Figure 1. Effects of Cd on protein level of p53 and expression level of the genes related on p53 stability in HK-2 cells.

(a) Cell viability of HK-2 cells after treatment with Cd for 6 or 24 h using the Alamar Blue assay. HK-2 cells were grown in 96-well plates at a density of 2.0 × 10⁴ cells/cm² and cultured for 48 h. Culture medium was discarded and the cells were treated with Cd (CdCl₂) in serum-free culture medium for 6 or 24 h. Values are the mean ± S.D. (n = 3). (b) Western blot analysis of p53 and MDM2 in HK-2 cells after treatment with Cd at 20 or 40 μM for 6 h. Anti-GAPDH antibody was used as a loading control. Upper panel, p53; middle panel, MDM2; lower panel, GAPDH. The three blots were run under the same experimental conditions. Uncropped images are provided in Supplementary Fig. 1a. (c) Western blot analysis of p53 and MDM2 in HK-2 cells after treatment with Cd at 40 μM for 0, 1, 3 and 6 h. Anti-GAPDH antibody was used as a loading control. Upper panel, p53; middle panel, MDM2; lower panel, GAPDH. The three blots were run under the same experimental conditions. Uncropped images are provided in Supplementary Fig. 1b. Real-time RT-PCR of UBE2D1 (d), UBE2D2 (e), UBE2D3 (f), UBE2D4 (g), p53 (h), MDM2 (i), OTUB1 (j) and USP7 (k) gene expression. HK-2 cells were grown in six-well plates at a density of 2.0 × 10⁴ cells/cm² and cultured for 48 h. Culture medium was discarded and the cells were treated with Cd in serum-free culture medium for the indicated time. mRNA levels were normalized to GAPDH. Values are the mean ± S.D. (n = 3). *Significantly different from the corresponding control group, P < 0.05.