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    Biosynth Carbosynth generating hmgb1
    Figure 7. <t>HMGB1</t> Is Secreted from Dying Tumor Cells and Is Required for Ad-Flt3L and Ad-TK Mediated Brain Tumor Regression (A) GL26 cells were infected with Ad-TK and incubated with 25 lM GCV. A western blot was performed using an antibody specific for HMGB1. The graph displays a quantification of the total amount of HMGB1 in the media of cells 24 h (yellow bars), 48 h (orange bars), or 72 h (black bars) after GCV treatment. (B) GL26 cells were incubated with Ad-TK (with and without GCV and glycyrrhizin), and media was overlaid on HEK293 reporter cells transfected with either a plasmid encoding TLR2 (þpTLR2; black) or with a control plasmid (-pTLR2; red). NFjB activity was determined by quantifying the activity of Firefly Luciferase (under the control of the NFjB promoter), *, p , 0.05 (Kruskal-Wallis followed by Dunn’s test). Inset: cells were incubated with 100 ng/ ml PAM3 (PAM3CSK4) as a positive control. (C) The levels of HMGB1 in mouse serum were quantified by ELISA 7 d after treatment of brain tumors with Ad-Flt3L and Ad-TK. *, p , 0.05 versus saline (Mann-Whitney U-test). (D) GL26 cells were implanted into C57BL/6 mice (n ¼ 5 mice/treatment group) and 17 d later were treated with saline, or with AdFlt3L and Ad-TK (F/T). Glycyrrhizin (Glyc), HMGB1-depleting (aHMGB1), or rabbit IgG isotype control antibodies were administered IP 2 d, 5 d, and 10 d after treatment, *, p , 0.05 Ad-Flt3L and Ad-TK versus Ad-Flt3L and Ad-TK þ glycyrrhizin; ^p , 0.05 Ad-Flt3L and Ad-TK þ isotype versus Ad-Flt3L and Ad-TK þ aHMGB1; Mantel log-rank test. doi:10.1371/journal.pmed.1000010.g007
    Generating Hmgb1, supplied by Biosynth Carbosynth, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/generating+hmgb1/HMGB1+antibody/pm19143470-220-0-8
    Average 90 stars, based on 2 article reviews
    generating hmgb1 - by Bioz Stars, 2026-09
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    1) Product Images from "HMGB1 mediates endogenous TLR2 activation and brain tumor regression."

    Article Title: HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

    Journal: PLoS medicine

    doi: 10.1371/journal.pmed.1000010

    Figure 7. HMGB1 Is Secreted from Dying Tumor Cells and Is Required for Ad-Flt3L and Ad-TK Mediated Brain Tumor Regression (A) GL26 cells were infected with Ad-TK and incubated with 25 lM GCV. A western blot was performed using an antibody specific for HMGB1. The graph displays a quantification of the total amount of HMGB1 in the media of cells 24 h (yellow bars), 48 h (orange bars), or 72 h (black bars) after GCV treatment. (B) GL26 cells were incubated with Ad-TK (with and without GCV and glycyrrhizin), and media was overlaid on HEK293 reporter cells transfected with either a plasmid encoding TLR2 (þpTLR2; black) or with a control plasmid (-pTLR2; red). NFjB activity was determined by quantifying the activity of Firefly Luciferase (under the control of the NFjB promoter), *, p , 0.05 (Kruskal-Wallis followed by Dunn’s test). Inset: cells were incubated with 100 ng/ ml PAM3 (PAM3CSK4) as a positive control. (C) The levels of HMGB1 in mouse serum were quantified by ELISA 7 d after treatment of brain tumors with Ad-Flt3L and Ad-TK. *, p , 0.05 versus saline (Mann-Whitney U-test). (D) GL26 cells were implanted into C57BL/6 mice (n ¼ 5 mice/treatment group) and 17 d later were treated with saline, or with AdFlt3L and Ad-TK (F/T). Glycyrrhizin (Glyc), HMGB1-depleting (aHMGB1), or rabbit IgG isotype control antibodies were administered IP 2 d, 5 d, and 10 d after treatment, *, p , 0.05 Ad-Flt3L and Ad-TK versus Ad-Flt3L and Ad-TK þ glycyrrhizin; ^p , 0.05 Ad-Flt3L and Ad-TK þ isotype versus Ad-Flt3L and Ad-TK þ aHMGB1; Mantel log-rank test. doi:10.1371/journal.pmed.1000010.g007
    Figure Legend Snippet: Figure 7. HMGB1 Is Secreted from Dying Tumor Cells and Is Required for Ad-Flt3L and Ad-TK Mediated Brain Tumor Regression (A) GL26 cells were infected with Ad-TK and incubated with 25 lM GCV. A western blot was performed using an antibody specific for HMGB1. The graph displays a quantification of the total amount of HMGB1 in the media of cells 24 h (yellow bars), 48 h (orange bars), or 72 h (black bars) after GCV treatment. (B) GL26 cells were incubated with Ad-TK (with and without GCV and glycyrrhizin), and media was overlaid on HEK293 reporter cells transfected with either a plasmid encoding TLR2 (þpTLR2; black) or with a control plasmid (-pTLR2; red). NFjB activity was determined by quantifying the activity of Firefly Luciferase (under the control of the NFjB promoter), *, p , 0.05 (Kruskal-Wallis followed by Dunn’s test). Inset: cells were incubated with 100 ng/ ml PAM3 (PAM3CSK4) as a positive control. (C) The levels of HMGB1 in mouse serum were quantified by ELISA 7 d after treatment of brain tumors with Ad-Flt3L and Ad-TK. *, p , 0.05 versus saline (Mann-Whitney U-test). (D) GL26 cells were implanted into C57BL/6 mice (n ¼ 5 mice/treatment group) and 17 d later were treated with saline, or with AdFlt3L and Ad-TK (F/T). Glycyrrhizin (Glyc), HMGB1-depleting (aHMGB1), or rabbit IgG isotype control antibodies were administered IP 2 d, 5 d, and 10 d after treatment, *, p , 0.05 Ad-Flt3L and Ad-TK versus Ad-Flt3L and Ad-TK þ glycyrrhizin; ^p , 0.05 Ad-Flt3L and Ad-TK þ isotype versus Ad-Flt3L and Ad-TK þ aHMGB1; Mantel log-rank test. doi:10.1371/journal.pmed.1000010.g007

