) Midbrain lacZ expression, driven by (K) EE1, (L) EE2 and (M) EE3. Black arrow indicates neuronal population from EE1 embryo stained in (N). Scale bars 100 µm.


( N ) EE1 drives expression in oculomotor ; immunofluorescent co - with antibodies against ISL1 / 2 ( motoneurons of oculomotor ; red ) β - Gal ( green ) . LacZ - of region in ( k ) ; black arrow . Scale bars 50 µm .

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(N) EE1 drives expression in oculomotor complex; immunofluorescent co-staining with antibodies against ISL1/2 (motoneurons of oculomotor complex; red) and β-Gal (green). LacZ-staining of the region in (k); black arrow. Scale bars 50 µm.


Sagittal section of adult mouse brain showing lacZ expression driven by EE2 . HC , hippocampus .

Sagittal section of adult mouse brain showing lacZ expression driven by EE2. HC, hippocampus.


EE2 - driven expression in rostral migratory stream ( black arrow ) in subventricular zone ( arrow ) . Scale bar 760 µm .

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EE2-driven expression in rostral migratory stream (black arrow) and in subventricular zone (white arrow). Scale bar 760 µm.


Adult mouse spinal cord , EE2 ( D ) EE3 expression pattern . Dorsal horns , laminae I - III ( black arrows ) central canal ( arrowheads ) . LacZ . Dashed lines in ( C ) indicates region of dorsal horn shown in ( D ) , ( F ) ( G ) . Inlets in ( C ) ( E ) show POLG immunohistochemistry of dorsal horn central canal ( grey arrow ) , respectively . Scale bars : 100 µm if not indicated otherwise .

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Adult mouse spinal cord, EE2 and (D) EE3 expression pattern. Dorsal horns, laminae I-III (black arrows) and central canal (arrowheads). LacZ staining. Dashed lines in (C) indicates the region of dorsal horn shown in (D), (F) and (G). Inlets in (C) and (E) show POLG immunohistochemistry of dorsal horn and central canal (grey arrow), respectively. Scale bars: 100 µm if not indicated otherwise.


( F ) - ( G ) Calbindin ( red ) β - Gal ( green ) expression in interneurons of dorsal horn laminae I - III of ( F ) EE2 ( G ) EE3 transgenic adult mice . Immunofluorescence . Scale bars 20 µm .

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(F)-(G) Calbindin (red) and β-Gal (green) expression in interneurons of the dorsal horn laminae I-III of (F) EE2 and (G) EE3 transgenic adult mice. Immunofluorescence. Scale bars 20 µm.


number of distal DNAse I hypersensitive sites ( DHSs ) of mtDNA maintenance genes genes in genomic POLG locus correlating > 0 . 85 with target promoter DHS across 125 lines . Abbreviations : TWNK , Twinkle ; POLG , Polymerase gamma , catalytic subunit ; POLG2 , Polymerase gamma , accessory subunit ; SSBP1 , Single stranded binding 1 ; TFAM , mitochondrial transcription factor A ; FANCI , Fanconi anemia group I ; RHCG , Ammonium transporter Rh type C ; TICRR , Treslin .

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The number of distal DNAse I hypersensitive sites (DHSs) of mtDNA maintenance genes and genes in the genomic POLG locus correlating >0.85 with target promoter DHS across 125 cell lines. Abbreviations: TWNK, Twinkle protein; POLG, DNA Polymerase gamma, catalytic subunit; POLG2, DNA Polymerase gamma, accessory subunit; SSBP1, Single stranded DNA binding protein 1; TFAM, mitochondrial transcription factor A; FANCI, Fanconi anemia group I protein; RHCG, Ammonium transporter Rh type C; TICRR, Treslin.


Distribution histogram of DHSs for mtDNA maintenance genes .

Distribution histogram of DHSs for mtDNA maintenance genes.


Genomic distribution of POLG DHSs . Black arrow shows a cluster upstream from POLG coding region .

Genomic distribution of POLG DHSs. Black arrow shows a cluster upstream from POLG coding region.


Expression of LINC00925 POLG in different human tissues types . Quantitative PCR amplification of cDNA . Abbreviations : iPS , induced pluripotent ; SH5Y , neuroblastoma ; HepG2 , liver hepatocellular carcinoma ; U2OS bone osteosarcoma . Error bars indicate standard deviation in 3 technical replicates . AU ; arbitrary units .

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Expression of LINC00925 and POLG in different human tissues and cell types. Quantitative PCR amplification of cDNA. Abbreviations: iPS, induced pluripotent stem cell; SH5Y, neuroblastoma line; HepG2, liver hepatocellular carcinoma line; U2OS bone osteosarcoma line. Error bars indicate standard deviation in 3 technical replicates. AU; arbitrary units.


Polg long - non - coding Ai854517 ( mouse homologue of human LINC00925 ) correlate tightly in mouse cerebellar development . Time points : E18 , postnatal days 0 , 3 , 6 , 9 ; three mice per time point . Expression calculated as cap of gene expression ( CAGE ) hits in transcription start site ( CTSS ) .

