Figure 4.

HCA causes an increase in ROS levels. (A) Representative confocal images of MIA PaCa-2 cells cultured for 48 h in the presence or absence of HCA (200 and 225 µM). After then, the cells were incubated with 10 µM dichlorofluorescein diacetate for 30 min at 37 °C. ROS species are shown in green. (B) Ros levels were measured by flow cytometry. MIA PaCa-2 cells were pretreated with ROS scavengers α-tocopherol (100 µM, Vit. E) or Ferrostatin-1 (1 µM) for 1.5 h before exposure to HCA (200 µM) for 48 h. Positive control H₂O₂ (100 µM) (the bars correspond to the mean ± SEM of three independent experiments). Student’s t-test: * p < 0.05 with respect to the controls. HCA: 2-hydroxycervonic acid. DCFH-DA: 2′, 7′-Dichlorofluorescin diacetate.