    Techniques Used: Infection, Incubation, Western Blot, Transfection, Plasmid Preparation, Control, Activity Assay, Luciferase, Positive Control, Enzyme-linked Immunosorbent Assay, Saline, MANN-WHITNEY

    Figure 8. Western Blot Analysis Reveals HMGB1 Is Released from GL26, LLc1, GL261, and B16-F10 Cell Lines Following Treatment with Ad-TK (þGCV), Irradiation, or Temozolomide (TMZ) (A) LLc1, GL261, or B16-F10 cells were infected with Ad-TK or Ad-0 at an MOI¼500. Noninfected cells were used as control. Ad-TK treated cells were also incubated with 25 lM GCV. 48 h later, supernatant was collected and HMGB1 release was assessed by western blotting. (B) GL26, LLc1, GL261, or B16-F10 cells were irradiated with 20 Gy for 30 min. Nonirradiated cells were used as control. 72 h later, supernatant was collected and HMGB1 release was assessed by western blotting. (C) GL26, LLc1, GL261, or B16-F10 cells were incubated with temozolomide (TMZ) (200 lM). Control cells were incubated without TMZ (mock). 48 h later, supernatant was collected and HMGB1 release was assessed by western blotting. doi:10.1371/journal.pmed.1000010.g008
    Figure Legend Snippet: Figure 8. Western Blot Analysis Reveals HMGB1 Is Released from GL26, LLc1, GL261, and B16-F10 Cell Lines Following Treatment with Ad-TK (þGCV), Irradiation, or Temozolomide (TMZ) (A) LLc1, GL261, or B16-F10 cells were infected with Ad-TK or Ad-0 at an MOI¼500. Noninfected cells were used as control. Ad-TK treated cells were also incubated with 25 lM GCV. 48 h later, supernatant was collected and HMGB1 release was assessed by western blotting. (B) GL26, LLc1, GL261, or B16-F10 cells were irradiated with 20 Gy for 30 min. Nonirradiated cells were used as control. 72 h later, supernatant was collected and HMGB1 release was assessed by western blotting. (C) GL26, LLc1, GL261, or B16-F10 cells were incubated with temozolomide (TMZ) (200 lM). Control cells were incubated without TMZ (mock). 48 h later, supernatant was collected and HMGB1 release was assessed by western blotting. doi:10.1371/journal.pmed.1000010.g008

