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 .

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

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 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 -

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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 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 .

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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 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 .

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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 .

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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 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 .

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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 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 - .

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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 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 ) .

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(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 ) .

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

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(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 ) .

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(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 -

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 .

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(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 ) .

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(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 .

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(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.


( C ) Titration of SUMO - 1 . Denaturing nickel purification of HTTex1p ( 46QP - H4 ) following transfection with decreasing amounts of SUMO - 1 reduces amount of SUMO - modified HTT to undetectable . Ni - NTA blot displayed in gray scale shows purified HTTex1 SUMO - modified HTTex1 using HTT antibody . Note that 0 . 5 μg of SUMO - 1 ( ¼ amount of SUMO - 1 cDNA ) was used for identifying SUMO - 1 E3 ligase for HTTex1p . Graph depicts quantitation of western blot using Odyssey Infrared Imaging System to calculate of HTT purified versus HTT modified by SUMO multiplied by 100 . Note that experiments were performed in triplicate , a representative figure is shown .

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(C) Titration of SUMO-1. Denaturing nickel purification of HTTex1p (46QP-H4) following transfection with decreasing amounts of SUMO-1 reduces the amount of SUMO-modified HTT to undetectable levels. The Ni-NTA blot displayed in the gray scale shows purified HTTex1 and SUMO-modified HTTex1 using HTT antibody. Note that 0.5 μg of SUMO-1 (¼ the amount of SUMO-1 cDNA ) was used for identifying the SUMO-1 E3 ligase for HTTex1p. Graph depicts quantitation of western blot using the Odyssey Infrared Imaging System to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100. Note that all experiments were performed in triplicate, and a representative figure is shown.


( A ) Under limiting SUMO conditions ( 1 / 4 SUMO - 1 , lanes 3 - 9 ) , PIAS1 increases HTT - SUMO modification above 1 / 4 SUMO alone . Purified HTT ( arrow ) SUMO - HTT ( boxed region ) were detected using anti - HTT . Graph depicts quantitation of Ni - NTA western blot using Odyssey Infrared Imaging Software ( LI - COR ) to calculate of HTT purified versus HTT modified by SUMO multiplied by 100 .

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(A) Under limiting SUMO conditions (1/4 SUMO-1, lanes 3-9), PIAS1 increases HTT-SUMO modification above 1/4 SUMO alone. Purified HTT (arrow) and SUMO-HTT (boxed region) were detected using anti-HTT. Graph depicts quantitation of the Ni-NTA western blot using the Odyssey Infrared Imaging Software (LI-COR) to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100.


( B ) Western of overexpression of HTTex1 ( 46QP - H4 or 3R ) , SUMO - 2 ( GFP - SUMO - 2 ) , PIAS ( PIAS1 , PIASxα , PIASxβ , PIAS3 , PIASy ) . Under nonlimiting SUMO - 2 conditions , PIAS1 enhances SUMO modification of HTT . WC TCA shows overall myc - actin ( transfection control ) HTT . Graph quantitating of HTT purified versus HTT modified using Odyssey ( LICOR ) .

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(B) Western analysis of overexpression of HTTex1 (46QP-H4 or 3R), SUMO-2 (GFP-SUMO-2), and all the PIAS proteins (PIAS1, PIASxα, PIASxβ, PIAS3, and PIASy). Under nonlimiting SUMO-2 conditions, PIAS1 enhances SUMO modification of HTT. WC TCA shows overall myc-actin (transfection control) and HTT levels. Graph quantitating the ratio of HTT purified versus the HTT modified using the Odyssey (LICOR).


( C ) Left panel is autoradiography results of a GST pull - down showing that radiolabeled human PIAS1 interacts with HTTex1p . Right panel is a phosphorimager of GST pull - downs , performed in triplicate , showing of 35S - labeled PIAS that bound GST : GST alone , unexpanded HTT with without proline - rich region ( 20QP 20Q ) , expanded HTT with without proline - rich region ( 51QP 51Q ) , proline - rich region alone ( Pro ) . Error bars were calculated as an estimate of SE = STDEV ( n ) / SQRT ( n - 1 ) .