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Polg and long-non-coding RNA Ai854517 (mouse homologue of human LINC00925) correlate tightly in mouse cerebellar development. Time points: E18, postnatal days 0, 3, 6, 9; three mice per time point. Expression calculated as cap analysis of gene expression (CAGE) hits in the transcription start site (CTSS).


Ai854517 Polg transcripts colocalize in adult mouse brain ; in situ hybridization . HC , hippocampus ; cerebellar molecular layer ( M ) , Purkinje layer ( PC ) , granular layer ( GC ) . Scale bars : hippocampus 760 µm , cerebellum 300 µm .

Ai854517 and Polg transcripts colocalize in adult mouse brain; in situ hybridization. HC, hippocampus; cerebellar molecular layer (M), Purkinje cell layer (PC), granular cell layer (GC). Scale bars: hippocampus 760 µm, cerebellum 300 µm.


Expression of MIR9 - 3 Ai854517 correlate in mouse cerebellar development . Time points : E18 , postnatal day 0 , 3 , 6 , 9 ; three mice per time point . . Expression calculated as cap of gene expression ( CAGE ) hits in transcription start site ( CTSS ) .

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Expression levels of MIR9-3 and Ai854517 correlate in mouse cerebellar development. Time points: E18, postnatal day 0, 3, 6, 9; three mice per time point.. Expression calculated as cap analysis of gene expression (CAGE) hits in the transcription start site (CTSS).


expressions of MIR9 , NR2E2 , LDLDRAP1 MTHFD2 in HEK293 untransfected controls in transfected with pre - MIR9 or scrambled . Shown is mean with standard error of mean . Statistical testing was performed using 1 - way ANOVA with Dunnet ' s correction for multiple comparisons . Abbreviations : AU ; arbitrary units .

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RNA expressions of MIR9, NR2E2, LDLDRAP1 and MTHFD2 in HEK293 untransfected controls and in cells transfected with pre-MIR9 or scrambled RNA. Shown is mean with standard error of the mean. Statistical testing was performed using 1-way ANOVA with Dunnet's correction for multiple comparisons. Abbreviations: AU; arbitrary units.


Spinal cord ( C8 - level ) of MIRAS , neurofilament . Dorsal columns , especially gracile , show pallor ( star ) ; motoneurons in anterior horns are preserved ( black arrow ) . Scale bar 1 mm .

Spinal cord (C8-level) of MIRAS patient, neurofilament staining. Dorsal columns, especially the gracile, show pallor (star); motoneurons in the anterior horns are preserved (black arrow). Scale bar 1 mm.


Oculomotor of POLG with progressive ophthalmoplegia ; midbrain level . Hematoxylin - eosin . whole shows spongiotic degeneration . Scale bar 50 µm .

Oculomotor complex of POLG patient with progressive ophthalmoplegia; midbrain level. Hematoxylin-eosin staining. The whole area shows spongiotic degeneration. Scale bar 50 µm.


( a ) Western blotting was performed to determine FoxO1 expression , p62 degradation LC3 - II accumulation in H1299 , HCT116 HeLa in response to serum starvation ( SS ) for 24 h or 0 . 5 mM H2O2 for 6 h in presence or absence of E64 pepstatin A ( PEPS A ) .

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(a) Western blotting was performed to determine FoxO1 expression, p62 degradation and LC3-II accumulation in H1299, HCT116 and HeLa cells in response to serum starvation (SS) for 24 h or 0.5 mM H2O2 for 6 h in the presence or absence of E64 and pepstatin A (PEPS A).


( b , c ) Stable FoxO1 knockdown HCT116 , or with an empty RNAi plasmid , were incubated in a serum - free medium for up to 36 h ( b ) or treated with 0 . 5 mM H2O2 for up to 12 h ( c ) ; western blotting was performed to detect changes in FoxO1 , p62 , LC3 - II or β - actin .

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(b, c) Stable FoxO1 knockdown HCT116 cells, or cells with an empty RNAi plasmid, were incubated in a serum-free medium for up to 36 h (b) or treated with 0.5 mM H2O2 for up to 12 h (c); western blotting was performed to detect changes in FoxO1, p62, LC3-II or β-actin.


( d ) HCT116 were labelled with [ 3H ] leucine for 48 h , washed incubated for 24 h , before being subjected to serum starvation for 24 h or 0 . 5 mM H2O2 for 12 h in presence or absence of 3 - MA . At 12 or 24 h after treatment , relative degradation of long - lived was measured by counting radioactivity . Data in d are means ± s . d . ( n = 3 ) . Two asterisks , P 0 . 01 ; asterisk , P 0 . 05 ; NS , not significant , P > 0 . 05 ; Student ' s t - .

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

(d) HCT116 cells were labelled with [3H]leucine for 48 h, washed and incubated for 24 h, before being subjected to serum starvation for 24 h or 0.5 mM H2O2 for 12 h in the presence or absence of 3-MA. At 12 or 24 h after treatment, the relative degradation of long-lived proteins was measured by counting radioactivity. Data in d are means ± s.d. (n = 3). Two asterisks, P 0.01; asterisk, P 0.05; NS, not significant, P > 0.05; Student's t-test.


