Figure 3.

Autophagy protects ovarian CAF1 cells against oxidative stress by inhibiting ROS production. (A) The representative images of CAF1 cells with knockdown of indicated siRNA and treated with H₂O₂. Nc: negative control siRNA. (B) The effect of blockage of autophagy on cell viability and H₂O₂-induced cell death of CAF1 cells. CAF1 cells were reverse transfected with the indicated siRNA for 48 h in 96-well plate in triplicate, treated with or without 1 mM H₂O₂ for 2 h. The cell viability was measured with MTT assay (mean ± SD, n = 3). (C) Cell cycle analysis of CAF1 cells transfected with Nc or Atg5 siRNA for 48 h_. (D) The cell cycle phase distribution in CAF1 cells transfected with Nc or Atg5 siRNA for 48 h (as in C). (E) ROS measurement of CAF1 cells transfected with Nc, Atg5 or Beclin siRNA for 48 h by flow cytometry. (F) NAC decreased ROS production in Beclin knockdown CAF1 cells. Cells were transfected with Nc or Beclin siRNA for 24 h, incubated with or without NAC (5 mg/ml) for 2 h, and further cultured in complete media for another 24 h. Cells were stained with H2DCF (20 μM) and lysed with RIPA buffer. The fluorescence was read with a fluorometer and normalized to protein concentration (mean ± SD, n = 4). (G) NAC restored cell viability of Beclin knockdown CAF1 cells. Cells were prepared the same way as in (F). Cell viability was measured by MTT assay (mean ± SD, n = 4). (H) The effect of blockage of autophagy on cisplatin-induced cell death of CAFs. CAF1 cells were reverse transfected with the indicated siRNA for 24 h in 96-well plate in triplicate, treated with 100 μM cisplatin for another 24 h. The Cell viability was measured with MTT assay (mean ± SD, n = 3).