Preventing endogenous GILZ expression accelerates interleukin-2 deprivation–mediated apoptosis in CTLL-2 cells through the up-regulation of Bim expression. (A) Selected pSUPER-control or pSUPER-GILZ clones were deprived of IL-2 for the indicated period of time. Cells were then stained with propidium iodide and analyzed for DNA hypodiploidy by flow cytometry. The data shown are representative of 2 independent experiments. Of 3 representative experiments, 1 is shown. Right hand panel: Western blot analysis of GILZ expression in the various pSUPER clones cultured in the absence of IL-2 for 4 hours. (B-C) GILZ siRNA expression accelerates IL-2 deprivation–induced Bim expression. pSUPER-GILZ clones (30 and 31) and pSUPER control clones (B-C) were deprived of IL-2 and lysed at the indicated period of time. (B) Western blot was performed using an anti-Bim antibody. After stripping, membranes were reblotted with an anti–β Tubulin antibody as a loading control. Bim expression was quantified by densitometric analysis and normalized to the densitometric value of the β Tubulin (ratio Bim/β Tubulin). (C) Total RNA was extracted and used as a template for RT-PCR using primers specific for Bim or β actin. Bim expression was quantified by densitometric analysis and normalized to the densitometric value of the β actin (ratio Bim/β actin).