Figure 3.

Knockdown of ATG5 sensitizes chemoresistant breast cancer cells

(A) Stable lentiviral knockdowns of ATG5 were established in both the BT-549Par and BT-549^rDOX²⁰ cells and (B) also in the MDA-MB-468Par and MDA-MB-468^r5-FU²⁰⁰⁰ cells. Stable lentiviral transduction of control vector was used as control (Ctrl). Knockdowns were confirmed by western blot. Knockdown of ATG5 reduced LC3-II protein expression in BT-549^rDOX²⁰ and MDA-MB-468^r5-FU²⁰⁰⁰ cells. GAPDH was used as loading control in western blot. (C) Down regulation of ATG5 reduces the protein expression of Mcl-1 and other anti-apoptotic proteins, whereas augments the expression of pro-apoptotic proteins likes Bak and Bax in BT-549^rDOX²⁰and (D) MDA-MB-468^r5-FU²⁰⁰⁰ cells respectively. GAPDH served as loading control in western blot. (E) BT-549^rDOX²⁰/ATG5 KD cell exhibited significantly higher levels of total cell death compared to the BT-549Par cells after 72 hours of DOX treatment in a dose dependent manner. 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 are means ± SEM of three different experiments each performed in triplicate. * p<0.05 ** p<0.01, *** p<0.001 and ns not significant compared to respective controls (Ctrls); # p<0.05, ## p<0.01 and ns not significant of ATG5 KD compared to respective sh Ctrls.