Figure 6

BAG3 is required for enhanced autophagic flux and resistance to oxidative stress in SK-ERα and MCF-7 cells. (a–c) SK-01, SK-ERα and MCF-7 cells were transfected with non-sense siRNA (siNS) and BAG3 siRNA (siBAG3) for 48 h, as indicated. Protein extracts from vehicle or BafA₁-treated cells were subjected to western blot analysis and calculation of autophagic flux using anti-LC3 antibodies. Tubulin was used as a loading control. Autophagic flux was determined by the accumulation of LC3-II in a 6 h treatment period with 500 nM BafA₁. Therefore, normalized LC3-II levels in the absence of the lysosomal inhibitor were subtracted from corresponding levels obtained in the presence of BafA₁. (d–f) SK-01, SK-ERα and MCF-7 cells were transfected with non-sense siRNA (siNS) and BAG3 siRNA (siBAG3) for 48 h and treated with vehicle control or 400 μM and 600 μM H₂O₂ for 24 h, as indicated. Cell death was quantified by flow cytometry after propidium iodide staining. Values of three (SK-01), four (SK-ERα) and three (MCF-7) independent experiments in each panel are expressed as mean±S.E.M. and representative data from the experiments are shown in FACS dot plot profiles. (*) on bars represents statistical significance of P<0.05 comparing two groups and n.s. displays no statistical significant difference.