( e ) Autophagic vesicles or autophagosomes in HCT116 or stable FoxO1 - RNAi HCT116 , were observed by electron microscopy after 24 h of serum starvation . bottom picture enlargement showing clearer autophagic vesicles indicated by an arrow . graph shows a statistical of autophagic vesicles in HCT116 with or without knockdown of FoxO1 in response to serum starvation . Data in e are means ± s . d . ( n = 3 ) .

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(e) Autophagic vesicles or autophagosomes in HCT116 cells or stable FoxO1-RNAi HCT116 cells, were observed by electron microscopy after 24 h of serum starvation. The bottom picture enlargement showing clearer autophagic vesicles indicated by an arrow. The graph shows a statistical analysis of autophagic vesicles in HCT116 cells with or without knockdown of FoxO1 in response to serum starvation. Data in e are means ± s.d. (n = 3).


( f ) LC3 - immunopositive punctate signals were observed in HCT116 or in stable FoxO1 - RNAi HCT116 in response to serum starvation for 24 h or 0 . 5 mM H2O2 treatment . ( g ) of LC3 - positive punctate in f . Data in g are means ± s . d . ( n = 3 ) .

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(f) LC3-immunopositive punctate signals were observed in HCT116 or in stable FoxO1-RNAi HCT116 cells in response to serum starvation for 24 h or 0.5 mM H2O2 treatment. (g) Quantification of LC3-positive punctate cells in f. Data in g are means ± s. d. (n = 3).


( h ) A rescue experiment using a specific mutated FoxO1 plasmid to validate activity of FoxO1 in induction of autophagy in HCT116 in presence or absence of serum , with or without E64 pepstatin A .

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(h) A rescue experiment using a specific mutated FoxO1 plasmid to validate the activity of FoxO1 in the induction of autophagy in HCT116 cells in the presence or absence of serum, with or without E64 and pepstatin A.


( a ) A Flag - tagged FoxO1 ( WT ) , FoxO1 ( ΔDB ) , FoxO1 ( ΔDB3A ) or an empty plasmid was individually transfected into GFP - LC3 pre - transfected H1299 , localization of FoxO1 ( upper panels ) or GFP - LC3 punctate signals ( lower panels ) was observed with a confocal microscope . Scale bars , 10 μm . ( b ) numbers of punctate GFP - LC3 are represented by of GFP - LC3 - labelled punctate to overall survived H1299 ( at least 200 were counted ) . Data in b are means ± 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A Flag-tagged FoxO1(WT), FoxO1(ΔDB), FoxO1(ΔDB3A) or an empty plasmid was individually transfected into GFP-LC3 pre-transfected H1299 cells, and the cellular localization of FoxO1 (upper panels) or GFP-LC3 punctate signals (lower panels) was observed with a confocal microscope. Scale bars, 10 μm. (b) The numbers of punctate GFP-LC3 cells are represented by the ratio of GFP-LC3-labelled punctate cells to overall survived H1299 cells (at least 200 cells were counted). Data in b are means ± s.d. (n = 3).


( c ) p62 degradation LC3 - II accumulation were evaluated by western blotting in H1299 transfected with exogenous FoxO1 - expressing plasmids mentioned above .

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(c) p62 degradation and LC3-II accumulation were evaluated by western blotting in H1299 cells transfected with exogenous FoxO1-expressing plasmids mentioned above.


( d ) H1299 were labelled with [ 3H ] leucine for 48 h , then transfected with a control plasmid or various Flag - FoxO1 plasmids , in presence or absence of 3 - MA or E64 pepstatin A . Degradation of long - lived was measured at indicated time , 24 h after transfection . Data in d are means ± s . d . ( n = 3 ) .

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(d) H1299 cells were labelled with [3H]leucine for 48 h, then transfected with a control plasmid or various Flag-FoxO1 plasmids, in the presence or absence of 3-MA or E64 and pepstatin A. Degradation of long-lived proteins was measured at the indicated time, 24 h after transfection. Data in d are means ± s.d. (n = 3).


( e ) A GFP - tagged FoxO1 ( 3A ) or an empty plasmid was co - transfected with a FoxO1 - promoter luciferase plasmid into H1299 . After 24 h , luciferase reporter activity of FoxO1 was measured . RLU , relative luciferase units . Data are means ± s . d . ( n = 3 ) .

(e) A GFP-tagged FoxO1(3A) or an empty plasmid was co-transfected with a FoxO1-promoter luciferase plasmid into H1299 cells. After 24 h, luciferase reporter activity of FoxO1 was measured. RLU, relative luciferase units. Data are means ± s.d. (n = 3).


( f ) A set of RNAi - resistant rescue forms of FoxO1 plasmids were transfected into stable FoxO1 - RNAi HCT116 in presence or absence of E64 pepstatin A . Western blotting was performed to detect p62 degradation LC3 - II accumulation .

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

(f) A set of RNAi-resistant rescue forms of FoxO1 plasmids were transfected into stable FoxO1-RNAi HCT116 cells in the presence or absence of E64 and pepstatin A. Western blotting was performed to detect p62 degradation and LC3-II accumulation.