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(C) Left panel is the autoradiography results of a GST pull-down assay showing that radiolabeled human PIAS1 interacts with HTTex1p. Right panel is a phosphorimager analysis of GST pull-downs, performed in triplicate, showing the percentage of 35S-labeled PIAS protein that bound the GST proteins: GST alone, unexpanded HTT with and without the proline-rich region (20QP and 20Q), expanded HTT with and without the proline-rich region (51QP and 51Q), and the proline-rich region alone (Pro). Error bars were calculated as an estimate of SE = STDEV(n)/SQRT(n-1).


( B ) Longer HTT polypeptides are modified by SUMO - 1 . Western blot overexpressing HTTex1p ( 46QP - H4 or 3R ) , unexpanded HTT - 586 fragment ( 25Q - 586 or 25Q - 3R - 586 aa ) , expanded HTT - 586 fragment ( 137Q - 586 or 137Q - 3R - 586 aa ) with SUMO - 1 ( GFP - SUMO - 1 ) . lysates were subjected to HTT immunoprecipitation ( IP ) using HTT antibody . Western performed with Odyssey ( LI - COR ) allows detection of HTT ( data not shown ) SUMO simultaneously shows that forms of HTT are SUMO - 1 modified using anti - GFP antibody . HTTex1p is covalently SUMO - 1 modified ( lane 3 ) , modification disappears when Lys are mutated to Arg ( 3R ) ( lane 4 ) . Both unexpanded ( lane 4 5 ) expanded HTT - 586 fragments ( lane 6 7 ) are covalently SUMO - 1 modified in both presence absence of three Lys in N - terminal region of HTT ( 3R ) . Free SUMO - 1 is indicated with arrow , SUMO - modified HTT is indicated by boxes . Inset on right , from a replicate experiment , shows comigration of expanded HTT ( anti - HTT ) SUMO - 1 ( anti - GFP ) displayed in gray scale in color when two antibodies are merged ( HTT in red , SUMO - 1 in green , yellow when colocalizing ) .

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(B) Longer HTT polypeptides are modified by SUMO-1. Western blot overexpressing HTTex1p (46QP-H4 or 3R), unexpanded HTT-586 fragment (25Q-586 or 25Q-3R-586 aa), and expanded HTT-586 fragment (137Q-586 or 137Q-3R-586 aa) with SUMO-1 (GFP-SUMO-1). Cell lysates were subjected to HTT immunoprecipitation (IP) using HTT antibody. Western analysis performed with the Odyssey (LI-COR) allows detection of HTT (data not shown) and SUMO simultaneously and shows that all forms of HTT are SUMO-1 modified using the anti-GFP antibody. HTTex1p is covalently SUMO-1 modified (lane 3), and the modification disappears when Lys are mutated to Arg (3R) (lane 4). Both unexpanded (lane 4 and 5) and expanded HTT-586 fragments (lane 6 and 7) are covalently SUMO-1 modified in both the presence and absence of the three Lys in the N-terminal region of HTT (3R). Free SUMO-1 is indicated with the arrow, and SUMO-modified HTT is indicated by the boxes. Inset on the right, from a replicate experiment, shows comigration of expanded HTT (anti-HTT) and SUMO-1 (anti-GFP) displayed in the gray scale and in color when the two antibodies are merged (HTT in red, SUMO-1 in green, and yellow when colocalizing).


( C ) Bait plasmids ( HTT - 586 - 25Q or HTT - 586 - 73Q aa ) were transformed into L40ccua MATa yeast . Yeast clones encoding bait were individually mated against a matrix of MATα yeast clones encoding 16 , 888 prey ( with Gal4 activation domain fusions ) using pipetting spotting robots . Diploid yeasts were spotted onto SDIV ( - Leu - Trp - Ura - His ) agar plates for selection of PPIs as well as nylon membranes placed on SDIV agar plates for β - galactosidase assays . After 5 - 6 days of incubation at 30ºC , digitized of agar plates nylon membranes were assessed for growth β - galactosidase activity using software Visual Grid ( GPC Biotech ) .