    Techniques Used: Western Blot, Irradiation, Infection, Control, Incubation

    Figure 9. HMGB1 Is Released into the Supernatant of GL26, LLc1, GL261, and B16-F10 Tumor Cell Lines in Response to Treatment with Ad-TK (þGCV), Irradiation, or Temozolomide (A–D) GL26, LLc1, GL261, or B16-F10 cells were infected with Ad-TK or Ad-0. Noninfected cells were used as control (mock). Ad-TK treated cells were incubated with 25 lM GCV. 48 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with Ad-TK (þGCV) significantly increased HMGB1 release when compared to corresponding mock and Ad0 treatment groups from each cell line, *, p , 0.05 versus mock and Ad-0 (Kruskal-Wallis test followed by Dunn’s test). (E–H) GL26, LLc1, GL261, or B16-F10 cells were irradiated with 20 Gy for 30 min. Nonirradiated cells were used as controls (mock). 72 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with irradiation significantly increased HMGB1 release when compared to corresponding control groups from each cell line, *, p , 0.05 versus mock (Mann-Whitney U-test). (I–L) GL26, LLc1, GL261, or B16-F10 cells (2.53105 cells per flask) were incubated with or without temozolomide (TMZ) (200 lM). 48 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with TMZ significantly increased HMGB1 release when compared to control groups from each cell type. *, p , 0.05 versus mock (Mann-Whitney U-test). doi:10.1371/journal.pmed.1000010.g009
    Figure Legend Snippet: Figure 9. HMGB1 Is Released into the Supernatant of GL26, LLc1, GL261, and B16-F10 Tumor Cell Lines in Response to Treatment with Ad-TK (þGCV), Irradiation, or Temozolomide (A–D) GL26, LLc1, GL261, or B16-F10 cells were infected with Ad-TK or Ad-0. Noninfected cells were used as control (mock). Ad-TK treated cells were incubated with 25 lM GCV. 48 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with Ad-TK (þGCV) significantly increased HMGB1 release when compared to corresponding mock and Ad0 treatment groups from each cell line, *, p , 0.05 versus mock and Ad-0 (Kruskal-Wallis test followed by Dunn’s test). (E–H) GL26, LLc1, GL261, or B16-F10 cells were irradiated with 20 Gy for 30 min. Nonirradiated cells were used as controls (mock). 72 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with irradiation significantly increased HMGB1 release when compared to corresponding control groups from each cell line, *, p , 0.05 versus mock (Mann-Whitney U-test). (I–L) GL26, LLc1, GL261, or B16-F10 cells (2.53105 cells per flask) were incubated with or without temozolomide (TMZ) (200 lM). 48 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with TMZ significantly increased HMGB1 release when compared to control groups from each cell type. *, p , 0.05 versus mock (Mann-Whitney U-test). doi:10.1371/journal.pmed.1000010.g009

