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A, CNS-1 cells were infected with Ads expressing pro-apoptotic transgenes, i.e. HSV1-thymidine kinase (Ad-TK), TNF-α (Ad-TNF-α), FasL (Ad-FasL) or TRAIL (Ad-TRAIL). 24h after infection, cells infected with Ad-TK were incubated with GCV. Untreated cells and cells infected with an Ad containing no transgene (Ad0) were used as controls. Cell death was determined 72h after infection or addition of GCV by flow cytometric analysis of Annexin V-PI-stained cells. B, Release of HMGB1 was assessed in the cell culture supernatant by ELISA. *p<0.05 vs mock (One-way ANOVA followed by Tukey's test). Inset: <t>Pearson</t> correlation analysis was used to determine the correlation coefficient (R2) between the concentration of HMGB1 in the cell supernatant and the percentage of cell death in vitro in CNS-1 cells infected with the pro-apoptotic Ads. *p<0.05 C, Kaplan Meier survival curves of rats implanted with CNS-1 cells in the brain and treated 4 days later with intratumoral injection of saline (n=9), Ad-TK (n=7), Ad-TNF-α (n=5), Ad-FasL (n=9) or Ad-TRAIL (n=8). Ad-TK-treated rats received GCV. *p<0.05 vs saline, ^p<0.05 vs Ad-FasL (Mantel log-rank test). Representative microphotographs show the appearance of the tumor at the time of treatment (day 4), as assessed by vimentin staining. Tumor volume is indicated; scale bar: 1 mm. D, Serum levels of HMGB1 were determined by ELISA 5 days after the treatment. *p<0.05 vs saline (One-way ANOVA followed by Tukey's test).
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A, CNS-1 cells were infected with Ads expressing pro-apoptotic transgenes, i.e. HSV1-thymidine kinase (Ad-TK), TNF-α (Ad-TNF-α), FasL (Ad-FasL) or TRAIL (Ad-TRAIL). 24h after infection, cells infected with Ad-TK were incubated with GCV. Untreated cells and cells infected with an Ad containing no transgene (Ad0) were used as controls. Cell death was determined 72h after infection or addition of GCV by flow cytometric analysis of Annexin V-PI-stained cells. B, Release of HMGB1 was assessed in the cell culture supernatant by ELISA. *p<0.05 vs mock (One-way ANOVA followed by Tukey's test). Inset: <t>Pearson</t> correlation analysis was used to determine the correlation coefficient (R2) between the concentration of HMGB1 in the cell supernatant and the percentage of cell death in vitro in CNS-1 cells infected with the pro-apoptotic Ads. *p<0.05 C, Kaplan Meier survival curves of rats implanted with CNS-1 cells in the brain and treated 4 days later with intratumoral injection of saline (n=9), Ad-TK (n=7), Ad-TNF-α (n=5), Ad-FasL (n=9) or Ad-TRAIL (n=8). Ad-TK-treated rats received GCV. *p<0.05 vs saline, ^p<0.05 vs Ad-FasL (Mantel log-rank test). Representative microphotographs show the appearance of the tumor at the time of treatment (day 4), as assessed by vimentin staining. Tumor volume is indicated; scale bar: 1 mm. D, Serum levels of HMGB1 were determined by ELISA 5 days after the treatment. *p<0.05 vs saline (One-way ANOVA followed by Tukey's test).
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A, CNS-1 cells were infected with Ads expressing pro-apoptotic transgenes, i.e. HSV1-thymidine kinase (Ad-TK), TNF-α (Ad-TNF-α), FasL (Ad-FasL) or TRAIL (Ad-TRAIL). 24h after infection, cells infected with Ad-TK were incubated with GCV. Untreated cells and cells infected with an Ad containing no transgene (Ad0) were used as controls. Cell death was determined 72h after infection or addition of GCV by flow cytometric analysis of Annexin V-PI-stained cells. B, Release of HMGB1 was assessed in the cell culture supernatant by ELISA. *p<0.05 vs mock (One-way ANOVA followed by Tukey's test). Inset: <t>Pearson</t> correlation analysis was used to determine the correlation coefficient (R2) between the concentration of HMGB1 in the cell supernatant and the percentage of cell death in vitro in CNS-1 cells infected with the pro-apoptotic Ads. *p<0.05 C, Kaplan Meier survival curves of rats implanted with CNS-1 cells in the brain and treated 4 days later with intratumoral injection of saline (n=9), Ad-TK (n=7), Ad-TNF-α (n=5), Ad-FasL (n=9) or Ad-TRAIL (n=8). Ad-TK-treated rats received GCV. *p<0.05 vs saline, ^p<0.05 vs Ad-FasL (Mantel log-rank test). Representative microphotographs show the appearance of the tumor at the time of treatment (day 4), as assessed by vimentin staining. Tumor volume is indicated; scale bar: 1 mm. D, Serum levels of HMGB1 were determined by ELISA 5 days after the treatment. *p<0.05 vs saline (One-way ANOVA followed by Tukey's test).
