Fig. 5.

Role of Smad3 in ALK4/5-mediated HK-2 cell death induced by cadmium. a–e Cells transfected with control siRNA, ALK4 siRNA-1, ALK4 siRNA-2 a, b, d, Smad3 siRNA-1, or Smad3 siRNA-2 c, e were incubated with or without 25 μm CdCl₂ (Cd) for 15 h a–c or 24 h d, e. Total RNAs were subjected to quantitative RT-PCR to determine ALK4 mRNA levels a. Cell lysates were subjected to western blotting using the indicated antibodies b, c. The viability of cells was determined by trypan blue exclusion assay d, e. f Cells were incubated with 0.1% DMSO, or 5 μm SIS3 for 1 h and then incubated with or without 25 μm CdCl₂ (Cd) for 30 h. The viability of cells was determined by trypan blue exclusion assay. g, h Cells were incubated with 25 μm CdCl₂ (Cd) for the indicated time. Total RNAs were subjected to quantitative RT-PCR to determine Inhibin-bA mRNA levels g. Cell lysates were subjected to western blotting using the indicated antibodies h. i Cells were incubated with or without 25 μm CdCl₂ (Cd) for 12 h. Concentration of Activin A per 1.0 × 10⁵ cells in the media was measured and reflects the mean ± SD of six experiments. j, k Cells transfected with control siRNA, Inhibin-bA siRNA-1, or Inhibin-bA siRNA-2 were incubated with or without 25 μm CdCl₂ (Cd) for 15 h j or 24 h k. Cell lysates were subjected to western blotting using the indicated antibodies j. The viability of cells was determined by trypan blue exclusion assay k. Data of quantitative RT-PCR was normalized to β-actin expression and reflects the mean ± SD of at least three experiments. 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 < 0.05, **P < 0.01, significant difference between the samples