Figure 6. GANT61-induced apoptosis and ROS production is dependent on mitochondria.

(A) LO68 cells were pretreated with 200 nM rotenone (ROT), an inhibitor of mitochondrial complex 1, for 1 h, and further incubated with 20 μM GANT61 for 48 h. The level of intracellular ROS was monitored using CH₂DCFDA and the fluorescence measured by flow cytometry. Data represent the mean ± SEM of three independent experiments. ***, p < 0.001, compared to GANT61-only treated cells. (B) Apoptosis was measured by annexin V/7AAD assay. Data represent the mean ± SEM of three independent experiments. ***, p < 0.001, compared to GANT61-only treated cells. (C) Mitochondria-targeted superoxide dismutase mimetic mitoTEMPO (MT) attenuates GANT61-induced apoptosis. LO68 cells were pretreated with MT (100 μM) for 1 h and further incubated with 20 μM GANT61 for 48 h. Apoptosis was measured by annexin-V/7AAD assay. Data represent the mean ± SEM of three independent experiments. ***, p < 0.001, compared to GANT61-only treated cells. (D) GANT61 induces mitochondrial superoxide production in LO68 cells. Cells were pretreated with NAC (20 mM) for 1 h and further treated with 20 μM GANT61 or vehicle for 48 h before subjected to mitoSOX red flow cytometric analysis. Bar graph represents the increase in the mean fluorescence intensity of mitoSOX red-positive cells measured in each experimental condition. Values are the average of independent measurements (mean ± SEM; n = 3). ***, p < 0.001, compared to GANT61-treated cells.