Research Paper Volume 16, Issue 11 pp 9410—9436

Figure 3. PIK3CA-E545K induced L-OHP resistance is mediated by the modulation of APOA5. (A) Western blot analysis was performed to evaluate the expression levels of APOA5 in PIK3CA-E545K infected HCT116 and SW480 cells. (B) After treatment with inhibitor LY294002, Western blot analysis was performed to evaluate the expression of APOA5 in PIK3CA-E545K infected HCT116 cells. (C) Ectopic APOA5 expression was conformed in HCT116 and SW480 cells with Western blot assays. (D) The IC₅₀ of control and HCT116/APOA5 cells were analyzed with cell viability with gradient L-OHP treatment. The IC₅₀ were 11.20 μM and 1.08 μM, respectively. (E) Flow cytometry was performed to determine cell apoptosis of APOA5 overexpressed HCT116 and SW480 cells which were treated with L-OHP (1 μM). (F) Caspase 3 activity was measured by pNA concentrations in APOA5 overexpressing cells with L-OHP (2 μM) treatment. (G) Western blotting was performed to detect caspase 3, cleaved caspase-3 (C-Caspase 3), full-length PARP and cleaved PARP (C-PARP) in APOA5 overexpressing cells after L-OHP treatment. β-actin was used as a loading control. (H) Cell cycle distribution in APOA5 overexpressed HCT116 and SW480 cells with L-OHP treatment (2 μM for 48 h). The proportion of cells in cell cycle phases was measured by flow cytometry and quantified by FlowJo software. Results are representative of at least three independent experiments. p-values < 0.05 are represented as a^*.