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 )

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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 cells using CRISPR / Cas9 technology . Monoclones ( # 3 and # 14 ) were selected .

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


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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 , 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 .

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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 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 .

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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 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 .

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

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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 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 .

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

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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 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 .

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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 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 .

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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.


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cTAZ and STAT1/2 expression levels of cells


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 .

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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 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 .

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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 . 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 .

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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 . 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 .

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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 . 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 and responses to STAT1 / 2 expression were measured . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; Student ' s t - test .

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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 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 .

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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.


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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

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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 - tailed t - test 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

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 and 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 , and E - cadherin protein 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 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 .

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

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


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 .

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

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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 protein 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 cell proliferation . Data information : data are representative of three independent experiments . Error bars , ± SD . * , p < 0 . 05 , by two - tailed t - test in G .

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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 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 .

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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 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 .

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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 protein ( B ) expression in 1123 GSCs .

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 .

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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 and luciferase reporters containing ZNF596 5 ' - UTR WT , mutant , or empty vector ( EV ) .

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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 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

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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 , and STAT3 phosphorylation in 1123 and 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 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 .

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

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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 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

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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 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 -

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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 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 -

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

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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 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 .

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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 , and LINC00115 from a separate cohort of a total of 75 paraffin - embedded clinical GBM samples . Statistical analysis was performed by Chi - square test .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 ) and dorsal root ganglia ( black arrow ) , and ( 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 and ( M ) EE3 . Black arrow indicates neuronal population from EE1 embryo 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 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 .

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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 ) and in subventricular zone ( white 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 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 .

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

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 ) 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 .

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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.


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 .

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

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 .

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


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 .

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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 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 ) .

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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 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 .

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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 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 ) .

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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).


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 .

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

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

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

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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 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 ) .

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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 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 .

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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 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 .

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(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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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 and LC3 - II accumulation were evaluated by western blotting in H1299 cells 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 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 ) .

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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 cells . After 24 h , luciferase reporter activity of FoxO1 was measured . RLU , relative luciferase units . Data are means ± s . d . ( n = 3 ) .

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(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 cells in the presence or absence of E64 and pepstatin A . Western blotting was performed to detect p62 degradation and LC3 - II accumulation .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR 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 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 ) .

O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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 ) 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 .

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

(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 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 ) .

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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 cells and a ChIP assay was performed to measure the binding activity of these mutated FoxO1 to the 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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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

(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 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 .

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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 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 -

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

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 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 .

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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 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 .

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

(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 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 -

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 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 .

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

(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 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 ) .

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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 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 . .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 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 .

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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 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 ) .

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

(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 ) 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 ) .

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

(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 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 ) .

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

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

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

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


( 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 .

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

(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 ) 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 .

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

(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 ) 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 ) .

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

(e) 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 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 .

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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 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 .

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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 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 .

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

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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.