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(C) Bait plasmids (HTT-586-25Q or HTT-586-73Q aa) were transformed into the L40ccua MATa yeast strain. Yeast clones encoding bait proteins were individually mated against a matrix of MATα yeast clones encoding 16,888 prey proteins (with Gal4 activation domain fusions) using pipetting and spotting robots. Diploid yeasts were spotted onto SDIV (-Leu-Trp-Ura-His) agar plates for selection of PPIs as well as nylon membranes placed on SDIV agar plates for β-galactosidase assays. After 5-6 days of incubation at 30ºC, digitized images of the agar plates and nylon membranes were assessed for growth and β-galactosidase activity using the software Visual Grid (GPC Biotech).


( D ) Overexpression of PIAS1 alone , with unexpanded HTT constructs ( 25Q - 586 aa ) plus SUMO - 1 ( GFP - SUMO - 1 ) , shows that PIAS1 binds both full - HTT HTT - 586 fragment ( 25Q - 586 aa ) . WT HTT was used in these experiments to preclude confounding aggregation effects . Western detection was performed using Odyssey is displayed in gray scale but is shown in color on merge ( HTT is red , PIAS1 is green ) . WB , western blot .

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(D) Overexpression of PIAS1 alone, with unexpanded HTT constructs (25Q-586 aa) plus SUMO-1 (GFP-SUMO-1), shows that PIAS1 binds both full-length HTT and HTT-586 fragment (25Q-586 aa). WT HTT was used in these experiments to preclude confounding aggregation effects. Western analysis detection was performed using Odyssey and is displayed in the gray scale but is shown in color on the merge (HTT is red, and PIAS1 is green). WB, western blot.


( E ) HeLa overexpressing either expanded 586 aa - HTT or phosphomimetic ( S13 , 16D = DD ) with SUMO - 2 plus minus PIAS1 . HTT was purified by IP using hydrazide beads ( Bioclone ) crosslinked to HTT ( Enzo ) antibody subjected to western using anti - HTT ( MAB5490 ) . Arrows indicate SUMO - 2 - modified HTT .

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(E) HeLa cells overexpressing either expanded 586 aa-HTT or the phosphomimetic (S13,16D = DD) with SUMO-2 plus and minus PIAS1. HTT was purified by IP using hydrazide beads (Bioclone) crosslinked to HTT (Enzo) antibody and subjected to western analysis using anti-HTT (MAB5490). Arrows indicate SUMO-2-modified HTT.


( F ) IP of HTT with hydrazide - linked beads shows that both unexpanded expanded HTT 586 aa phosphomimetics ( S13 , 16D - 586 aa ) are modified by SUMO - 2 . Arrows indicate SUMO - conjugated HTT . Note that experiments including Y2H were done in triplicate ; representative experiments are shown . Arrows indicate SUMO - 2 - modified HTT .

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(F) IP of HTT with hydrazide-linked beads shows that both unexpanded and expanded HTT 586 aa phosphomimetics (S13, 16D-586 aa) are modified by SUMO-2. Arrows indicate SUMO-conjugated HTT. Note that all experiments including the Y2H assay were done in triplicate; representative experiments are shown. Arrows indicate SUMO-2-modified HTT.


( A ) Western of whole - lysates from HeLa transfected with His - SUMO - 1 or SUMO - 2 / or 97Q - HTT exon 1 treated with 5 μM MG132 for 18 hr . Lysates were separated using differential centrifugation into a detergent - soluble fraction ( SOLUBLE ) with 1 % Triton X - 100 a detergent - insoluble fraction ( INSOLUBLE ) with 4 % SDS . Western blot probed with anti - HTT shows full - endogenous HTT in SOLUBLE fraction ( upper arrow ) 97Q - HTTex1 ( lower arrow ) ( left panel ) . In INSOLUBLE fraction , HTT HMW species are indicated by bracket asterisks ( right panel ) .

