Silencing or pharmacologic inhibition of Rac1 reduces intracellular and intramitochondrial O2∸ levels in Bcl-2-overexpressing cells. (A) Intracellular O2∸ levels were determined in HeLa/Neo and HeLa/Bcl-2 cells using lucigenin-based chemiluminescence. (B) Intracellular levels were determined in HeLa/Neo and HeLa/Bcl-2 cells silenced with control scrambled siRNA (Negsi) Rac1 siRNA using lucigenin-based chemiluminescence assay. (B-C) Error bars represent the mean ± SD (n = 4). (C) Western blot analysis of Rac1, Bcl-2, and β-actin expression in HeLa/Neo and HeLa/Bcl-2 cells transfected with control scrambled siRNA (Neg) or siRNA against Rac1. (D-F) Intramitochondrial O2∸ levels were determined after cells (D-E) or isolated mitochondria (F) loading with MitoSOX as described in “Determination of O2∸.” The mean fluorescence intensity values are expressed as mean ± SE. The histograms are representative of 3 independent experiments.
