Figure 3. VPA/HU-treatment specifically induces the BCL-2 family protein BIM modulating cell proliferation and apoptosis.

A) Cells were drug treated (1.5 mM; 20 h) and expression levels of the indicated proteins were visualized by immunoblot. Actin served to control equal loading of cell lysates. B) Cell death induction by ectopic expression of BIM_EL-GFP. BIM_EL-GFP was visualized 24 h post transfection in FaDu cells by direct and indirect immunofluorescence using α-BIM Ab. C) Downregulation of BIM in HNSCCUM-03T cells stably transfected with BIM- (shBIM) versus scrambled-shRNA (shCtl) verified by immunoblot. Counting revealed that cells with attenuated endogenous BIM levels displayed enhanced proliferation. D) Decreased VPA/HU-induced apoptosis (1.5 mM each, 24 h) in BIM-depleted cells shown by immunoblot analyses for BIM and cleaved Caspase-3 (left), as well as by quantification of enzymatic Caspase-3 activity in cell lysates (right). E) Immunoblot revealed that VPA/HU (1.5 mM each) induced BIM in a time-dependent manner. F) VPA/HU-mediated transcriptional activation was monitored by analyzing luciferase activity. FaDu cells transfected with a BIM reporter were treated with VPA/HU (1.5 mM each). G) In contrast to the strong induction of BIM levels by VPA/HU, correlating with Caspase-3 cleavage, no enhanced expression of PUMA and BAX was induced by VPA/HU. Actin and Tubulin served as loading controls. Columns, mean; bars, ±SD from three independent experiments.