    Techniques Used: Irradiation, Infection, Control, Incubation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Figure 10. Treatment of Animals Bearing Intracranial Glioma (GL261) or Melanoma (B16-F10) with Ad-Flt3L and Ad-TK (þGCV) Results in Long-Term Survival and Increased Serum Levels of HMGB1 (A) GL261 cells were implanted into the striatum of C57/Bl6 mice. 17 d later animals were treated with Ad-Flt3L (n ¼ 5), Ad-TK (þGCV) (n ¼ 6), Ad-Flt3L plus Ad-TK (þGCV) (n ¼ 5), or saline (n ¼ 5). Treatment with Ad-Flt3L and Ad-TK significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test), Ad-Flt3L, or Ad-TK alone treatment groups (^, p , 0.05; Mantel log-rank test). Treatment with Ad-Flt3L, or Ad-TK alone significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test). (B) 7 d after treatment, tumor-bearing animals were humanely killed and HMGB1 release was assessed in the serum. Treatment of GL261 tumor-bearing animals with Ad-Flt3L and Ad-TK (þGCV) significantly increased the levels of HMGB1 in the serum when compared to tumor-bearing animals treated with saline. *, p , 0.05 versus saline (Mann-Whitney U-test). (C) B16-F10 cells were implanted into the striatum of C57/Bl6 mice. 17 d later animals were treated with Ad-Flt3L, Ad-TK (þGCV), Ad-Flt3L plus Ad-TK (þGCV), or saline. Treatment with Ad-Flt3L and Ad-TK significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test), Ad- Flt3L, or Ad-TK alone treatment groups (^, p , 0.05; Mantel log-rank test). Treatment with Ad-Flt3L, or Ad-TK alone, significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test). (D) 7 d after treatment, tumor-bearing animals were humanely killed and HMGB1 release was assessed in the serum. Treatment of B16-F10 tumor- bearing animals with Ad-Flt3L and Ad-TK (þGCV) significantly increased the levels of HMGB1 in the serum when compared to tumor-bearing animals treated with saline. *, p , 0.05 versus saline (Mann-Whitney U-test). doi:10.1371/journal.pmed.1000010.g010
    Figure Legend Snippet: Figure 10. Treatment of Animals Bearing Intracranial Glioma (GL261) or Melanoma (B16-F10) with Ad-Flt3L and Ad-TK (þGCV) Results in Long-Term Survival and Increased Serum Levels of HMGB1 (A) GL261 cells were implanted into the striatum of C57/Bl6 mice. 17 d later animals were treated with Ad-Flt3L (n ¼ 5), Ad-TK (þGCV) (n ¼ 6), Ad-Flt3L plus Ad-TK (þGCV) (n ¼ 5), or saline (n ¼ 5). Treatment with Ad-Flt3L and Ad-TK significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test), Ad-Flt3L, or Ad-TK alone treatment groups (^, p , 0.05; Mantel log-rank test). Treatment with Ad-Flt3L, or Ad-TK alone significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test). (B) 7 d after treatment, tumor-bearing animals were humanely killed and HMGB1 release was assessed in the serum. Treatment of GL261 tumor-bearing animals with Ad-Flt3L and Ad-TK (þGCV) significantly increased the levels of HMGB1 in the serum when compared to tumor-bearing animals treated with saline. *, p , 0.05 versus saline (Mann-Whitney U-test). (C) B16-F10 cells were implanted into the striatum of C57/Bl6 mice. 17 d later animals were treated with Ad-Flt3L, Ad-TK (þGCV), Ad-Flt3L plus Ad-TK (þGCV), or saline. Treatment with Ad-Flt3L and Ad-TK significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test), Ad- Flt3L, or Ad-TK alone treatment groups (^, p , 0.05; Mantel log-rank test). Treatment with Ad-Flt3L, or Ad-TK alone, significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test). (D) 7 d after treatment, tumor-bearing animals were humanely killed and HMGB1 release was assessed in the serum. Treatment of B16-F10 tumor- bearing animals with Ad-Flt3L and Ad-TK (þGCV) significantly increased the levels of HMGB1 in the serum when compared to tumor-bearing animals treated with saline. *, p , 0.05 versus saline (Mann-Whitney U-test). doi:10.1371/journal.pmed.1000010.g010

    Techniques Used: Saline, MANN-WHITNEY

    Figure 11. Model Illustrating the Role of TLR2 and HMGB1 in Initiating T Cell-Dependent Brain Tumor Regression by Flt3L and TK Treatment of tumor cells with TK þ GCV releases endogenous TLR2 ligands including HMGB1. DC infiltration into tumors and subsequent activation require TLR2 signalling. DC phagocytose tumor antigen and migrate to the dLN where they stimulate T cell clonal expansion resulting in T cell- dependent brain tumor regression and long-term immunological memory. doi:10.1371/journal.pmed.1000010.g011
    Figure Legend Snippet: Figure 11. Model Illustrating the Role of TLR2 and HMGB1 in Initiating T Cell-Dependent Brain Tumor Regression by Flt3L and TK Treatment of tumor cells with TK þ GCV releases endogenous TLR2 ligands including HMGB1. DC infiltration into tumors and subsequent activation require TLR2 signalling. DC phagocytose tumor antigen and migrate to the dLN where they stimulate T cell clonal expansion resulting in T cell- dependent brain tumor regression and long-term immunological memory. doi:10.1371/journal.pmed.1000010.g011

    Techniques Used: Activation Assay

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    Article Title: HMGB1 mediates endogenous TLR2 activation and brain tumor regression.
    Article Snippet: .. Generating HMGB1-Depleting Antibodies Antibodies were custom generated by New England Peptides and raised in New Zealand White rabbits against the N-terminal 11 amino acids of HMGB1 (MGKGDPKKPRG) (see Figure S3) [39]. ..