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(A) Western analysis of whole-cell lysates from HeLa cells transfected with His-SUMO-1 or SUMO-2 and/or 97Q-HTT exon 1 and treated with 5 μM MG132 for 18 hr. Lysates were separated using differential centrifugation into a detergent-soluble fraction (SOLUBLE) with 1% Triton X-100 and a detergent-insoluble fraction (INSOLUBLE) with 4% SDS. Western blot probed with anti-HTT shows full-length endogenous HTT in the SOLUBLE fraction (upper arrow) and 97Q-HTTex1 (lower arrow) (left panel). In the INSOLUBLE fraction, HTT HMW species are indicated by the bracket and asterisks (right panel).


( B ) MG132 SUMO - 2 cause mutant HTT to accumulate as HMW species ( bracke asterisk ) . Western blot showing IP with HTT antibody crosslinked beads from detergent - insoluble fraction probed with anti - HTT antibody .

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(B) MG132 and SUMO-2 cause mutant HTT to accumulate as HMW species (bracke and asterisk). Western blot showing IP with HTT antibody crosslinked beads from the detergent-insoluble fraction probed with the anti-HTT antibody.


( C ) Mutant HTT ( 97Q - Httex1 ) fibrils are detected with anti - HTT in insoluble fraction with treatment of MG132 or addition of exogenous SUMO - 2 .

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(C) Mutant HTT (97Q-Httex1) fibrils are detected with anti-HTT in the insoluble fraction with treatment of MG132 or addition of exogenous SUMO-2.


( D ) Western blot with increasing concentrations of SUMO - 2 detected with anti - His antibody ( left panel ) . Middle panel is same western blot probed with anti - HTT showing soluble forms of HTT . Right panel presents western blot from detergent - insoluble fraction with monomeric HTT ( 97Q ) at 55 kDa , asterisk ( * ) indicates HMW species . Note that experiments were performed in triplicate ; representative figures are shown .

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(D) Western blot with increasing concentrations of SUMO-2 detected with anti-His antibody (left panel). Middle panel is the same western blot probed with anti-HTT showing soluble forms of HTT. Right panel presents western blot from detergent-insoluble fraction with monomeric HTT (97Q) at 55 kDa, and the asterisk (*) indicates the HMW species. Note that all experiments were performed in triplicate; representative figures are shown.


( A ) Western blot of HeLa over - expressing exogenous PIAS1 in presence of mutant HTT ( 97Q ) when separated into detergent - soluble detergent - insoluble fractions . No difference is detected in monomeric HTT ( top panel , Soluble ) , but HMW HTT increase with PIAS1 overexpression . Anti - PIAS1 antibody ( Invitrogen ) was used to detect PIAS1 .

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(A) Western blot analysis of HeLa cells over-expressing exogenous PIAS1 in the presence of mutant HTT (97Q) when separated into detergent-soluble and detergent-insoluble fractions. No difference is detected in monomeric HTT (top panel, Soluble), but HMW HTT levels increase with PIAS1 overexpression. Anti-PIAS1 antibody (Invitrogen) was used to detect PIAS1.


( B ) Acute knockdown of PIAS1 decreases HMW HTT species in detergent - insoluble fraction . PIAS1 knockdown is detected in detergent - soluble - insoluble fractions using anti - PIAS1 antibody .

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(B) Acute knockdown of PIAS1 decreases HMW HTT species in the detergent-insoluble fraction. PIAS1 knockdown is detected in detergent-soluble and -insoluble fractions using anti-PIAS1 antibody.


( C ) Drosophila melanogaster expressing mutant HTTex1p ( 93Q ) in a reduced Su ( var ) 2 - 10 / dPIAS genetic background exhibits statistically significantly reduced photoreceptor neuron degeneration ( left panel , p = 0 . 033 ) when comparing dPIAS / + to + / + flies increased overall survival ( right panel , p = 0 . 047 ) when comparing dPIAS1 / + to + / + flies . Significance was measured by Student ' s t .

