Fig. 3.

Curcumin-generated ROS production is involved in autophagy induction. (A) HCT116 cells were treated as in Fig. 1 and stained with DCF-DA to detect intracellular ROS. ROS generation was determined using flow cytometry. The data shown are representative of three independent experiments. (B) GFP-LC3 expressing HCT116 cells were treated with indicated concentrations of hydrogen peroxide for 20 h. The level of GFP-LC3, endogenous LC3-II, and SQSTM1 protein was determined by Western blot analysis and GAPDH was used as a loading control. (C) Cells were exposed to 20µM curcumin with or without pretreatment of 10 mM NAC and stained with DCF-DA. Intracellular ROS level was determine using flow cytometry. The data shown are representative of three independent experiments. (D) HCT116 cells were treated with indicated concentrations of NAC for 2 h and subsequently exposed to a range of curcumin for 20 h. Western blot analysis was conducted to detect GFP-LC3, endogenous LC3, and SQSTM1 protein. GAPDH was used as a loading control. (E) LC3 mRNA expression was determined with total RNAs isolated from cells after treatment and normalized to the level of GAPDH. Results are from three independent experiments. The data shown are representative of three independent experiments.