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    Biosynth Carbosynth generating hmgb1
    Figure 7. <t>HMGB1</t> Is Secreted from Dying Tumor Cells and Is Required for Ad-Flt3L and Ad-TK Mediated Brain Tumor Regression (A) GL26 cells were infected with Ad-TK and incubated with 25 lM GCV. A western blot was performed using an antibody specific for HMGB1. The graph displays a quantification of the total amount of HMGB1 in the media of cells 24 h (yellow bars), 48 h (orange bars), or 72 h (black bars) after GCV treatment. (B) GL26 cells were incubated with Ad-TK (with and without GCV and glycyrrhizin), and media was overlaid on HEK293 reporter cells transfected with either a plasmid encoding TLR2 (þpTLR2; black) or with a control plasmid (-pTLR2; red). NFjB activity was determined by quantifying the activity of Firefly Luciferase (under the control of the NFjB promoter), *, p , 0.05 (Kruskal-Wallis followed by Dunn’s test). Inset: cells were incubated with 100 ng/ ml PAM3 (PAM3CSK4) as a positive control. (C) The levels of HMGB1 in mouse serum were quantified by ELISA 7 d after treatment of brain tumors with Ad-Flt3L and Ad-TK. *, p , 0.05 versus saline (Mann-Whitney U-test). (D) GL26 cells were implanted into C57BL/6 mice (n ¼ 5 mice/treatment group) and 17 d later were treated with saline, or with AdFlt3L and Ad-TK (F/T). Glycyrrhizin (Glyc), HMGB1-depleting (aHMGB1), or rabbit IgG isotype control antibodies were administered IP 2 d, 5 d, and 10 d after treatment, *, p , 0.05 Ad-Flt3L and Ad-TK versus Ad-Flt3L and Ad-TK þ glycyrrhizin; ^p , 0.05 Ad-Flt3L and Ad-TK þ isotype versus Ad-Flt3L and Ad-TK þ aHMGB1; Mantel log-rank test. doi:10.1371/journal.pmed.1000010.g007
    Generating Hmgb1, supplied by Biosynth Carbosynth, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/generating+hmgb1/HMGB1+antibody/pm19143470-220-0-8
    Average 90 stars, based on 1 article reviews
    generating hmgb1 - by Bioz Stars, 2026-09
    90/100 stars
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    Figure 7. HMGB1 Is Secreted from Dying Tumor Cells and Is Required for Ad-Flt3L and Ad-TK Mediated Brain Tumor Regression (A) GL26 cells were infected with Ad-TK and incubated with 25 lM GCV. A western blot was performed using an antibody specific for HMGB1. The graph displays a quantification of the total amount of HMGB1 in the media of cells 24 h (yellow bars), 48 h (orange bars), or 72 h (black bars) after GCV treatment. (B) GL26 cells were incubated with Ad-TK (with and without GCV and glycyrrhizin), and media was overlaid on HEK293 reporter cells transfected with either a plasmid encoding TLR2 (þpTLR2; black) or with a control plasmid (-pTLR2; red). NFjB activity was determined by quantifying the activity of Firefly Luciferase (under the control of the NFjB promoter), *, p , 0.05 (Kruskal-Wallis followed by Dunn’s test). Inset: cells were incubated with 100 ng/ ml PAM3 (PAM3CSK4) as a positive control. (C) The levels of HMGB1 in mouse serum were quantified by ELISA 7 d after treatment of brain tumors with Ad-Flt3L and Ad-TK. *, p , 0.05 versus saline (Mann-Whitney U-test). (D) GL26 cells were implanted into C57BL/6 mice (n ¼ 5 mice/treatment group) and 17 d later were treated with saline, or with AdFlt3L and Ad-TK (F/T). Glycyrrhizin (Glyc), HMGB1-depleting (aHMGB1), or rabbit IgG isotype control antibodies were administered IP 2 d, 5 d, and 10 d after treatment, *, p , 0.05 Ad-Flt3L and Ad-TK versus Ad-Flt3L and Ad-TK þ glycyrrhizin; ^p , 0.05 Ad-Flt3L and Ad-TK þ isotype versus Ad-Flt3L and Ad-TK þ aHMGB1; Mantel log-rank test. doi:10.1371/journal.pmed.1000010.g007

