Figure 4.

Depletion of BAG3 sensitizes the chemoresistant breast cancer cells by attenuating protective autophagy

(A) Stable lentiviral knockdowns of BAG3 were established in both the BT-549-Par and BT-549^rDOX²⁰ cells and (B) also in the MDA-MB-468Par and MDA-MB-468^r5-FU²⁰⁰⁰ cells. Stable lentiviral transduction of empty vector was used as control (Ctrl). Knockdowns were confirmed by western blot. Knockdown of BAG3 reduced LC3-II protein expression in BT-549^rDOX²⁰ and MDA-MB-468^r5-FU²⁰⁰⁰ cells. GAPDH served as loading control in western blot. (C) Relative BAG3 mRNA expression was also determined by quantitative PCR (qPCR) after stable lentiviral knockdowns of BAG3 in BT-549Par and BT-549^rDOX²⁰ cell lines and (D) similarly in MDA-MB-468 cells respectively. qPCR data represent means of three independent experiments± SEM (n = 3). Significant BAG3 mRNA expression compared to parental sh Ctrls are marked by asterisks: * p<0.05, ** p<0.01 and ns not significant. Significant differences between BAG3 KD and respective sh Ctrls are denoted by hashtags: # p<0.05, ##p<0.01 and ns not significant. (E) Knockdown of BAG3 reduced the protein expression of HSP70, Mcl-1 and other anti-apoptotic proteins whereas augments the expression of pro-apoptotic proteins like Bak and Bax in BT-549^rDOX²⁰ and (F) MDA-MB-468^r5-FU²⁰⁰⁰ cells respectively. GAPDH served as loading control. (G) BT-549^rDOX²⁰/BAG3 KD and (H) MDA-MB-468^r5-FU²⁰⁰⁰/BAG3 KD cells exhibited significantly higher levels of total cell death compared to their parental counterparts after 72 hours of DOX and 5-FU treatment in a dose dependent manner respectively. 0.1% DMSO for 72 h was used as control. Cell death was determined by Annexin V/PI double staining followed by flow cytometry. Data represent means of three independent experiments ± SEM (n = 3). *p<0.05 and ns not significant of BAG3 KD compared to respective sh Ctrls.