Figure 1

Silencing of apoptosis-inducing factor (AIF) by siRNA in human breast adenocarcinoma cells. Human breast adenocarcinoma cells were transfected with 20 nM small interfering RNA (siRNA) oligos for AIF as previously reported [26]. A, (a) Immunoblotting detection of AIF in MDA-MB-231 cell extracts 48 h after siRNA transfection using polyclonal anti-AIF antibody (Rockland Immunochemcials) (1:1000 dilution). Controls were provided by untransfected cells (Con) and cells transfected with scrambled siRNA oligos (Scr). Densitometric quantification (b) of AIF protein bands from (a) was then performed. Values represent AIF/actin ratios of the densitometric intensities of the protein bands. *P < 0.01 between Scr and AIF (one-way ANOVA and unpaired Student’s t test). Error bars represent the standard error of the mean (SEM). B, Immunoblotting detection (a) and densitometric quantification of AIF in MCF-7 cell extracts 48 h after siRNA transfection. C, MDA-MB-231 cells were transfected with siRNA oligos, treated with 0.5 mM N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) for 45 min, then analyzed by immunoblot for levels of nuclear AIF. Controls for cell fractionations were provided by mitochondrial manganese superoxide dismutase (MnSOD) and nuclear lamin B2. PN, post-nuclear fraction (cytoplasm + mitochondria); Nuc, nuclear fraction. All experiments were repeated at least three times with similar results.