Figure 5

Apoptosis effectors genes. (A) p53 siRNA transfected and non- transfected cells were treated with MTA (0.84 μM) for 48 h before RNA extraction. Later, the expression of p53, and of the p53-related genes PUMA (BBC3), PIDD (LRDD) and Mcl-1 were analysed by RT-PCR. Results are represented in a heatmap showing the fold change of expression in three human melanoma cell lines, where upregulation is shown in red and downregulation in blue as shown on the rule on the right. We found that MTA induced the upregulation of PUMA, PIDD and Mcl-1 mRNA in all cell lines. However, when silencing p53 by siRNA, p53 was downregulated and there was a decreased upregulation of PIDD and Mcl-1 expression comparing with non-p53 silenced cells, pointing to a role of p53 in their regulation. PUMA expression did not change when p53 was silenced comparing with its expression in non-silenced cells, pointing to a p53-independent regulation. (B) Immunoblotting of cell lysates from cells treated with 0.84 μM MTA for the indicated time periods using antibodies specific for the indicated proteins. No changes in PUMA protein levels were observed and Mcl-1 protein appeared to fall after MTA treatment, suggesting that MTA may also exert its effect via postranscriptional modifications. (C) Immunoblotting of cell lysates from A375 cells with or without transiently silenced p53 by siRNA treated with 0.84 μM MTA for 48 h. On top, MTA induced the increment of p53 protein and around a 60% p53 reduction was silenced by siRNA assay. Middle, MTA induced a p53-dependent downregulation of Mcl-1 protein. Under each immunoblot ratios are shown, which the result of the normalisation of the raw volume of each sample with the corresponding actin’s value and the subsequent relativisation to the control. The blots were stripped and reprobed with actin which served as loading control.