A - F Immunostaining of HL with H2A . B ( brown , indicated by black arrows ) with a counterstain ( blue ) . Human testis ( positive control ) ( A ) , Human tonsils ( negative control ) ( B ) , Nodular sclerosis classical cHL , double with CD15 ( pink ) to identify HRS H2A . B ( brown ) ( C ) , Mixed cellularity cHL ( D ) , Lymphocyte rich cHL ( E ) , Nodular sclerosis HL ( F ) . Scale bar for panels A , B , D - F is 50 µm ; scale bar for panel C is 15 µm .

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A-F Immunostaining of primary HL with H2A.B (brown, indicated by black arrows) with a nuclear counterstain (blue). Human testis (positive control) (A), Human tonsils (negative control) (B), Nodular sclerosis classical cHL, double staining with CD15 (pink) to identify HRS cells and H2A.B (brown) (C), Mixed cellularity cHL (D), Lymphocyte rich cHL (E), Nodular sclerosis HL (F). Scale bar for panels A, B, D-F is 50 µm; scale bar for panel C is 15 µm.


A Relative mRNA expression of H2A . B in L1236 , L428 , HDLM2 HL lines Germinal centre B lymphocytes ( GC ) compared to Ramos B lymphocyte . results are presented as mean ± SD of three biological replicates .

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A Relative mRNA expression levels of H2A.B in L1236, L428, HDLM2 HL cell lines and Germinal centre B lymphocytes (GC) compared to the Ramos B lymphocyte cell line. The results are presented as the mean ± SD of three biological replicates.


D H2A . B interacts with Pol I . Western blot showing co - immunoprecipitation of RPA194 with H2A . B . Pol I subunit RPA194 was immunoprecipitated from L1236 ( left panel ) L428 ( right panel ) extracts . pull - down by RPA194 antibody ( IP ) , pull - down by empty beads or IgG isotype control , 1 % input were loaded for Western blot detection using anti - H2A . B anti - RPA194 antibodies .

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D H2A.B interacts with Pol I. Western blot showing co-immunoprecipitation of RPA194 with H2A.B. The Pol I subunit RPA194 was immunoprecipitated from L1236 (left panel) and L428 (right panel) nuclear extracts. The pull-down by RPA194 antibody (IP), pull-down by empty beads or IgG isotype control, and 1% input protein were loaded for Western blot detection using anti-H2A.B and anti-RPA194 antibodies.


E H2A . B interacts with RPA194 in live . Western blot was performed to detect RPA194 in BioID samples from L1236 ( left panel ) , L428 ( right panel ) . samples were prepared using pull - down with streptavidin beads from lysate of empty vector , BirA * - H2A . B H2A . B - BirA * transduced , which were preincubated with 50 µM biotin for 24 h . positive control was loaded as 1 % input .

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E H2A.B interacts with RPA194 in live cells. Western blot analysis was performed to detect RPA194 in the BioID samples from L1236 cells (left panel), and L428 cells (right panel). The samples were prepared using pull-down with streptavidin beads from the cell lysate of the empty vector, BirA*-H2A.B and H2A.B-BirA* transduced cells, which were preincubated with 50 µM biotin for 24 h. The positive control was loaded as 1% input.


B proliferation in presence ( shNTC ) or during depletion of H2A . B ( shH2A . B ) . results are presented as mean ± SD for five biological replicates . significance of difference was determined by Student ' s t denoted as ns ( not significant ) ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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B Cell proliferation assay in the presence (shNTC) or during depletion of H2A.B (shH2A.B). The results are presented as mean ± SD for five biological replicates. The significance of the difference was determined by Student's t test and denoted as ns (not significant); **, p < 0.01; ***, p<0.001.


E Genome browser screen shots of gene examples ( RPs RPLP1 RPL23A , HDAC5 TAF10 ) that contain H2A . B at their promoters identified by CUT & RUN peak calling whose expression decreased significantly following H2A . B knockdown in - seq . Green purple indicates L1236 L428cells , respectively .

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E Genome browser screen shots of gene examples (RPs RPLP1 and RPL23A, and HDAC5 and TAF10) that contain H2A.B at their promoters identified by the CUT&RUN peak calling and whose expression decreased significantly following H2A.B knockdown in the RNA-seq analysis. Green and purple indicates L1236 and L428cells, respectively.


B Differential exon usage of CD44 gene . C . Differential exon usage of RPL17 gene .

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B Differential exon usage of the CD44 gene. C. Differential exon usage of the RPL17 gene.


D , E Two examples show increased accessibility at promoter peak ( NXF1 ) ( D ) decreased accessibility at peak across intron - exon boundary ( KDM4A ) ( E ) , respectively .

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D, E Two examples show increased accessibility at the promoter peak (NXF1) (D) and decreased accessibility at the peak across the intron-exon boundary (KDM4A) (E), respectively.


Growth inhibitory ( GI50 ) of CY bendamustine in NCI60 panel .

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Growth inhibitory concentration (GI50) of CY and bendamustine in the NCI60 cell line panel.


Nontoxic dose of Cpd A enhances bendamustine cytotoxicity in cancer . were taken at 48 h posttreatment .

