Fig. 2

IC overexpression and cell proliferation. a RasB8, U87-EGFRvIII or b U87 were either serum-starved or maintained in 10% FBS, lysed and immunoblotted with indicated antibodies (top panel) or BrdU incorporation assay conducted (bottom panel). c Representative immunohistochemistry images using anti-Ki67 (scale bar, 500 μm) or anti-CIC (scale bar, 1 mm) antibody of high-grade tumors from RasB8 transgenic mice. d RasB8 transfected with or without increasing HA-CIC plasmid lysed and immunoblotted with indicated antibodies. e Quantitative real-time PCR analysis of RasB8 transfected with or without HA-CIC. The graph depicts fold changes in ETV1 expression relative to control. RasB8 transfected with or without HA-CIC plated equally plated and cell viability was assessed at indicated time points (in hours) by trypan blue exclusion or f alamar blue assay (g). h U87-EGFRvIII transfected with or without increasing concentrations of HA-CIC lysed and protein immunoblotted with indicated antibodies. i Quantitative real-time PCR analysis of U87-EGFRvIII transfected with or without HA-CIC. The graph depicts fold changes in ETV1 or -5 expression relative to control. U87-EGFRvIII transfected with or without HA-CIC plated equally and cell viability assessed at indicated time points (in hours) by trypan blue exclusion or j alamar blue assay k. l U87-EGFRvIII transfected with GFP-CIC labeled with eFluor 670 proliferation dye analyzed by flow cytometry. Graphs depict percentage of proliferating GFP-positive versus GFP-negative cells within the same population. Data are representative of at least three independent experiments. U87-Flag-CIC or control cells lysed and m protein lysates immunoblotted with indicated antibodies, n quantitative real-time PCR analysis conducted assessing ETV- or -5 expression or o BrdU incorporation assay. GL261-Flag-CIC or controls were lysed and p immunoblotted with indicated antibodies or q BrdU incorporation assay conducted. Anchorage-independent growth assay of GL261 (r) or U87 (s) cells expressing Flag-CIC showing fold changes relative to control (bottom panel) or phase contrast microscopy images (top panel). All graphs represent mean ± s.e.m. of three independent experiments performed either in octuplet for viability assays or in triplicate for real-time analysis. *P < 0.05 Student’s t-test compared with control. The immunoblot data are representative of at least three separate experiments