Figure 4.

Cross-talk between the Notch1 and PI3K/Akt signaling pathways in HK-2 cells exposed to CdCl₂. (a) Cells were incubated with 20 μM CdCl₂ (Cd) for the indicated time. The untreated control is labeled 0 h. Cell lysates were subjected to western blotting using antibodies against phospho-EGFR, total EGFR, phospho-Akt, total Akt, phospho-GSK-3α, phospho-GSK-3β, total GSK-3α/β, phospho-S6K, and actin. (b–e) Cells were incubated with 0.1% DMSO, 25 μM LY294002 (b and d), or 10 μM MK2206 (c and e) for 1 h and then incubated with or without 20 μM CdCl₂ (Cd) for 24 h (b and c) or 30 h (d and e). Phase-contrast micrographs were taken (b and c). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. **P<0.01 versus CdCl₂-treated cells incubated with DMSO (d and e). (f) Cells transfected with control siRNA or Notch1 siRNA-1 were incubated with or without 20 μM CdCl₂ (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against phospho-EGFR, total EGFR, phospho-Akt, total Akt, phospho-S6K, Notch1-NICD, and actin. Data are from the same experiment shown in Figure 3f. (g) Cells were incubated with 0.1% DMSO, 2.5 μM AG1478, 5 μM PPP, or 25 μM LY294002 for 1 h and then incubated with or without 20 μM CdCl₂ (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, phospho-Akt, total Akt, and actin. Immunoblots shown are representative of at least three independent experiments