Fig 1: Chemoresistant MB cells secrete elevated levels of LIF, OSM, IL-11 and IL-6. Secreted cytokine analysis for (A) LIF, (B) IL-11, (C) OSM, and (D) IL-6 of conditioned media from Med8A-S and IL-6R-/- cells with or without cytokine conditioning (designated as -IL6+, -IL11+, -OSM+ or -LIF+) or exposure to co-culture with microglia (HMC3). The conditioned media of HMC3 cells was also assessed. Plotted is the mean ± SD of triplicate assays; *** p < 0.001, ordinary one-way ANOVA with Bonferroni’s multiple comparison test.
Fig 2: MB cells conditioned with IL-6 family cytokines enhance JAK1 and STAT3 activity and chemoresistance. (A) Med8A-S and IL-6R-/- cells untreated (Ctrl) or treated with bolus LIF, OSM, IL-11, or IL-6, and immunoblotted to detect pJAK1, JAK1, pY705-STAT3, and STAT3 expression. (B) Flow cytometry assay for cell surface expression of LIFR, OSMR, IL-11R, or IL-6R in Med8A-S and cytokine conditioned cells (LIF, OSM, IL-11, or IL-6), with corresponding IgG controls. Cell viability assay to assess the vincristine sensitivity of (C) Med8A-S and (D) IL-6R-/- cells not conditioned, or conditioned with IL-6, LIF, OSM or IL-11, as indicated. Plotted is the mean ± SD of triplicate assays, representative of 3 independent experiments. *** p < 0.001, ** < 0.01, * < 0.05, two-way ANOVA with Bonferroni’s multiple comparison test.
Fig 3: Response of IL-6 family cytokine-conditioned IL6R-/- cells to combined treatment with vincristine and ruxolitinib. Cell viability assay to assess the sensitivity of (A) IL-6R-/--IL6+, (B) IL-6R-/--IL11+, (C) IL-6R-/--LIF+, and (D) IL-6R-/--OSM+ cells to the indicated concentrations of vincristine with and without 2 µM ruxolitinib co-treatment. Plotted is the mean ± SD of triplicate assays, representative of three independent experiments. *** p < 0.001, ** < 0.01, two-way ANOVA with Bonferroni’s multiple comparison test.
Fig 4: Response of IL-6 family cytokine-conditioned Med8A-S cells to combined treatment with vincristine and ruxolitinib. Cell viability assay to assess the sensitivity of (A) Med8A-S, (B) Med8A-IL6+, (C) Med8A-IL11+, (D) Med8A-LIF+, and (E) Med8A-OSM+ cells to the indicated concentrations of vincristine with or without 2 µM ruxolitinib co-treatment. Plotted is the mean ± SD of triplicate assays, representative of three independent experiments. *** p < 0.001, ** < 0.01, two-way ANOVA with Bonferroni’s multiple comparison test.
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