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A, CNS-1 cells were infected with Ads expressing pro-apoptotic transgenes, i.e. HSV1-thymidine kinase (Ad-TK), TNF-α (Ad-TNF-α), FasL (Ad-FasL) or TRAIL (Ad-TRAIL). 24h after infection, cells infected with Ad-TK were incubated with GCV. Untreated cells and cells infected with an Ad containing no transgene (Ad0) were used as controls. Cell death was determined 72h after infection or addition of GCV by flow cytometric analysis of Annexin V-PI-stained cells. B, Release of HMGB1 was assessed in the cell culture supernatant by ELISA. *p<0.05 vs mock (One-way ANOVA followed by Tukey's test). Inset: <t>Pearson</t> correlation analysis was used to determine the correlation coefficient (R2) between the concentration of HMGB1 in the cell supernatant and the percentage of cell death in vitro in CNS-1 cells infected with the pro-apoptotic Ads. *p<0.05 C, Kaplan Meier survival curves of rats implanted with CNS-1 cells in the brain and treated 4 days later with intratumoral injection of saline (n=9), Ad-TK (n=7), Ad-TNF-α (n=5), Ad-FasL (n=9) or Ad-TRAIL (n=8). Ad-TK-treated rats received GCV. *p<0.05 vs saline, ^p<0.05 vs Ad-FasL (Mantel log-rank test). Representative microphotographs show the appearance of the tumor at the time of treatment (day 4), as assessed by vimentin staining. Tumor volume is indicated; scale bar: 1 mm. D, Serum levels of HMGB1 were determined by ELISA 5 days after the treatment. *p<0.05 vs saline (One-way ANOVA followed by Tukey's test).
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A, CNS-1 cells were infected with Ads expressing pro-apoptotic transgenes, i.e. HSV1-thymidine kinase (Ad-TK), TNF-α (Ad-TNF-α), FasL (Ad-FasL) or TRAIL (Ad-TRAIL). 24h after infection, cells infected with Ad-TK were incubated with GCV. Untreated cells and cells infected with an Ad containing no transgene (Ad0) were used as controls. Cell death was determined 72h after infection or addition of GCV by flow cytometric analysis of Annexin V-PI-stained cells. B, Release of HMGB1 was assessed in the cell culture supernatant by ELISA. *p<0.05 vs mock (One-way ANOVA followed by Tukey's test). Inset: <t>Pearson</t> correlation analysis was used to determine the correlation coefficient (R2) between the concentration of HMGB1 in the cell supernatant and the percentage of cell death in vitro in CNS-1 cells infected with the pro-apoptotic Ads. *p<0.05 C, Kaplan Meier survival curves of rats implanted with CNS-1 cells in the brain and treated 4 days later with intratumoral injection of saline (n=9), Ad-TK (n=7), Ad-TNF-α (n=5), Ad-FasL (n=9) or Ad-TRAIL (n=8). Ad-TK-treated rats received GCV. *p<0.05 vs saline, ^p<0.05 vs Ad-FasL (Mantel log-rank test). Representative microphotographs show the appearance of the tumor at the time of treatment (day 4), as assessed by vimentin staining. Tumor volume is indicated; scale bar: 1 mm. D, Serum levels of HMGB1 were determined by ELISA 5 days after the treatment. *p<0.05 vs saline (One-way ANOVA followed by Tukey's test).
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A, CNS-1 cells were infected with Ads expressing pro-apoptotic transgenes, i.e. HSV1-thymidine kinase (Ad-TK), TNF-α (Ad-TNF-α), FasL (Ad-FasL) or TRAIL (Ad-TRAIL). 24h after infection, cells infected with Ad-TK were incubated with GCV. Untreated cells and cells infected with an Ad containing no transgene (Ad0) were used as controls. Cell death was determined 72h after infection or addition of GCV by flow cytometric analysis of Annexin V-PI-stained cells. B, Release of HMGB1 was assessed in the cell culture supernatant by ELISA. *p<0.05 vs mock (One-way ANOVA followed by Tukey's test). Inset: <t>Pearson</t> correlation analysis was used to determine the correlation coefficient (R2) between the concentration of HMGB1 in the cell supernatant and the percentage of cell death in vitro in CNS-1 cells infected with the pro-apoptotic Ads. *p<0.05 C, Kaplan Meier survival curves of rats implanted with CNS-1 cells in the brain and treated 4 days later with intratumoral injection of saline (n=9), Ad-TK (n=7), Ad-TNF-α (n=5), Ad-FasL (n=9) or Ad-TRAIL (n=8). Ad-TK-treated rats received GCV. *p<0.05 vs saline, ^p<0.05 vs Ad-FasL (Mantel log-rank test). Representative microphotographs show the appearance of the tumor at the time of treatment (day 4), as assessed by vimentin staining. Tumor volume is indicated; scale bar: 1 mm. D, Serum levels of HMGB1 were determined by ELISA 5 days after the treatment. *p<0.05 vs saline (One-way ANOVA followed by Tukey's test).
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A, CNS-1 cells were infected with Ads expressing pro-apoptotic transgenes, i.e. HSV1-thymidine kinase (Ad-TK), TNF-α (Ad-TNF-α), FasL (Ad-FasL) or TRAIL (Ad-TRAIL). 24h after infection, cells infected with Ad-TK were incubated with GCV. Untreated cells and cells infected with an Ad containing no transgene (Ad0) were used as controls. Cell death was determined 72h after infection or addition of GCV by flow cytometric analysis of Annexin V-PI-stained cells. B, Release of HMGB1 was assessed in the cell culture supernatant by ELISA. *p<0.05 vs mock (One-way ANOVA followed by Tukey's test). Inset: Pearson correlation analysis was used to determine the correlation coefficient (R2) between the concentration of HMGB1 in the cell supernatant and the percentage of cell death in vitro in CNS-1 cells infected with the pro-apoptotic Ads. *p<0.05 C, Kaplan Meier survival curves of rats implanted with CNS-1 cells in the brain and treated 4 days later with intratumoral injection of saline (n=9), Ad-TK (n=7), Ad-TNF-α (n=5), Ad-FasL (n=9) or Ad-TRAIL (n=8). Ad-TK-treated rats received GCV. *p<0.05 vs saline, ^p<0.05 vs Ad-FasL (Mantel log-rank test). Representative microphotographs show the appearance of the tumor at the time of treatment (day 4), as assessed by vimentin staining. Tumor volume is indicated; scale bar: 1 mm. D, Serum levels of HMGB1 were determined by ELISA 5 days after the treatment. *p<0.05 vs saline (One-way ANOVA followed by Tukey's test).