( g ) Flag - tagged FoxO1 ( WT ) r , FoxO1 ( 3A ) r , FoxO1 ( 3A - NLSm ) r , FoxO1 ( NESm ) r or an empty plasmid were individually transfected into GFP - LC3 pre - transfected HCT116 ( FoxO1 - RNAi treated ) , localization of FoxO1 ( upper panels ) or GFP - LC3 punctate signals ( lower panels ) were observed with a confocal microscope . ( h ) numbers of punctate GFP - LC3 are represented by of GFP - LC3 - labelled punctate to overall survived HCT116 . Data in h are means ± 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) Flag-tagged FoxO1(WT)r, FoxO1(3A)r, FoxO1(3A-NLSm)r, FoxO1(NESm)r or an empty plasmid were individually transfected into GFP-LC3 pre-transfected HCT116 cells (FoxO1-RNAi treated), and the cellular localization of FoxO1 (upper panels) or GFP-LC3 punctate signals (lower panels) were observed with a confocal microscope. (h) The numbers of punctate GFP-LC3 cells are represented by the ratio of GFP-LC3-labelled punctate cells to overall survived HCT116 cells. Data in h are means ± s.d. (n = 3).


( a ) Different FoxO1 - expressing plasmids , namely FoxO1 ( WT ) , FoxO1 ( 3A ) , FoxO1 ( ΔDB ) , FoxO1 ( ΔDB3A ) , FoxO1 ( NESm ) FoxO1 ( 3A - NLSm ) , were transfected into H1299 fractionation was performed to analyse localization ( N , nucleus ; C , cytoplasm ) of these FoxO1 mutants . PCAF is a loading control for , actin is loading control for .

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(a) Different FoxO1-expressing plasmids, namely FoxO1(WT), FoxO1(3A), FoxO1(ΔDB), FoxO1(ΔDB3A), FoxO1(NESm) and FoxO1(3A-NLSm), were transfected into H1299 cells and cell fractionation was performed to analyse cellular localization (N, nucleus; C, cytoplasm) of these FoxO1 mutants. PCAF is a loading control for nuclear protein, and actin is the loading control for cytoplasmic protein.


( b ) Quantitative PCR was performed to measure transactivity of autophagy - related genes in transfected H1299 with different FoxO1 - expressing plasmids ( upper panel ) or in stable FoxO1 - RNAi or non - RNAi - treated HCT116 in response to serum starvation for 24 h or 0 . 5 mM H2O2 for 12 h ( lower panel ) . data are means ± s . d . ( n = 3 ) .

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(b) Quantitative PCR was performed to measure the transactivity of autophagy-related genes in transfected H1299 cells with different FoxO1-expressing plasmids (upper panel) or in stable FoxO1-RNAi or non-RNAi-treated HCT116 cells in response to serum starvation for 24 h or 0.5 mM H2O2 for 12 h (lower panel). All data are means ± s.d. (n = 3).


( c ) Different FoxO1 - mutant - expressing plasmids were transfected into H1299 a ChIP was performed to measure binding activity of these mutated FoxO1 to p27 promoter .

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(c) Different FoxO1-mutant-expressing plasmids were transfected into H1299 cells and a ChIP assay was performed to measure the binding activity of these mutated FoxO1 to the p27 promoter.


( a ) HCT116 were treated with serum starvation for 24 h ( upper panels ) or 0 . 5 mM H2O2 for 12 h ( lower panels ) ; were extracted for co - immunoprecipitation with anti - FoxO1 probed with anti - acetylated lysine ( Ace - lyn ) . Both bottom panels display FoxO1 . IP , immunoprecipitation .

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(a) HCT116 cells were treated with serum starvation for 24 h (upper panels) or 0.5 mM H2O2 for 12 h (lower panels); cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 and probed with anti-acetylated lysine (Ace-lyn). Both bottom panels display cytosolic FoxO1. IP, immunoprecipitation.


( b ) HCT116 were serum - starved for 24 h were extracted for co - immunoprecipitation with anti - FoxO1 or anti - SIRT2 , followed by probing with anti - SIRT2 or anti - FoxO1 , respectively ( top middle panels ) . FoxO1 ( WT ) was also transfected into H1299 was extracted , followed by co - immunoprecipitation with anti - FoxO1 probing with anti - SIRT2 ( bottom panels ) .

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(b) HCT116 cells were serum-starved for 24 h and the cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 or anti-SIRT2, followed by probing with anti-SIRT2 or anti-FoxO1, respectively (top and middle panels). FoxO1(WT) was also transfected into H1299 cells and the cytosolic protein was extracted, followed by co-immunoprecipitation with anti-FoxO1 and probing with anti-SIRT2 (bottom panels).


( c ) HCT116 were transfected with SIRT2 RNAi plasmid or an empty control plasmid . At 48 h after transfection , lysate was extracted for probing with SIRT2 or FoxO1 ( upper panels ) , or for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - acetylated lysine ( lower panels ) . IB , immunoblot .

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(c) HCT116 cells were transfected with SIRT2 RNAi plasmid or an empty control plasmid. At 48 h after transfection, the cytosolic lysate was extracted for probing with SIRT2 or FoxO1 (upper panels), or for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine (lower panels). IB, immunoblot.