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Nontoxic dose of Cpd A enhances bendamustine cytotoxicity in cancer cells. Images were taken at 48 h posttreatment.


Cpd A augments damage caused by bendamustine . H1650 were treated with 100 μM Bend , 50 μM Cpd A , 100 μM Bend plus 50 μM Cpd A , or 10 μM CY for 12 h , lysates were blotted for γ - H2AX . Actin was used as a loading control .

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Cpd A augments DNA damage caused by bendamustine. H1650 cells were treated with 100 μM Bend, 50 μM Cpd A, 100 μM Bend plus 50 μM Cpd A, or 10 μM CY for 12 h, and cell lysates were blotted for γ-H2AX. Actin was used as a loading control.


Upper panel , in vitro HDAC1 inhibition using bendamustine CY . Lower panel , summary of CY IC50 against other HDACs .

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Upper panel, in vitro HDAC1 inhibition assay using bendamustine and CY. Lower panel, summary of CY IC50 against other HDACs.


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CY CpdA , but not bendamustine or CpdB , inhibited HDAC activities in cancer . H1650 were treated with 20 μM CY , 20 μM CpdA , 200 μM CpdB , 200 μM Bend , or 10 μM SAHA for 12 h , lysates were blotted for histone acetylation markers . Actin was used as a loading control .

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CY and CpdA, but not bendamustine or CpdB, inhibited HDAC activities in cancer cells. H1650 cells were treated with 20 μM CY, 20 μM CpdA, 200 μM CpdB, 200 μM Bend, or 10 μM SAHA for 12 h, and cell lysates were blotted for histone acetylation markers. Actin was used as a loading control.


Alteration of CY side chain ( Cpd B ) caused loss of HDAC inhibitory activity resulted in reduced ability to induce damage . were treated for 12 h . treated with 200 μM Cpd B 20 μM CY exhibited similar of γ - H2AX . Actin was used as a loading control .

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Alteration of CY side chain (Cpd B) caused loss of HDAC inhibitory activity and resulted in reduced ability to induce DNA damage. Cells were treated for 12 h. Cells treated with 200 μM Cpd B and 20 μM CY exhibited similar levels of γ-H2AX. Actin was used as a loading control.


Summary of lethal dose 50 ( LD50 ) of bendamustine , CY , Cpd A ( HDAC inhibition only ) , Cpd B ( nitrogen mustard only ) against 11 human cancer lines .

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Summary of lethal dose 50 (LD50) of bendamustine, CY, Cpd A (HDAC inhibition only), and Cpd B (nitrogen mustard only) against 11 human cancer cell lines.


Cpd A enhances anticancer activity of bendamustine . H1650 were treated with various doses of bendamustine with or without 40 μM Cpd A ( nontoxic dose , see Fig1C Supplementary Fig S2 ) for 48 h subjected to MTT viability assays .

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Cpd A enhances the anticancer activity of bendamustine. H1650 cells were treated with various doses of bendamustine with or without 40 μM Cpd A (nontoxic dose, see Fig1C and Supplementary Fig S2) for 48 h and subjected to MTT viability assays.


CY , but not bendamustine treatment , led to suppression of CBP , TIP60 , MORF , MSL1 in H1650 . were treated with DMSO , bendamustine ( 350 μM ) , CY ( 15 μM ) , or SAHA ( 10 μM ) for 6 h , mRNA was extracted for qPCR . Data represent mean ± from three independent experiments , statistical significance was determined by unpaired two - t - . * * * P < 0 . 01 .

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CY, but not bendamustine treatment, led to suppression of CBP, TIP60, MORF, and MSL1 in H1650 cells. Cells were treated with DMSO, bendamustine (350 μM), CY (15 μM), or SAHA (10 μM) for 6 h, and mRNA was extracted for qPCR analysis. Data represent mean ± SEM from three independent experiments, and statistical significance was determined by unpaired two-tailed t-test. ***P < 0.01.


Drug treatment up to 10 h at concentrations indicated in ( A ) did not cause apoptosis as judged by PARP cleavage . Actin was used as a loading control .

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Drug treatment up to 10 h at the concentrations indicated in (A) did not cause apoptosis as judged by PARP cleavage. Actin was used as a loading control.


ShRNA - mediated suppression of CBP , TIP60 , MORF , MSL1 at mRNA level . Data represent mean ± from three independent experiments , statistical significance was determined by unpaired two - t - . * P < 0 . 1 , * * P < 0 . 05 , * * * P < 0 . 01 .

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ShRNA-mediated suppression of CBP, TIP60, MORF, and MSL1 at mRNA level. Data represent mean ± SEM from three independent experiments, and statistical significance was determined by unpaired two-tailed t-test. *P < 0.1, **P < 0.05, ***P < 0.01.


Suppression of CBP , TIP60 , MORF , MSL1 sensitized to bendamustine . Y - axis represents relative resistance calculated from results of GFP competition assays , statistical significance was determined by unpaired two - t - . Data represent mean ± from two independent experiments . * * P < 0 . 05 , * * * P < 0 . 01 .