    Journal: PLoS medicine

    Article Title: HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

    doi: 10.1371/journal.pmed.1000010

    Figure Lengend Snippet: Figure 7. HMGB1 Is Secreted from Dying Tumor Cells and Is Required for Ad-Flt3L and Ad-TK Mediated Brain Tumor Regression (A) GL26 cells were infected with Ad-TK and incubated with 25 lM GCV. A western blot was performed using an antibody specific for HMGB1. The graph displays a quantification of the total amount of HMGB1 in the media of cells 24 h (yellow bars), 48 h (orange bars), or 72 h (black bars) after GCV treatment. (B) GL26 cells were incubated with Ad-TK (with and without GCV and glycyrrhizin), and media was overlaid on HEK293 reporter cells transfected with either a plasmid encoding TLR2 (þpTLR2; black) or with a control plasmid (-pTLR2; red). NFjB activity was determined by quantifying the activity of Firefly Luciferase (under the control of the NFjB promoter), *, p , 0.05 (Kruskal-Wallis followed by Dunn’s test). Inset: cells were incubated with 100 ng/ ml PAM3 (PAM3CSK4) as a positive control. (C) The levels of HMGB1 in mouse serum were quantified by ELISA 7 d after treatment of brain tumors with Ad-Flt3L and Ad-TK. *, p , 0.05 versus saline (Mann-Whitney U-test). (D) GL26 cells were implanted into C57BL/6 mice (n ¼ 5 mice/treatment group) and 17 d later were treated with saline, or with AdFlt3L and Ad-TK (F/T). Glycyrrhizin (Glyc), HMGB1-depleting (aHMGB1), or rabbit IgG isotype control antibodies were administered IP 2 d, 5 d, and 10 d after treatment, *, p , 0.05 Ad-Flt3L and Ad-TK versus Ad-Flt3L and Ad-TK þ glycyrrhizin; ^p , 0.05 Ad-Flt3L and Ad-TK þ isotype versus Ad-Flt3L and Ad-TK þ aHMGB1; Mantel log-rank test. doi:10.1371/journal.pmed.1000010.g007

    Article Snippet: Generating HMGB1-Depleting Antibodies Antibodies were custom generated by New England Peptides and raised in New Zealand White rabbits against the N-terminal 11 amino acids of HMGB1 (MGKGDPKKPRG) (see Figure S3) [39].

    Techniques: Infection, Incubation, Western Blot, Transfection, Plasmid Preparation, Control, Activity Assay, Luciferase, Positive Control, Enzyme-linked Immunosorbent Assay, Saline, MANN-WHITNEY

    Figure 8. Western Blot Analysis Reveals HMGB1 Is Released from GL26, LLc1, GL261, and B16-F10 Cell Lines Following Treatment with Ad-TK (þGCV), Irradiation, or Temozolomide (TMZ) (A) LLc1, GL261, or B16-F10 cells were infected with Ad-TK or Ad-0 at an MOI¼500. Noninfected cells were used as control. Ad-TK treated cells were also incubated with 25 lM GCV. 48 h later, supernatant was collected and HMGB1 release was assessed by western blotting. (B) GL26, LLc1, GL261, or B16-F10 cells were irradiated with 20 Gy for 30 min. Nonirradiated cells were used as control. 72 h later, supernatant was collected and HMGB1 release was assessed by western blotting. (C) GL26, LLc1, GL261, or B16-F10 cells were incubated with temozolomide (TMZ) (200 lM). Control cells were incubated without TMZ (mock). 48 h later, supernatant was collected and HMGB1 release was assessed by western blotting. doi:10.1371/journal.pmed.1000010.g008

    Journal: PLoS medicine

    Article Title: HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

    doi: 10.1371/journal.pmed.1000010

    Figure Lengend Snippet: Figure 8. Western Blot Analysis Reveals HMGB1 Is Released from GL26, LLc1, GL261, and B16-F10 Cell Lines Following Treatment with Ad-TK (þGCV), Irradiation, or Temozolomide (TMZ) (A) LLc1, GL261, or B16-F10 cells were infected with Ad-TK or Ad-0 at an MOI¼500. Noninfected cells were used as control. Ad-TK treated cells were also incubated with 25 lM GCV. 48 h later, supernatant was collected and HMGB1 release was assessed by western blotting. (B) GL26, LLc1, GL261, or B16-F10 cells were irradiated with 20 Gy for 30 min. Nonirradiated cells were used as control. 72 h later, supernatant was collected and HMGB1 release was assessed by western blotting. (C) GL26, LLc1, GL261, or B16-F10 cells were incubated with temozolomide (TMZ) (200 lM). Control cells were incubated without TMZ (mock). 48 h later, supernatant was collected and HMGB1 release was assessed by western blotting. doi:10.1371/journal.pmed.1000010.g008

    Article Snippet: Generating HMGB1-Depleting Antibodies Antibodies were custom generated by New England Peptides and raised in New Zealand White rabbits against the N-terminal 11 amino acids of HMGB1 (MGKGDPKKPRG) (see Figure S3) [39].