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(C) Drosophila melanogaster expressing mutant HTTex1p (93Q) in a reduced Su(var)2-10/dPIAS genetic background exhibits statistically significantly reduced photoreceptor neuron degeneration (left panel, p = 0.033) when comparing dPIAS/+ to +/+ flies and increased overall survival (right panel, p = 0.047) when comparing dPIAS1/+ to +/+ flies. Significance was measured by Student's t test.


( D ) HMW SUMO - 2 accumulates in postmortem HD striata . Western blot of insoluble fraction from three control three HD postmortem striata as described ( Experimental Procedures ) .

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(D) HMW SUMO-2 accumulates in postmortem HD striata. Western blot analysis of the insoluble fraction from three control and three HD postmortem striata as described (Experimental Procedures).


B ) Larval brains at 6 h ALH from grh - Gal4 ; UAS - CD8 - GFP were labeled with Sas - 4 , Dpn , GFP .

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B) Larval brains at 6 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with Sas-4, Dpn, and GFP.


C ) Larval brains at 0 h ALH from grh - Gal4 ; UAS - CD8 - GFP were labeled with γ - tubulin ( γ - tub ) , Asl , GFP imaged under super - resolution microscopy . D ) Larval brains at 0 h ALH from grh - Gal4 ; UAS - CD8 - GFP were labeled with Centrosomin ( CNN ) , Asl GFP imaged under spinning disc super - resolution microscopy .

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C) Larval brains at 0 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with γ-tubulin (γ-tub), Asl, and GFP and imaged under super-resolution microscopy. D) Larval brains at 0 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with Centrosomin (CNN), Asl and GFP and imaged under spinning disc super-resolution microscopy.


E ) Larval brains at 0 h ALH from Jupiter - GFP ( G147 ) were labeled with GFP , Msps , Dpn . F ) Larval brains at 24 h ALH from Jupiter - GFP were raised on normal food ( fed ) food depleted of ( nutritional restriction / NR ) labelled with GFP Dpn .

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E) Larval brains at 0 h ALH from Jupiter-GFP (G147) were labeled with GFP, Msps, and Dpn. F) Larval brains at 24 h ALH from Jupiter-GFP were raised on normal food (fed) and food depleted of amino acids (nutritional restriction/NR) and labelled with GFP and Dpn.


A ) Larval brains at 16 h ALH , in which kin - lacZ was expressed under control of insc - Gal4 , tub - Gal80ts , were labelled with β - Gal , Msps , Dpn . protrusion of qNSCs was marked by Msps . Quiescent NSCs at ventral nerve cord ( VNC ) are shown . Arrows indicate localization of Kin - lacZ in qNSCs . B ) Larval brains at 6 h ALH from insc - Gal4 ; UAS - nod - lacZ were labeled with β - Gal , Msps , Dpn . Quiescent NSCs at both CB VNC are shown . Arrows indicate localization of Nod - lacZ in qNSCs

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A) Larval brains at 16 h ALH, in which kin-lacZ was expressed under the control of insc-Gal4, tub-Gal80ts, were labelled with β-Gal, Msps, and Dpn. The primary protrusion of qNSCs was marked by Msps. Quiescent NSCs at the ventral nerve cord (VNC) are shown. Arrows indicate the localization of Kin-lacZ in qNSCs. B) Larval brains at 6 h ALH from insc-Gal4; UAS-nod-lacZ were labeled with β-Gal, Msps, and Dpn. Quiescent NSCs at both CB and VNC are shown. Arrows indicate the localization of Nod-lacZ in qNSCs


