Fig. 4

RuCUR compound induces NRF2 pathway in mutp53-carrying cancer cells. (a) Western blot analysis of the indicated protein was performed in T98 and SKBR3 cells untreated or treated with RuCUR (100 μM) for 24 h. Actin was used as protein loading control. Densitometry was performed using ImageJ software. Relative band intensities value were normalized to β-actin (loading control) and finally quantified with respect to untreated control arbitrarily set to 1.0. (b) Total mRNA was extracted from T98 and SKBR3 cells untreated or treated with RuCUR (100 μM) for 24 h. The indicated gene expression was assayed by semi-quantitative 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). (c) Oxidant species production in T98 and SKBR3 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) are the mean ± SD of three independent experiments. * (p ≤ 0.01). (d) Western blot analysis of the indicated protein levels was performed in T98 and SKBR3 cells untreated or treated with geldanamycin (gelda) (100 nM) for 24 h. The ratio of p53 and HO-1 levels vs β-actin, following densitometric analysis using ImageJ software, is shown. Representative images are shown