Fig. 5

Targeting NRF2 pathway increases cell death of mutp53-carrying cancer cells. (a) Cell viability was measured by trypan blue exclusion assay in T98 and SKBR3 cells pre-treated with brusatol (100 nM for 4 h) prior to adding RuCUR (100 μM) for 24 h and expressed as cell death percentage ± S.D. * (p ≤ 0.01) (single treatments compared to untreated cells). (b) Western blot analysis of the indicated protein in T98 cells pre-treated with brusatol (100 nM for 4 h) prior to adding RuCUR (100 μM) for 24 h. Actin was used as protein loading control. The ratio of protein levels vs β-actin, following densitometric analysis using ImageJ software, is reported. (c) Total mRNA was extracted from T98 cells pre-treated with brusatol (100 nM for 4 h) prior to adding RuCUR (100 μM) for 24 h. HO-1 gene expression was assayed by the polymerase chain reaction (PCR) of reverse-transcribed cDNA. Densitometric analysis was performed using ImageJ software to calculate the gene/28S ratio. Histograms represent the fold increase quantified with respect to controls set to 1.0, ± SD. * (p ≤ 0.01). (d) Western blot analysis of the indicated protein in NRF2-silenced and in siRNA-control (ctr) T98G cells, treated with RuCUR (100 μM) for 24 h. Actin was used as protein loading control. The ratio of the protein levels vs β-actin, following densitometric analysis using ImageJ software, is reported