( d ) HCT116 were treated with AGK2 for 24 h at different doses , as indicated . lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - acetylated lysine .

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(d) HCT116 cells were treated with AGK2 for 24 h at different doses, as indicated. The cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine.


( a , b ) H1299 were transfected with an empty control plasmid or with Flag - FoxO1 ( WT ) plasmid . At 24 h after transfection , lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - mTOR , Beclin 1 , Vps34 or Atg1 ( a ) with Atg3 , Atg4 , Atg10 , Atg7 or LC3 ( b ) .

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(a, b) H1299 cells were transfected with an empty control plasmid or with the Flag-FoxO1(WT) plasmid. At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-mTOR, Beclin 1, Vps34 or Atg1 (a) and with Atg3, Atg4, Atg10, Atg7 or LC3 (b).


( c ) H1299 were co - transfected with Myc - Atg7 , with Flag - FoxO1 ( WT ) or with an empty control plasmid . At 24 h after transfection , lysate was extracted for Flag with anti - Flag , followed by probing with anti - Myc .

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(c) H1299 cells were co-transfected with Myc-Atg7, with Flag-FoxO1(WT) or with an empty control plasmid. At 24 h after transfection, the cytosolic lysate was extracted for Flag with anti-Flag, followed by probing with anti-Myc.


HCT116 were serum - starved for 24 h ( d ) lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti -

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HCT116 cells were serum-starved for 24 h (d)and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7


( e ) Different Myc - tagged FoxO1 fragments Flag - tagged Atg7 were co - transfected into H1299 , lysate was extracted for co - immunoprecipitation with anti - Myc , followed by probing with anti - Flag .

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(e) Different Myc-tagged FoxO1 fragments and Flag-tagged Atg7 were co-transfected into H1299 cells, and the cytosolic lysate was extracted for co-immunoprecipitation with anti-Myc, followed by probing with anti-Flag.


( f ) HCT116 were transfected with an empty control plasmid , an SIRT2 ( WT ) - expressing plasmid or an SIRT2 ( 168A ) - expressing plasmid . At 24 h after transfection , were serum - starved for 24 h , lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - Atg7 .

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(f) HCT116 cells were transfected with an empty control plasmid, an SIRT2(WT)-expressing plasmid or an SIRT2(168A)-expressing plasmid. At 24 h after transfection, cells were serum-starved for 24 h, and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7.


HCT116 weretreated with AGK2 for 24 h ( g ) , lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti -

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HCT116 cells weretreated with AGK2 for 24 h (g), and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7


( a ) HCT116 or stable FoxO1 - RNAi HCT116 were treated with a non - specific RNAi control or SIRT2 - RNAi , LC3 - II accumulation or p62 degradation was detected in presence or absence of E64 pepstatin A .

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(a) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with a non-specific RNAi control or SIRT2-RNAi, and LC3-II accumulation or p62 degradation was detected in the presence or absence of E64 and pepstatin A.


( b ) HCT116 or stable FoxO1 - RNAi HCT116 were treated with 10 μM AGK2 for 24 h , in presence or absence of E64 pepstatin A . Changes in FoxO1 , p62 LC3 - II were determined by western blotting ( upper panels ) , or formation of LC3 punctate signals was observed ( lower panels ) . HCT116 were treated with 10 μM AGK2 for 24 h formation of LC3 punctate signals was observed . of of LC3 punctate to overall HCT116 is shown in lower right panel . Data are means ± s . d . ( n = 3 ) .

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(b) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with 10 μM AGK2 for 24 h, in the presence or absence of E64 and pepstatin A. Changes in FoxO1, p62 and LC3-II were determined by western blotting (upper panels), or the formation of LC3 punctate signals was observed (lower panels). HCT116 cells were treated with 10 μM AGK2 for 24 h and the formation of LC3 punctate signals was observed. Quantification of the ratio of LC3 punctate cells to overall HCT116 cells is shown in the lower right panel. Data are means ± s.d. (n = 3).


( c ) H1299 were transfected with Flag - FoxO1 ( WT ) , SIRT2 ( WT ) or SIRT2 ( 168A ) . At 24 h after transfection , lysate was extracted immunoblotted with anti - Flag , anti - SIRT2 , anti - LC3 , anti - p62 or anti - β - actin . .

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(c) H1299 cells were transfected with Flag-FoxO1(WT), SIRT2(WT) or SIRT2(168A). At 24 h after transfection, the cell lysate was extracted and immunoblotted with anti-Flag, anti-SIRT2, anti-LC3, anti-p62 or anti-β-actin..


( d ) HCT116 were transfected with a control plasmid , a SIRT2 ( WT ) - expressing plasmid or a SIRT2 ( 168A ) - expressing plasmid . At 24 h after transfection , were subjected to serum starvation for 24 h . lysate was extracted immunoblotted with anti - SIRT2 , anti - LC3 , anti - p62 or anti - β - actin .

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(d) HCT116 cells were transfected with a control plasmid, a SIRT2(WT)-expressing plasmid or a SIRT2(168A)-expressing plasmid. At 24 h after transfection, the cells were subjected to serum starvation for 24 h. The cell lysate was extracted and immunoblotted with anti-SIRT2, anti-LC3, anti-p62 or anti-β-actin.


