Research Paper Volume 16, Issue 11 pp 9410—9436

Figure 5. APOA5 alleviates ROS production in PIK3CA-E545K CRC cells with L-OHP treatment. (A) APOA5-silencing and control HCT116/E545K cells were lysed to measure APOA5 and PPARγ protein expression by Western blot analysis. (B) CCK-8 assays were performed to determine the growth inhibition rate in control and APOA5 silencing HCT116/E545K cells in the presence of increasing doses of L-OHP. (C) Established cells were treated for 48 h with L-OHP. Cell death was measured with the LDH assay as 100%^* (LDH _medium/(LDH _medium + LDH _lysate)). (D) Flow cytometry analysis was performed to evaluate the ROS production of APOA5 silenced and control cells with a gradient concentration of L-OHP treatment. Displayed as mean +/− SD (n = 3). (E) After combined treatment of L-OHP (2 μM), LY294002 (10 μM) and NAC (2 mM) as described, flow cytometry analysis was performed to evaluate ROS production of HCT116/E545K control and APOA5 silencing cells. (F) Caspase 3 activity was measured by pNA concentrations in HCT116/E545K control and APOA5 overexpressing cells, which was treated with L-OHP (10 μM) and LY294002 or NAC. (G) Flow cytometry was performed to determine cell apoptosis of HCT116/E545K control and APOA5 silencing cells, which was treated with L-OHP (2 μM) and LY294002 or NAC for 48 h. Results are representative of at least three independent experiments. p-values < 0.05 are represented as a^*.