Journal:

Article Title: Release of HMGB1 in response to pro-apoptotic glioma killing strategies: efficacy and neurotoxicity

doi: 10.1158/1078-0432.CCR-09-0155

Figure Lengend Snippet: A, CNS-1 cells were infected with Ads expressing pro-apoptotic transgenes, i.e. HSV1-thymidine kinase (Ad-TK), TNF-α (Ad-TNF-α), FasL (Ad-FasL) or TRAIL (Ad-TRAIL). 24h after infection, cells infected with Ad-TK were incubated with GCV. Untreated cells and cells infected with an Ad containing no transgene (Ad0) were used as controls. Cell death was determined 72h after infection or addition of GCV by flow cytometric analysis of Annexin V-PI-stained cells. B, Release of HMGB1 was assessed in the cell culture supernatant by ELISA. *p<0.05 vs mock (One-way ANOVA followed by Tukey's test). Inset: Pearson correlation analysis was used to determine the correlation coefficient (R2) between the concentration of HMGB1 in the cell supernatant and the percentage of cell death in vitro in CNS-1 cells infected with the pro-apoptotic Ads. *p<0.05 C, Kaplan Meier survival curves of rats implanted with CNS-1 cells in the brain and treated 4 days later with intratumoral injection of saline (n=9), Ad-TK (n=7), Ad-TNF-α (n=5), Ad-FasL (n=9) or Ad-TRAIL (n=8). Ad-TK-treated rats received GCV. *p<0.05 vs saline, ^p<0.05 vs Ad-FasL (Mantel log-rank test). Representative microphotographs show the appearance of the tumor at the time of treatment (day 4), as assessed by vimentin staining. Tumor volume is indicated; scale bar: 1 mm. D, Serum levels of HMGB1 were determined by ELISA 5 days after the treatment. *p<0.05 vs saline (One-way ANOVA followed by Tukey's test).

Article Snippet: Pearson test was used to determine correlation coefficient (R 2 ) between HMGB1 release and percentage of cell death (GraphPad Prism).

Techniques: Infection, Expressing, Incubation, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, In Vitro, Injection, Saline