Fig. 7.

Role of Nrf2 in ALK4/5-mediated HK-2 cell death induced by erastin. a, b Cells were incubated with 0.1% DMSO, 10 μm SB431542, 10 μm SB505124, 5 μm SIS3 a, or 10 µm MK2206 b for 1 h and then incubated with or without 5 μm erastin for 24 h. The viability of cells was determined by trypan blue exclusion assay. c Cells were incubated with 5 μm erastin for the indicated time. Cell lysates were subjected to western blotting using the indicated antibodies. d Cells transfected with control siRNA, Keap1 siRNA, or Nrf2 siRNA were incubated with or without 5 μm erastin for 24 h. The viability of cells was determined by trypan blue exclusion assay. e Cells were incubated with 0.1% DMSO, 10 µm SB431542, 10 µm SB505124, 5 µm SIS3, or 20 µm U0126 for 1 h and then incubated with or without 5 μm erastin for 20 h. Cell lysates were subjected to western blotting using the indicated antibodies. f, g Cells transfected with control siRNA or Nrf2 siRNA were incubated with 0.1% DMSO or 10 µm SB505124 for 1 h and then incubated with or without 5 μm erastin for 20 h f or for 24 h g. Cell lysates were subjected to western blotting using the indicated antibodies f. The viability of cells was determined by trypan blue exclusion assay g. Immunoblots shown are representative of at least three independent experiments. Each value of trypan blue exclusion assay is the percentage of trypan blue-positive cells and reflects the mean ± SD of at least three experiments with duplicate assays in each experiment. **P < 0.01, significant difference between the samples