Fig. 1: Mutant p53 induces miR-30d expression through HIF1α.

a miR-30d expression was evaluated by RT-qPCR, normalized to U6B RNA expression levels, in MDA-MB-231 cells upon silencing endogenous mut-p53 with a siRNA targeting the 3′UTR (sip53u); expression of mut-p53 R280K was rescued by transfecting a siRNA-resistant mut-p53 HA-R280K construct. Bottom: western blot analysis of p53 expression using HSP90 as loading control. b Expression levels of miR-30d were analyzed as in (a) upon silencing of mut-p53 in the indicated human breast cancer cell lines. c Endogenous wt-p53 was stably silenced in MCF10A mammary epithelial cells (shp53), and shRNA-resistant forms of the indicated p53 mutants were expressed by viral transduction where indicated. miR-30d expression was then evaluated as in (a). d mut-p53 was silenced in MDA-MB-231 cells as in (a); expression of pri-miR-30d and pre-miR-30d was then evaluated by RT-qPCR, normalized, respectively, to the expression of H3 and U6B RNA. e Expression of miR-30d was evaluated by RT-qPCR as in (a), upon silencing of either mut-p53 or HIF1α in MDA-MB-231 cells cultured either in normoxic (20% pO₂) or hypoxic conditions (2% pO₂) for 16 h. Right: western blot analysis of HIF1α and mut-p53 levels, using actin as a loading control. f Proximity ligation assay (PLA) with primary antibodies against p53 and HIF1α were performed in MDA-MB-231 cells cultured either under normoxic (20% pO₂) or hypoxic conditions (2% pO₂) for 16 h, or exposed for 16 h to 150 μM CoCl₂ as hypoxia-mimetic treatment. g–i Lysates of MDA-MB-231 cells cultured under the indicated conditions for 16 h upon silencing of either mut-p53 or HIF1α were subjected to chromatin immunoprecipitation (ChIP) analysis with anti-p53 FL-393 (g), HIF1α (h), and Acetyl-Histone H3 (Lys9) antibodies (i), or Protein A/G PLUS-Agarose as negative control. Binding to MIR30D promoter region was calculated as fraction of input chromatin bound. Binding to non-specific chromatin is shown in Supplementary Fig. 1o-p-q. Graphs represent the individual data points, the mean +/− SEM of three independent experiments. Blots are representative of n = 3 biological replicates. P value (*p < 0.05, **p < 0.01, ***p < 0.001) was calculated by two-tailed unpaired Student’s t-test.