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Suppression of CBP, TIP60, MORF, and MSL1 sensitized cells to bendamustine. Y-axis represents relative resistance calculated from results of GFP competition assays, and statistical significance was determined by unpaired two-tailed t-test. Data represent mean ± SEM from two independent experiments. **P < 0.05, ***P < 0.01.


A Determination of maximally tolerated dose ( MTD ) of CY at 60 mg / kg . Other drugs were used at MTD according to literature . For each drug , n = 5 mice survived treatment . Data represent mean ± .

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A Determination of maximally tolerated dose (MTD) of CY at 60 mg/kg. Other drugs were used at MTD according to the literature. For each drug, n = 5 and all mice survived treatment. Data represent mean ± SEM.


B CY exhibited enhanced activity against BCR - ABL Arf − / − murine in vitro .

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B CY exhibited enhanced activity against BCR-ABL Arf−/− murine ALL cells in vitro.


C , D CY showed superior antitumor activity compared with bendamustine , SAHA , other chemotherapeutic drugs in mice transplanted with BCR - ABL . P - values of CY versus other drugs : NT ( P = 5 . 02E - 06 ) ( C ) , SAHA ( P = 4 . 45E - 06 ) , Bend ( P = 5 . 18E - 06 ) , NT ( P = 1 . 20E - 05 ) ( D ) , VCR ( P = 1 . 13E - 05 ) , Dox ( P = 8 . 08E - 06 ) , AraC ( P = 7 . 62E - 06 ) , CTX ( P = 1 . 13E - 05 ) . Survival statistical was done with Mantel - Cox ( log - rank ) of GraphPad Prism .

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C, D CY showed superior antitumor activity compared with bendamustine, SAHA, and other chemotherapeutic drugs in mice transplanted with BCR-ABL ALL cells. P-values of CY versus other drugs: NT (P = 5.02E-06) (C), SAHA (P = 4.45E-06), Bend (P = 5.18E-06), NT (P = 1.20E-05) (D), VCR (P = 1.13E-05), Dox (P = 8.08E-06), AraC (P = 7.62E-06), and CTX (P = 1.13E-05). Survival statistical analysis was done with the Mantel-Cox (log-rank) test of GraphPad Prism.


E Therapeutic effects of CY ( weekly dose ) targeted drugs PP242 ( daily dose ) dasatinib ( daily dose ) in mice transplanted with BCR - ABL . P - values of CY versus other drugs : NT ( P = 3 . 94E - 05 ) , PP242 ( P = 1 . 20E - 05 ) , dasatinib ( P = 0 . 99 ) . Survival statistical was done with Mantel - Cox ( log - rank ) of GraphPad Prism .

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E Therapeutic effects of CY (weekly dose) and targeted drugs PP242 (daily dose) and dasatinib (daily dose) in mice transplanted with BCR-ABL ALL cells. P-values of CY versus other drugs: NT (P = 3.94E-05), PP242 (P = 1.20E-05), and dasatinib (P = 0.99). Survival statistical analysis was done with the Mantel-Cox (log-rank) test of GraphPad Prism.


F , G Antitumor activity of CY bendamustine in nude mice models . Mice were inoculated with human NSCLC H460 . When volume reached 200 mm3 , mice were treated with single dose of bendamustine ( 40 mg / kg , MTD ) or CY ( 20 , 40 , 60 mg / kg ) . In ( G ) , tumors were dissected out after 15 days posttreatment .

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F, G Antitumor activity of CY and bendamustine in nude mice models. Mice were inoculated with human NSCLC cell line H460. When tumor volume reached 200 mm3, mice were treated with single dose of bendamustine (40 mg/kg, MTD) or CY (20, 40, 60 mg/kg). In (G), tumors were dissected out after 15 days posttreatment.


Dose curve of CY , fludarabine , bendamustine using fresh CLL samples . Data represent mean ± SD .

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Dose curve of CY, fludarabine, and bendamustine using fresh CLL samples. Data represent mean ± SD.


CY preferentially killed CLL over healthy B . Data represent mean ±

CY preferentially killed CLL cells over healthy B cells. Data represent mean ± SD


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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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 using CRISPR / Cas9 technology . Monoclones ( # 3 # 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 of expression of WT , cTAZ - / - , cTAZ - / - / GFP - cTAZ put - back RKO . Genes down - up - regulated more than 2 folds ( P < 0 . 05 ) in cTAZ KO were included in cluster . Green red indicate down - up - regulated genes respectively .

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

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

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

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

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

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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 interaction between STAT1 STAT2 . RKO lines ( WT # 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 importin α5 ( Im - α5 ) . Following transfection , HEK293A 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 accumulation of STAT1 upon IFN - α stimulation . RKO ( WT cTAZ - / - ) treated with or without IFN - α ( 50 ng / ml ) for 1 hour , fixed , subjected to IF using STAT1 antibody ( green ) . was labeled by DAPI ( blue ) . Scale bar , 50 μm . was performed using ImageJ software ( right ) . N > C : ; N = C : plus ; N < C : localization .

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

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

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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.


cTAZ and STAT1/2 expression levels of cells


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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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

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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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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 .

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 B-MEASURED_VAR O B-MEASURED_VAR O O

(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.