Fig. 6

RuCUR compound induces NRF2 pathway in wtp53-carrying cancer cells. (a) Oxidant species production in RKO and HCT116 cells after RuCUR (100 μM) treatment for 16 h evaluated by 2′,7′-dichlorofluorescein diacetate (DC-FDA) staining and assessed by Fluorescence-Activated Cell Sorting (FACS) analysis. Histograms of the mean fluorescence intensity (MFI) represent the mean ± SD. * (p ≤ 0.01). (b) Western blot analysis of the indicated protein levels was performed in (left panel) RKO and HCT116 cells untreated or treated with RuCUR (100 μM) for 24 h and in MCF7 and U87 cells (right panel) 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 the protein levels vs β-actin, following densitometric analysis using ImageJ software, is reported (c) Western blot analysis of the indicated protein levels in RKO and HCT116 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. (d) Total mRNA was extracted from RKO and HCT116 cells pre-treated with brusatol (100 nM for 4 h) prior to adding RuCUR (100 μM) for 24 h. The indicated 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). (e) Cell viability was measured by trypan blue exclusion assay in RKO and HCT116 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)