Fig. 2

Metformin facilitates the pro-apoptotic effects of ATO on ICC cells through increasing the ROS production induced by ATO. a After treatment with 10 mmol/L metformin, 3 μmol/L ATO, or a combination of both, ICC cells were examined using Annexin V/PI staining, and the distribution of apoptotic cells was measured by flow cytometry. The percentages of early apoptotic cells plus late apoptotic/necrotic cells are shown in the bar graph. b Cells were treated with 10 mmol/L metformin, 3 μmol/L ATO, or a combination of both for 24 h. The intracellular ROS were measured by flow cytometric analysis using an oxidation-sensitive fluorescent probe, DCFH-DA, which is oxidized to DCF by ROS (the negative control was not treated with DCFH-DA). The mean volumes of DCF are shown in the bar graph as the means ± SD from three independent experiments. c Caspase3/7 activity in ICC cells treated with 10 mmol/L metformin, 3 μmol/L ATO, or a combination of both. d Cleaved caspase-3 and cleaved PARP were monitored using western blot analysis. (Combination vs metformin or combination vs ATO ^* P < 0.05)