    Techniques: Western Blot, Irradiation, Infection, Control, Incubation

    Figure 9. HMGB1 Is Released into the Supernatant of GL26, LLc1, GL261, and B16-F10 Tumor Cell Lines in Response to Treatment with Ad-TK (þGCV), Irradiation, or Temozolomide (A–D) GL26, LLc1, GL261, or B16-F10 cells were infected with Ad-TK or Ad-0. Noninfected cells were used as control (mock). Ad-TK treated cells were incubated with 25 lM GCV. 48 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with Ad-TK (þGCV) significantly increased HMGB1 release when compared to corresponding mock and Ad0 treatment groups from each cell line, *, p , 0.05 versus mock and Ad-0 (Kruskal-Wallis test followed by Dunn’s test). (E–H) GL26, LLc1, GL261, or B16-F10 cells were irradiated with 20 Gy for 30 min. Nonirradiated cells were used as controls (mock). 72 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with irradiation significantly increased HMGB1 release when compared to corresponding control groups from each cell line, *, p , 0.05 versus mock (Mann-Whitney U-test). (I–L) GL26, LLc1, GL261, or B16-F10 cells (2.53105 cells per flask) were incubated with or without temozolomide (TMZ) (200 lM). 48 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with TMZ significantly increased HMGB1 release when compared to control groups from each cell type. *, p , 0.05 versus mock (Mann-Whitney U-test). doi:10.1371/journal.pmed.1000010.g009

    Journal: PLoS medicine

    Article Title: HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

    doi: 10.1371/journal.pmed.1000010

    Figure Lengend Snippet: Figure 9. HMGB1 Is Released into the Supernatant of GL26, LLc1, GL261, and B16-F10 Tumor Cell Lines in Response to Treatment with Ad-TK (þGCV), Irradiation, or Temozolomide (A–D) GL26, LLc1, GL261, or B16-F10 cells were infected with Ad-TK or Ad-0. Noninfected cells were used as control (mock). Ad-TK treated cells were incubated with 25 lM GCV. 48 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with Ad-TK (þGCV) significantly increased HMGB1 release when compared to corresponding mock and Ad0 treatment groups from each cell line, *, p , 0.05 versus mock and Ad-0 (Kruskal-Wallis test followed by Dunn’s test). (E–H) GL26, LLc1, GL261, or B16-F10 cells were irradiated with 20 Gy for 30 min. Nonirradiated cells were used as controls (mock). 72 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with irradiation significantly increased HMGB1 release when compared to corresponding control groups from each cell line, *, p , 0.05 versus mock (Mann-Whitney U-test). (I–L) GL26, LLc1, GL261, or B16-F10 cells (2.53105 cells per flask) were incubated with or without temozolomide (TMZ) (200 lM). 48 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with TMZ significantly increased HMGB1 release when compared to control groups from each cell type. *, p , 0.05 versus mock (Mann-Whitney U-test). doi:10.1371/journal.pmed.1000010.g009

    Article Snippet: Generating HMGB1-Depleting Antibodies Antibodies were custom generated by New England Peptides and raised in New Zealand White rabbits against the N-terminal 11 amino acids of HMGB1 (MGKGDPKKPRG) (see Figure S3) [39].

    Techniques: Irradiation, Infection, Control, Incubation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Figure 10. Treatment of Animals Bearing Intracranial Glioma (GL261) or Melanoma (B16-F10) with Ad-Flt3L and Ad-TK (þGCV) Results in Long-Term Survival and Increased Serum Levels of HMGB1 (A) GL261 cells were implanted into the striatum of C57/Bl6 mice. 17 d later animals were treated with Ad-Flt3L (n ¼ 5), Ad-TK (þGCV) (n ¼ 6), Ad-Flt3L plus Ad-TK (þGCV) (n ¼ 5), or saline (n ¼ 5). Treatment with Ad-Flt3L and Ad-TK significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test), Ad-Flt3L, or Ad-TK alone treatment groups (^, p , 0.05; Mantel log-rank test). Treatment with Ad-Flt3L, or Ad-TK alone significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test). (B) 7 d after treatment, tumor-bearing animals were humanely killed and HMGB1 release was assessed in the serum. Treatment of GL261 tumor-bearing animals with Ad-Flt3L and Ad-TK (þGCV) significantly increased the levels of HMGB1 in the serum when compared to tumor-bearing animals treated with saline. *, p , 0.05 versus saline (Mann-Whitney U-test). (C) B16-F10 cells were implanted into the striatum of C57/Bl6 mice. 17 d later animals were treated with Ad-Flt3L, Ad-TK (þGCV), Ad-Flt3L plus Ad-TK (þGCV), or saline. Treatment with Ad-Flt3L and Ad-TK significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test), Ad- Flt3L, or Ad-TK alone treatment groups (^, p , 0.05; Mantel log-rank test). Treatment with Ad-Flt3L, or Ad-TK alone, significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test). (D) 7 d after treatment, tumor-bearing animals were humanely killed and HMGB1 release was assessed in the serum. Treatment of B16-F10 tumor- bearing animals with Ad-Flt3L and Ad-TK (þGCV) significantly increased the levels of HMGB1 in the serum when compared to tumor-bearing animals treated with saline. *, p , 0.05 versus saline (Mann-Whitney U-test). doi:10.1371/journal.pmed.1000010.g010