( e ) H1299 were transfected with Myc - Atg7 , Flag - FoxO1 ( WT ) or mutated Flag - FoxO1 ( KR ) . At 24 h after transfection , lysate was extracted for co - immunoprecipitation with anti - Myc , followed by probing with anti - Flag .

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(e) H1299 cells were transfected with Myc-Atg7, Flag-FoxO1(WT) or mutated Flag-FoxO1(KR). At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-Myc, followed by probing with anti-Flag.


( f ) H1299 were transfected with a control plasmid , Flag - FoxO1 ( WT ) , Flag - FoxO1 ( K262R ) , Flag - FoxO1 ( K265R ) , Flag - FoxO1 ( K274R ) or Flag - FoxO1 ( KR ) . At 24h after transfection in presence or absence of E64 pepstatin A immunoblotted , lysate was extracted immunoblotted with anti - Flag , anti - LC3 , anti - p62 or anti - β - actin .

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(f) H1299 cells were transfected with a control plasmid, Flag-FoxO1(WT), Flag-FoxO1(K262R), Flag-FoxO1(K265R), Flag-FoxO1(K274R) or Flag-FoxO1(KR). At 24h after transfection in the presence or absence of E64 and pepstatin A and immunoblotted, the cell lysate was extracted and immunoblotted with anti-Flag, anti-LC3, anti-p62 or anti-β-actin.


( g ) Left : H1299 were co - transfected with GFP - LC3 , Flag - FoxO1 ( WT ) or Flag - FoxO1 ( KR ) . formation of GFP - LC3 - labelled punctate signals was observed 24 h after transfection . Right : of of GFP - LC3 - labelled punctate to overall survived H1299 . Data in g are means ± s . d . ( n = 3 ) . Scale bars , 25 μm ( b ) , 10 μm ( g ) .

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(g) Left: H1299 cells were co-transfected with GFP-LC3, Flag-FoxO1(WT) or Flag-FoxO1(KR). The formation of GFP-LC3-labelled punctate signals was observed 24 h after transfection. Right: quantification of the ratio of GFP-LC3-labelled punctate cells to overall survived H1299 cells. Data in g are means ± s.d. (n = 3). Scale bars, 25 μm (b), 10 μm (g).


( a ) H1299 were transfected with a control plasmid , Flag - FoxO1 ( WT ) , Flag - FoxO1 ( 3A ) , Flag - FoxO1 ( ΔDB ) or Flag - FoxO1 ( ΔDB3A ) , in presence or absence of 10 mM 3 - MA ( upper panel ) or in presence or absence of 50 μM Z - VAD - fmk ( lower panel ) ; were stained with propidium iodide ( PI ) 48 h after transfection . PI - stained ( dead ) were counted by flow cytometry loss was calculated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O 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) H1299 cells were transfected with a control plasmid, Flag-FoxO1(WT), Flag-FoxO1(3A), Flag-FoxO1(ΔDB) or Flag-FoxO1(ΔDB3A), in the presence or absence of 10 mM 3-MA (upper panel) or in the presence or absence of 50 μM Z-VAD-fmk (lower panel); cells were stained with propidium iodide (PI) 48 h after transfection. PI-stained (dead) cells were counted by flow cytometry and the percentage cell loss was calculated.


( b ) FoxO1 ( WT ) or Flag - FoxO1 ( ΔDB ) was transfected into H1299 ; 48 h later autophagic vesicles were observed under an electron microscope . Enlarged ( right ) show clearer autophagic vesicles indicated by arrows . Scale bars , 2 μm .

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(b) FoxO1(WT) or Flag-FoxO1(ΔDB) was transfected into H1299 cells; 48 h later the autophagic vesicles were observed under an electron microscope. Enlarged images (right) show clearer autophagic vesicles indicated by arrows. Scale bars, 2 μm.


( c ) 48 h with or without Atg5 RNAi were transfected with FoxO1 ( WT ) , was determined by with PI at 48 h after transfection . Data in c are means ± s . d . ( n = 3 ) .

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(c) 48 h cells with or without Atg5 RNAi were transfected with FoxO1(WT), and cell death was determined by staining with PI at 48 h after transfection. Data in c are means ± s.d. (n = 3).


( d ) stable Atg5 knockdown H1299 was further transfected with a control plasmid , Flag - FoxO1 ( WT ) , or Flag - FoxO1 ( ΔDB ) , loss was quantified by flow cytometry at 48 h after transfection . Data in d are means ± s . d . ( n = 3 ) .

(d) The stable Atg5 knockdown H1299 cells was further transfected with a control plasmid, Flag-FoxO1(WT), or Flag-FoxO1(ΔDB), and the percentage cell loss was quantified by flow cytometry at 48 h after transfection. Data in d are means ± s.d. (n = 3).


