Figure 6. Acidosis oxidizes PTEN and presence of PTEN is required for acidosis induced AKT activation in breast cancer cells.

(A) MCF-7 and MDA-MB-231 cells were cultured under acidic conditions or treated with 1 mM H₂O₂ for the indicated time. The whole cell extract was then alkylated with NEM and subjected to nonreducing SDS-PAGE followed by Western blot analysis using indicated antibodies. For PTEN blot, upper band indicates the reduced form while lower band indicates the oxidized form of PTEN. (B) BT-549 cells transiently expressing wild-type and mutant (C⁷¹S, C¹²⁴S) PTEN plasmids were exposed to acidosis for 30 minutes, whole cell extracts were prepared and analyzed for indicated proteins by Western blot analysis. (C) PTEN deficient cells (BT-549, MDA-MB-468) were exposed to acidosis for the indicated times, whole cell extracts were prepared and analyzed for indicated proteins by Western blot analysis. (D) The generation of ROS under acidic conditions is mediated through NOX in breast cancer cells. Cells were cultured at pH 7.4 or pH 6.6 without and with 20 μM DPI and analyzed for intracellular ROS levels using DCFH-DA by flow cytometry. (E) A schematic diagram showing the signaling pathways by which acidosis promotes breast cancer invasion.