    Journal: PLoS medicine

    Article Title: HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

    doi: 10.1371/journal.pmed.1000010

    Figure Lengend Snippet: Figure 10. Treatment of Animals Bearing Intracranial Glioma (GL261) or Melanoma (B16-F10) with Ad-Flt3L and Ad-TK (þGCV) Results in Long-Term Survival and Increased Serum Levels of HMGB1 (A) GL261 cells were implanted into the striatum of C57/Bl6 mice. 17 d later animals were treated with Ad-Flt3L (n ¼ 5), Ad-TK (þGCV) (n ¼ 6), Ad-Flt3L plus Ad-TK (þGCV) (n ¼ 5), or saline (n ¼ 5). Treatment with Ad-Flt3L and Ad-TK significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test), Ad-Flt3L, or Ad-TK alone treatment groups (^, p , 0.05; Mantel log-rank test). Treatment with Ad-Flt3L, or Ad-TK alone significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test). (B) 7 d after treatment, tumor-bearing animals were humanely killed and HMGB1 release was assessed in the serum. Treatment of GL261 tumor-bearing animals with Ad-Flt3L and Ad-TK (þGCV) significantly increased the levels of HMGB1 in the serum when compared to tumor-bearing animals treated with saline. *, p , 0.05 versus saline (Mann-Whitney U-test). (C) B16-F10 cells were implanted into the striatum of C57/Bl6 mice. 17 d later animals were treated with Ad-Flt3L, Ad-TK (þGCV), Ad-Flt3L plus Ad-TK (þGCV), or saline. Treatment with Ad-Flt3L and Ad-TK significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test), Ad- Flt3L, or Ad-TK alone treatment groups (^, p , 0.05; Mantel log-rank test). Treatment with Ad-Flt3L, or Ad-TK alone, significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test). (D) 7 d after treatment, tumor-bearing animals were humanely killed and HMGB1 release was assessed in the serum. Treatment of B16-F10 tumor- bearing animals with Ad-Flt3L and Ad-TK (þGCV) significantly increased the levels of HMGB1 in the serum when compared to tumor-bearing animals treated with saline. *, p , 0.05 versus saline (Mann-Whitney U-test). doi:10.1371/journal.pmed.1000010.g010

    Article Snippet: Generating HMGB1-Depleting Antibodies Antibodies were custom generated by New England Peptides and raised in New Zealand White rabbits against the N-terminal 11 amino acids of HMGB1 (MGKGDPKKPRG) (see Figure S3) [39].

    Techniques: Saline, MANN-WHITNEY

    Figure 11. Model Illustrating the Role of TLR2 and HMGB1 in Initiating T Cell-Dependent Brain Tumor Regression by Flt3L and TK Treatment of tumor cells with TK þ GCV releases endogenous TLR2 ligands including HMGB1. DC infiltration into tumors and subsequent activation require TLR2 signalling. DC phagocytose tumor antigen and migrate to the dLN where they stimulate T cell clonal expansion resulting in T cell- dependent brain tumor regression and long-term immunological memory. doi:10.1371/journal.pmed.1000010.g011

    Journal: PLoS medicine

    Article Title: HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

    doi: 10.1371/journal.pmed.1000010

    Figure Lengend Snippet: Figure 11. Model Illustrating the Role of TLR2 and HMGB1 in Initiating T Cell-Dependent Brain Tumor Regression by Flt3L and TK Treatment of tumor cells with TK þ GCV releases endogenous TLR2 ligands including HMGB1. DC infiltration into tumors and subsequent activation require TLR2 signalling. DC phagocytose tumor antigen and migrate to the dLN where they stimulate T cell clonal expansion resulting in T cell- dependent brain tumor regression and long-term immunological memory. doi:10.1371/journal.pmed.1000010.g011

    Article Snippet: Generating HMGB1-Depleting Antibodies Antibodies were custom generated by New England Peptides and raised in New Zealand White rabbits against the N-terminal 11 amino acids of HMGB1 (MGKGDPKKPRG) (see Figure S3) [39].

    Techniques: Activation Assay