( a ) Stable H1299 with or without Atg7 knockdown were transfected with a control plasmid ( group 1 ) , Flag - FoxO1 ( ΔDB ) ( group 2 ) or Flag - FoxO1 ( ΔDB - 3A ) ( group 3 ) ; 107 were then injected into 18 nude mice , with 6 mice per group . Seven later mice were killed tumours were weighed analysed ( bottom graph ) . Group 1 : tumour weights were 1 . 22 ± 0 . 78 g ( without Atg7 RNAi ) 1 . 31 ± 0 . 43 g ( with Atg7 RNAi ) , P = 0 . 426 . Group 2 : tumour weights were 0 . 49 ± 0 . 18 g ( without Atg7 RNAi ) 0 . 80 ± 0 . 20 g ( with Atg7 RNAi ) , P = 0 . 017 . Group 3 : tumour weights were 1 . 00 ± 0 . 40 g ( without Atg7 RNAi ) 1 . 10 ± 0 . 36 g ( with Atg7 RNAi ) , P = 0 . 500 . Data are means ± s . d . ( n = 6 ) .

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(a) Stable H1299 cells with or without Atg7 knockdown were transfected with a control plasmid (group 1), Flag-FoxO1(ΔDB) (group 2) or Flag-FoxO1(ΔDB-3A) (group 3); 107 cells were then injected into 18 nude mice, with 6 mice per group. Seven weeks later the mice were killed and the tumours were weighed and analysed (bottom graph). Group 1: tumour weights were 1.22 ± 0.78 g (without Atg7 RNAi) and 1.31 ± 0.43 g (with Atg7 RNAi), P = 0.426. Group 2: tumour weights were 0.49 ± 0.18 g (without Atg7 RNAi) and 0.80 ± 0.20 g (with Atg7 RNAi), P = 0.017. Group 3: tumour weights were 1.00 ± 0.40 g (without Atg7 RNAi) and 1.10 ± 0.36 g (with Atg7 RNAi), P = 0.500. Data are means ± s.d. (n = 6).


( b ) Expression of FoxO1 Atg7 conjugate in mice tumours were determined by western blotting .

(b) Expression levels of FoxO1 and Atg7 conjugate in mice tumours were determined by western blotting.


( c ) HCT116 ( 107 ) or stable FoxO1 - knockdown HCT116 ( 107 ) were injected into left or right hind legs of three nude mice . Six later mice were killed tumours were weighed analysed ( bottom graph ) . Tumour weights were 0 . 08 ± 0 . 10 g ( without FoxO1 RNAi ) 0 . 48 ± 0 . 06 g ( with FoxO1 RNAi ) , P = 0 . 020 . Data are means ± sd . n = 3 .

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(c) HCT116 cells (107) or stable FoxO1-knockdown HCT116 cells (107) were injected into the left or right hind legs of three nude mice. Six weeks later the mice were killed and the tumours were weighed and analysed (bottom graph). Tumour weights were 0.08 ± 0.10 g (without FoxO1 RNAi) and 0.48 ± 0.06 g (with FoxO1 RNAi), P = 0.020. Data are means ± sd. n = 3.


( d ) was extracted from one paired colon tumour ( T ) or adjacent normal tissue ( N ) from one , western blotting was performed to detect changes in FoxO1 or p62 .

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(d) Protein was extracted from one paired colon tumour (T) or adjacent normal tissue (N) from one patient, and western blotting was performed to detect changes in FoxO1 or p62.


( e ) were extracted from 16 paired samples from for western blotting with anti - FoxO1 or with anti - p62 antibody . bands were scanned with a phosphorimager relative band was normalized to β - actin band . FoxO1 of normal tissue , 0 . 37 ; FoxO1 of tumour tissue , 0 . 10 ; P = 0 . 001 , two - sided t - . p62 of normal tissue , 0 . 32 ; p62 of tumour tissue , 1 . 00 ; P = 0 . 009 , two - sided t - . Data in e are means ± s . d . ( n = 16 ) .

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(e) Proteins were extracted from 16 paired samples from patients for western blotting with anti-FoxO1 or with anti-p62 antibody. Protein bands were scanned with a phosphorimager and relative band intensity was normalized to the β-actin band. FoxO1 of normal tissue, 0.37; FoxO1 of tumour tissue, 0.10; P = 0.001, two-sided t-test. p62 of normal tissue, 0.32; p62 of tumour tissue, 1.00; P = 0.009, two-sided t-test. Data in e are means ± s.d. (n = 16).


( B ) Time course of SUMO - 1 modification of His - tagged HTTex1p ( 25Q ) - purified ( WT , K6R , K9R , K6 , 9R , K6 , 9 , 15R , S13 , 16D ) was performed in vitro . SUMOylation was visualized using anti - His antibody . WT HTTex1p is SUMOylated within 16 min , K6R or K9R mutations delay SUMOylation , combined mutations ( K6 , 9R or K6 , 9 , 15R ) greatly reduce SUMOylation . Mutations that mimic phosphorylation ( S13 , 16D ) alter kinetics of SUMO - 1 modification with SUMO modification observed beginning by 4 min .

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(B) Time course of SUMO-1 modification of His-tagged HTTex1p (25Q)-purified proteins (WT, K6R, K9R, K6,9R, K6,9,15R, and S13,16D) was performed in vitro. SUMOylation was visualized using anti-His antibody. WT HTTex1p is SUMOylated within 16 min, K6R or K9R mutations delay SUMOylation, and combined mutations (K6,9R or K6,9,15R) greatly reduce SUMOylation. Mutations that mimic phosphorylation (S13,16D) alter kinetics of SUMO-1 modification with SUMO modification observed beginning by 4 min.


