Figure 7.
Carboplatin-induced ROS is associated with higher induction of DNA damage and apoptotic cell death in CPT1A-KO versus CPT1A WT cells
(A) The basal level of ROS (detected with 2,7’–dichlorofluorescin diacetate [DCFDA] dye) was compared between PEO4 CPT1A-KO cells and the parental PEO4 cell line (n = 3; ∗p < 0.05). Relative changes in ROS production for both PEO4 WT and PEO4 CPT1A-KO cells upon carboplatin exposure were plotted for both 24 h and 48 h.
(B) Representative western blot showing the baseline and effects of carboplatin exposure on NRF2 and γH2AX expression (48 h). Quantification of NRF2 and γH2AX proteins was done using NIH ImageJ software and plotted as line graphs. Data are expressed as mean ± SEM (n = 3; ∗p < 0.05; ∗∗p < 0.001).
(C) RNA expression levels of NQO1, PRDX1, ME1, and PIR at 0 h and 8 h after carboplatin treatment were compared for PEO1S and PEO4R cells.
(D) Detection of carboplatin-induced apoptosis via Annexin V staining in PEO4-WT and PEO4-KO cells. Cells were treated with different concentrations of carboplatin for 48 h and incubated with AV-fluorescein isothiocyanate (FITC) and phosphatidylinositol (PI). Stained cells were analyzed by flow cytometry. Percentage of intact cells (AV−/PI−) and different stage apoptotic cells (AV+/PI−, AV+/PI+, and AV−/PI+) are presented. Data represent mean ± SEM (n = 3; ∗p < 0.001).
(E) Western blot showing the effects of carboplatin treatment (160 μM; 48 h) in PEO4-WT versus PEO4-KO on caspase-3 cleavage as an indicator of cell death via apoptosis. Data are expressed as mean ± SEM (n = 3; ∗p < 0.05; ∗∗p < 0.001).