Supplementary Figure 5: Additional data for Fig. 5.

(a) Mutant p53 co-immunoprecipitates (co-IP) with Nrf2 in MDA-MB-231 and MDA-MB-468 cell lysates but not in MCF-7 cell lysate (anti-Nrf2 antibody). Representative of 2 repeats; (b) Co-immunoprecipitation (co-IP) of Nrf2 (anti-Nrf2 antibody) and overexpressed WT or mutant (R175H and R280K) p53 in p53-null H1299 cells (representative of 2 repeats); (c) GST tagged mutant p53 variants (E.coli overexpressed) interact via DNA binding domain with overexpressed full length Nrf2 in the p53-null H1299 cell lysates. (FL- full length protein; DBD—DNA binding domain; N-term—amino terminal domain; C-term—carboxy terminal domain of p53). Below a ponceau-red stained membrane is shown with transferred GST-fusion constructs (representative of 3 repeats) and a scheme of the N-terminally GST-tagged p53 constructs used for the experiment; (d) The increased expression of PSMA2 and PSMC1 proteasome genes is blunted by silencing of TP53 or NRF2 in the presence of the overexpressed mutant p53 variants (R175H and R280K) in p53-null H1299 cells. The effect is absent in the WT p53 overexpressing cells (means of two independent experiments). Below—a western blot showing p53 and Nrf2 levels in H1299 on indicated silencing (representative of 2 repeats); (e) Nrf2 and p53 are present in the nuclei of MDA-MB-231 cells with or without oxidative stress. Cells optionally treated with Nrf2-targeting siRNA and/or for 6h with 50 μM of oxidative stress-inducing sodium arsenite (NaAsO₂). Representative of 3 repeats; (f) Mutant p53 co-immunoprecipitates with Nrf2 in the nuclear fraction of MDA-MB-231 (representative of 3 repeats); (g) Mutant p53 regulates transcription of Nrf2-dependent oxidative stress induced gene HO-1 in the opposite manner to the proteasome genes. Means of two independent experiments. (h) Nrf2 and p53 co-localize in the nuclei of MDA-MB-231 with or without oxidative stress. Cells optionally treated for 6h with 50 μM of oxidative stress-inducing sodium arsenite (NaAsO₂). Representative of 3 repeats); (i) Nrf2 and p53 co-localize in the nuclei of MCF10A control cells (WT p53) and MCF10A cells with silenced endogenous WT TP53 (sh p53) plus overexpressed mutant p53 variants (+p53 R280K. +p53 R175H). Representative of 3 repeats. Unprocessed original scans of blots are shown in Supplementary Fig. 9. Statistics source data for 5d, g provided in Supplementary Table 10.