Fig. 6
From: CIC protein instability contributes to tumorigenesis in glioblastoma
Effect of stable CIC on its function and ERK inhibition. a U87 transfected with indicated plasmids lysed and IP with anti-HA antibody. b HEK293 transfected cells lysed and Streptavidin agarose bound to biotinylated ETV5 oligonucleotide octameric motif pull-down assay was conducted. c Luciferase activity of HEK293 transfected cells, pGL3-ETV5 plasmid normalized to control. d HEK293 transfected cells lysed and immunoblotted with antibodies. e U87-HA-CIC or -CIC(ΔEBI) treated with or without cycloheximide lysed and immunoblotted with indicated antibodies. f U87 transfected cells lysed and IP with anti-HA antibody. g Alamar blue assay of U87-Flag-CIC, -CIC(ΔEBI) or control cells. Alamar blue assay of GSC h 7-2-, j 7-11-, l 8-11- or n 30-Flag-CIC, -CIC(ΔEBI) or controls cells. Representative images of 14 day old GSC i 7-2- k 7-11-, m 8-11- or o 30-Flag-CIC, -CIC(ΔEBI) or control cultures. Scale bar, 2 mm. GL261-Flag-CIC, -CIC(ΔEBI) or controls were p lysed and immunoblotted with indicated antibodies or q alamar blue assay conducted. r Alamar blue assay of GSC 8-18-Flag-CIC, -CIC(ΔEBI) or controls treated with or without DMSO, PD98509 or selumetinib. Alamar blue assay of GSC s 7-2- or t 7-11-Flag-CIC, -CIC(ΔEBI) or controls treated with or without DMSO or selumetinib. Anchorage-independent growth assay was u imaged (left) or quantified (right) of selumetinib treated GL261-Flag-CIC(ΔEBI). Scale bar, 200 μm. v Kaplan–Meier survival curves of selumetinib treated (7 days post injections) GL261-Flag-CIC(ΔEBI) or control xenograft mice. Log rank (Mantel–Cox) test, P < 0.0001. Eight-week-old mice, n = 7. T2 weighted anatomy MRI was w imaged 14 day post GL261-Flag-CIC(ΔEBI) injections (scale bar 2 mm) or x tumor volume quantified. y Kaplan–Meier survival curves of GSC 7-2-Flag-CIC(ΔEBI) mice treated with vehicle or selumetinib 7 days post injections. Log rank (Mantel–Cox) test, P < 0.0003. Eight-week-old mice, n = 7. z Quantitative T2 weighted anatomy MRI images of GSC 7-2-Flag-CIC(ΔEBI) or control mice treated with selumetinib 7 days post injections. Scale bar, 2 mm. WCE Whole-cell extract, strep-PD Streptavidin pull down, IP immunoprecipitated. Graphs represent mean ± s.e.m. of three independent experiments performed in octuplet for viability assays or in triplicate for real-time, luciferase or anchorage-independent growth assay. *P < 0.05 Student’s t-test versus control