( A ) qRT - PCR of SUMO - modifying enzymes in cortex striata of WT mice at 12 . Relative expression for SUMO enzymes is normalized to mouse β - actin .

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(A) qRT-PCR analysis of SUMO-modifying proteins and enzymes in cortex and striata of WT mice at 12 weeks. Relative expression for all the SUMO enzymes is normalized to mouse β-actin.


( B C ) qRT - PCR of SUMO mRNAs from 12 - week - old WT R6 / 2 mouse cortex ( B ) striatum ( C ) . SUMO enzyme mRNAs are differentially expressed in R6 / 2 versus control with statistically significant increases in SENP1 ( p = 0 . 01 ) , SENP3 ( p = 0 . 02 ) , SUMO - 1 ( p = 0 . 003 ) in cortex SENP1 ( p = 0 . 02 ) , SENP6 ( p = 0 . 04 ) , PIAS3 ( p = 0 . 007 ) , SUMO - 1 ( p = 0 . 02 ) , SUMO - 2 ( p = 0 . 02 ) in striatum . Samples were analyzed in quadruplicate normalized to mouse β - actin . Data are shown as R6 / 2 expression relative to WT set at 1 for each enzyme with ± SD ( n = 4 ) . * p < 0 . 05 . n . s . , not significant .

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(B and C) qRT-PCR of SUMO mRNAs from 12-week-old WT and R6/2 mouse cortex (B) and striatum (C). SUMO enzyme mRNAs are differentially expressed in R6/2 versus control with statistically significant increases in SENP1 (p = 0.01), SENP3 (p = 0.02), and SUMO-1 (p = 0.003) in cortex and SENP1 (p = 0.02), SENP6 (p = 0.04), PIAS3 (p = 0.007), SUMO-1 (p = 0.02), and SUMO-2 (p = 0.02) in striatum. Samples were analyzed in quadruplicate and normalized to mouse β-actin. Data are shown as R6/2 expression relative to WT levels set at 1 for each enzyme with ± SD (n = 4). *p < 0.05. n.s., not significant.


( D E ) Western blot of SUMO - 2 in 14 - week R6 / 2 cortex ( D ) striatum ( E ) versus aged - matched controls . SUMO - 2 is upregulated in R6 / 2 striatum versus control ( p = 0 . 026 ; n = 4 ) . is normalized to α - tubulin quantitated using ImageJ .

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(D and E) Western blot analysis of SUMO-2 in 14-week R6/2 cortex (D) and striatum (E) versus aged-matched controls. SUMO-2 is upregulated in R6/2 striatum versus control (p = 0.026; n = 4). Protein is normalized to α-tubulin and quantitated using ImageJ.


( A ) HeLa transfected with His - tagged HTTex1p ( 46Q ) or 46QP - K6 , 9 , 15R ( 3R ) along with GFP - SUMO - 1 or GFP - SUMO - 2 , lysed under denaturing conditions , nickel purified ( Ni - NTA ) . Unmodified HTT - 46Q is indicated by arrow SUMO - modified HTT by boxed region . lysine mutant ( 3R ) serves as a negative control . Ni - NTA represents nickel - purified His - tagged HTT , WC TCA represents 10 % of whole - lysate expression of HTT myc - actin ( transfection control ) . HTT is modified by SUMO - 1 ( left ) SUMO - 2 ( right ) .

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(A) HeLa cells transfected with His-tagged HTTex1p (46Q) or 46QP-K6,9,15R (3R) along with GFP-SUMO-1 or GFP-SUMO-2, lysed under denaturing conditions, and nickel purified (Ni-NTA). Unmodified HTT-46Q is indicated by the arrow and SUMO-modified HTT by the boxed region. The lysine mutant (3R) serves as a negative control. Ni-NTA represents nickel-purified His-tagged HTT, and WC TCA represents 10% of the whole-cell lysate expression of HTT and myc-actin (transfection control). HTT is modified by SUMO-1 (left) and SUMO-2 (right).


( B ) SUMO isopeptidases ( SENP1 , SENP2 , SENP3 , SENP5 , SENP6 ) modulate SUMO - modified HTT when overexpressed together with HTT ( 46QP - H4 or 3R ) SUMO - 1 ( GFP - SUMO - 1 ) . SENP1 , SENP2 , SENP6 decrease HTT SUMOylation . Graph depicts quantitation of western blot using Odyssey Infrared Imaging System ( LI - COR ) to calculate of HTT purified versus HTT modified by SUMO multiplied by 100 .

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(B) SUMO isopeptidases (SENP1, SENP2, SENP3, SENP5, and SENP6) modulate SUMO-modified HTT when overexpressed together with HTT (46QP-H4 or 3R) and SUMO-1 (GFP-SUMO-1). SENP1, SENP2, and SENP6 decrease HTT SUMOylation. Graph depicts quantitation of western blot using the Odyssey Infrared Imaging System (LI-COR) to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100.