Fig. 3

Mcl-1 is downregulated in IDH1-mutated gliomas. a Protein was extracted from human tumor samples of anaplastic astrocytoma expressing IDH1-WT or IDH1-R132H. Western blot analysis was performed for Mcl-1, Bcl-xL, Bcl-2, Noxa, and Bim. Actin served as loading control. b Scatter plot demonstrating Mcl-1 protein expression among IDH1-WT (n = 5) and IDH1-R132H (n = 6) expressing anaplastic astrocytoma samples. Densitometric analysis was performed using imageJ 1.47 v (http://imagej.nih.gov/ij). Pixel density was normalized to the respective Actin control. c Whole-cell extracts of U87MG IDH1-WT/IDH1-R132H cells were collected to determine base line protein expression of Mcl-1, Noxa, Bcl-2, Bcl-xL, and BIM by western blot analysis. Actin was used to ensure equal loading. d, e U87MG glioblastoma cells (d) and GBM12 patient-derived glioblastoma xenograft cells (e) were treated for 3 h (U87MG) or 24 h (GBM12) with increasing concentrations of 2-HG. Whole-cell extracts were collected prior to performing western blot analysis for Mcl-1, Bcl-2, and Bcl-xL. Western blot for Actin or 14-3-3 was performed to confirm equal loading. f, g U87MG (f) and LN229 (g) cells were treated with non-targeting (n.t.)-siRNA or 4 different Mcl-1-siRNAs and subjected to treatment with solvent or ABT263, for 24 h. Staining for propidium iodide was performed prior to flowcytometry. The fraction of subG1 cells was determined. Column: mean. Error bar: SD, n = 3. Western blot analysis was performed to confirm Mcl-1 knockdown. h, i Quantitative representation of LN229 cells treated with non-targeting (n.t.)-siRNA, Noxa-siRNA (h) or Bak-siRNA (i) prior to treatment with ABT263/2-HG or solvent. Staining for propidium iodide and flowcytometry were performed to determine the fraction of subG1 cells. Column: mean. Error bar: SD, n = 3. Whole-cell extracts were collected and western blot analysis was performed for Noxa and Bak to confirm appropriate knockdown