FIG. 1.

FGF-2 prevents Smac but not cytochrome c release by etoposide. H510 cells, pretreated with or without FGF-2 for 4 h, were incubated in the presence or absence of etoposide (VP-16) for 4 h (A, B, and D) or the times indicated (C). (A and C) Mitochondrial (M) and cytoplasmic (C) fractions were obtained by digitonin fractionation and analyzed by SDS-PAGE and Western blotting for the presence of Smac or cytochrome c (Cyto c). The absence of mitochondrial contamination of the cytoplasmic fraction was confirmed by detection of cytochrome oxidase (COX). (B) Smac and cytochrome c immunodetection, in fixed H510 cells, was visualized using FITC-labeled secondary antibodies (green). Cellular DNA was highlighted using DAPI staining (blue). (D) Cytochrome c and Smac colocalize with the mitochondria in untreated, but not in etoposide-treated, H510 cells. H510 cells were treated with 10 μM Mitotracker Red for 1 h to visualize the mitochondria, while FGF-2 and etoposide treatments were as described for panel A. Samples were then visualized by confocal microscopy. (E) FGF-2-rescued cells are still able to proliferate. Control untreated cells (open circles) and FGF-2-rescued cells (solid circles) were compared for their abilities to proliferate in RPMI containing 10% FCS. Results are representative of three independent experiments performed in quadruplicate. Error bars, standard errors of the means. For panels A through D, results are representative of at least three independent experiments.