Figure 6.

BAG3 depletion alters cell matrix adhesion in chemoresistant breast cancer cells

(A) Knockdown of BAG3 altered cellular morphology and actin cytoskeletal distribution in BT-549^rDOX²⁰ and (B) MDA-MB-468^r5-FU²⁰⁰⁰ cells compared to their respective parental counterparts in confocal imaging. For staining of F-actin, Texas Red-X phalloidin was used whereas nuclei were stained with DAPI. Scale bar 10 μm. (C) FAK phosphorylation was reduced in BT-549^rDOX²⁰/BAG3 KD and (D) MDA-MB-468^r5-FU²⁰⁰⁰/BAG3 KD cells respectively. GAPDH served as loading control in the western blot. Densitometric analysis of relative pFAK protein expression was performed in control and BAG3 KD of both BT-549 and MDA-MB-468 parental and chemoresistant cell lines. Columns represent means ± SEM. Statistical significance; * p<0.05, ** p<0.01, *** p<0.001 compared to controls (E) BT-549^rDOX²⁰/BAG3 KD cells cultured in suspension exhibited more sensitivity to DOX treatment in a dose dependent manner. Cell cultured dishes were coated with pHEMA to prevent cell adhesion. Water (0.1%, 72 h) was used as control (Ctrl) for the solvent. Cell death was determined by Annexin V/PI double staining followed by flow cytometry. Columns represent means of three independent experiments performed in triplicate ± SEM. Statistical significance: * p<0.05, ** p<0.01, *** p<0.001 and ns not significant compared to ctrls (0.1 % water); # p<0.05 and ns not significant with combined treatment of DOX and pHEMA compared to DOX treatment alone.