Fig. 1

Bag-1 increases cell survival and suppresses apoptotic cell death. a. Immunoblot analysis of Bag-1 expression in normal (N) and tumor (T) tissues of 30 breast cancer patients with different expression profiles of ER/PR and Her2 receptors. Bag-1 expression was normalized to vinculin. b. Immunoblot analysis of Bag-1 protein levels in MCF-7, MDA-MB-231 and MCF-10A cells. Cells were transfected with Bag-1 (Bag-1:TAP) expression vector, mock vector, Bag-1 siRNA (siBag-1), or non-silencing control siRNAs (nc-siRNA). Expression of Bag-1 isoforms was detected with Bag-1 antibody. β-actin was used as the loading control. c, d. Cell viability and apoptotic cell death following Bag-1 overexpression or Bag-1 silencing in MCF-7 cells. XTT cell viability assay (c) and Cell Death Detection ELISAPLUS assay (d) was performed 24, 48 and 72 h after transfection of MCF-7 cells with Bag-1 expression vector, Bag-1 siRNA, and their negative controls. All values are given relative to 24 h untransfected control in XTT assay. Data are represented as mean ± standard error from three independent experiments for XTT assay and two independent experiments for apoptosis assay. Two-way ANOVA was used to calculate p values. e. Effects of Bag-1 expression on Bax/Bcl-2 ratio in MCF-7 and MCF-10A cells. Expression levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins were determined by densitometric analysis on the immunoblots of MCF-7 and MCF-10A cell lysates after Bag-1 overexpression (48 h) or Bag-1 silencing (24 and 48 h). Bax and Bcl-2 levels were normalized according to β-actin levels, and Bax/Bcl-2 ratio was calculated to determine the apoptotic potential of cells at each condition