A ) Larval brains at 24 h ALH from control ( insc - Gal4 ; UAS - dicer2 / UAS - β - Gal RNAi ) msps RNAi ( VDRC # 21982 ) controlled under insc - Gal4 were analyzed for EdU incorporation . NSCs were marked by Dpn Mira . B ) graph of EdU - negative NSCs per brain lobe for genotypes in ( A ) . n = 13 BL for both control msps RNAi . * * * * p < 0 . 0001

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A) Larval brains at 24 h ALH from control (insc-Gal4; UAS-dicer2/UAS-β-Gal RNAi) and msps RNAi (VDRC#21982) controlled under insc-Gal4 were analyzed for EdU incorporation. NSCs were marked by Dpn and Mira. B) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (A). n=13 BL for both control and msps RNAi. ****p< 0.0001


C ) Larval brains at 24 h ALH from wild - type various msps loss - of - function alleles msps810 with a genomic rescue construct ( g - msps ) were analyzed for EdU incorporation . NSCs were marked by Dpn Mira . D ) graph of EdU - negative NSCs per brain lobe for genotypes in ( C ) . n = 13 BL for control ; n = 13 BL for msps810 ; n = 17 BL for msps924 ; n = 14 BL for mspsP18 ; n = 6 BL for mspsP ; n = 12 BL for g - msps ; msps924 . * * * * p < 0 . 0001 ; p = 0 . 7502 ( ns ) . E ) graph of diameter of in NSCs at 24 h ALH from various genotypes . n = 238 NSCs for control ; n = 219 NSC for msps924 ; n = 170 NSCs for mspsP . * * * * p < 0 . 0001 . F ) graph of of qNSCs with a protrusion in wild - type , msps924 , mspsP18 larval brains . protrusion was labelled by grh > CD8 - GFP . n = 5 BL for control ; n = 6 BL for msps924 ; n = 4 BL for mspsP . * * * * p < 0 . 0001

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

C) Larval brains at 24 h ALH from wild-type and various msps loss-of-function alleles and msps810 with a genomic rescue construct (g-msps) were analyzed for EdU incorporation. NSCs were marked by Dpn and Mira. D) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (C). n=13 BL for control; n=13 BL for msps810; n=17 BL for msps924; n=14 BL for mspsP18; n=6 BL for mspsP; n=12 BL for g-msps; msps924. ****p< 0.0001; p=0.7502 (ns). E) Quantification graph of the diameter of the cell body in NSCs at 24 h ALH from various genotypes. n=238 NSCs for control; n=219 NSC for msps924; n=170 NSCs for mspsP. ****p< 0.0001. F) Quantification graph of the percentage of qNSCs with a primary protrusion in wild-type, msps924, and mspsP18 larval brains. The protrusion was labelled by grh>CD8-GFP. n=5 BL for control; n=6 BL for msps924; n=4 BL for mspsP. ****p< 0.0001


G ) Larval brains at 24 h ALH from wild - type tacc loss - of - function tacc74 were examined for EdU incorporation . NSCs were marked by Dpn Mira . H ) graph of EdU - negative NSCs per brain lobe for genotypes in ( G ) . n = 17 BL for control ; n = 15 BL for tacc74 . * * * * p < 0 . 0001 . I ) graph of qNSCs retaining protrusion per brain lobe for genotypes in ( G ) . n = 15 BL for both control tacc74 . * * * * p < 0 . 0001 . J ) graph of NSCs are positive for mitotic marker PH3 per brain lobe for genotypes in ( G ) . n = 15 BL for both control tacc74 . * * * * p < 0 . 0001

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

G) Larval brains at 24 h ALH from wild-type and tacc loss-of-function tacc74 were examined for EdU incorporation. NSCs were marked by Dpn and Mira. H) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (G). n=17 BL for control; n=15 BL for tacc74. ****p< 0.0001. I) Quantification graph of qNSCs retaining primary protrusion per brain lobe for genotypes in (G). n=15 BL for both control and tacc74. ****p< 0.0001. J) Quantification graph of NSCs are positive for mitotic marker PH3 per brain lobe for genotypes in (G). n=15 BL for both control and tacc74. ****p< 0.0001