Figure legends
Supp Figure 1. Primary fibroblast WT and p53 KO cell death induced by H2O2 is comparable to MEFs.
Primary fibroblasts WT and p53 KO were treated for 24 hours with H2O2 1 mM, with or without a 16 hours preincubation with PARP inhibitor 4-ANI 2 M with or without the pan-caspase inhibitor qVD-OPH 10 M. Cells were stained with fluorescent conjugates of annexin-V and propidium iodide (PI) and analyzed by FACS. **p<0.01. Values indicate mean values ± SEM. All experiments were performed independently at least three times (N≥3).
Supp Figure 2. H2O2–induced cell death is dose and time-dependent.
(A) WT, DKO and p53 KO MEFs were treated for 24 hours with H2O2 0.5 and 0.75 mM, DKO and p53 KO, but not WT MEF, were resistant to both treatments, in contrast to the observations made at 1 mM. (B) Cell death timecourse in the first 6h after H2O21 mM treatment, with or without 4-ANI 2 M 16h preincubation. Cells were stained with fluorescent conjugates of annexin-V and propidium iodide (PI) and analyzed by FACS.(C) Representative FACS plots shown. Values indicate mean values ± SEM. All experiments were performed independently at least three times (N≥3).
Supp Figure 3. Cell death induced by H2O2 in WT MEF and Bax Bak DKO at 8h cannot be rescued by inhibiting apoptosis or necroptosis.
(A) WT MEF, Bax Bak DKO and p53 KO were treated for 8 hours with H2O2 1 mM, after pretreatment for 16h with the pan-caspase inhibitor qVD-OPH (10 M) or the RIPK1 inhibitor necrostatin-1 (10 M). Cells were stained with fluorescent conjugates of annexin-V and propidium iodide (PI) and analyzed by FACS. Viable cells are annexin-V negative and PI negative, and cell death is expressed as 100%-viable cells. Values indicate mean values ± StDev, at least three independent experiments were performed (N≥3).
Suppl. Figure 4.p53 KO cells are protected against apoptosis.
(A) Representative plots for cell cycle analysis of WT MEF, Bax Bak DKO and p53 KO treated for 24 h with H2O2 1 mM, with or without preincubation with 4-ANI 2 M for 16h. Percentatge of subG1 population is indicated. Experiments were performed at least three times with similar results.
All three cell lines were exposed to paclitaxel 1 M (B) or Etoposide 100 M for 24 hours and cell death was analyzed. p53 KO are resistant to apoptosis induced by these two agents. Cells were stained with fluorescent conjugates of annexin-V and/or propidium iodide (PI) and analyzed by FACS. * p<0.05 **p<0.01 (compared to WT MEF). All experiments were performed independently at least three times (N≥3).
Suppl. Figure 5. p53 KD protects against H2O2-induced cell death.
(A) Representative Western Blot, showing PARP-1 and p53 expression in the five shRNA constructs tested. shRNA4 was selected for its higher efficiency in downregulating p53. No downregulation in PARP-1 expression was observed in the KD. (B) WT MEF, p53 KD and p53 KO were treated for 24 hours with H2O2 1 mM. Cells were stained with fluorescent conjugates of annexin-V and propidium iodide (PI) and analyzed by FACS. Viable cells are annexin-V negative and PI negative, and cell death is expressed as 100%-viable cells. p53 KD and p53 KO are protected against cell death. *p<0.05 **p<0.01 (compared to WT MEFH2O2-treated). Values indicate mean values ± StDev, at least three independent experiments were performed (N≥3).
Suppl. Figure 6. p53 expression and functionality in HCT116 and MCF7.
(A) Western blot analysis p53 was performed in HCT116 WT and p53 KO after daunorubicin 0.22 M treatment for 24 h, to induce p53 expression. p53 was absent in HCT116 p53 KO. (B) Western blot analysis of p53 and p21 after daunorubicin 0.22 M treatment for 24 h. p53 expression was induced equally in MCF7 and MCF7 DD (dominant negative), while p21 expression was reduced in the latter. This indicates p53 loss of function in MCF7 DD because it is not promoting p21 expression. (C) Western blot analysis for PARP-1 overexpression in WT MEF and p53 KO. Actin was used as loading control.
Suppl. Figure 7. Cyclophilin D KO or cyclosporine A treatment cannot rescue cells from H2O2-induced cell death.
(A) WT MEF, Bax Bak DKO, p53 KO and Cyclophilin D KO MEFs were treated for 24 hours with H2O2 1 mM. CypD KO had a minor effect on improving cell viability. Cells were stained with fluorescent conjugates of annexin-V and propidium iodide (PI) and analyzed by FACS. Viable cells are annexin-V negative and PI negative, and cell death is expressed as 100%-viable cells. Values indicate mean values ± SEM. All experiments were performed independently at least three times (N≥3). **p<0.01.
Table 1. shRNA constructs to knockdown p53 in MEF.
List of the different constructs used to KD p53 in MEF. The fourth was proven the most effective to that purpose.