Fig. 3

RuCUR compound reduces proliferation and induces cell death in wild-type (wt) p53-carrying cancer cells. (a) Colon cancer HCT116 cell, were seeded on ultra-low attachment multiwell plates allowing for tumor spheroid formation. Four days after seeding, spheroids were formed (approximate size 300–500 μm) and then treated with different doses of RuCUR (10, 50, 100 μM) for the indicated times. Tumor spheroids volume was quantified according to the formula: V = a x (b2)/2, where a and b are, respectively, length and width. Representative images of spheroids are shown in left panel. Spheroid volume are reported on the right panel. Histograms represent the fold increase quantified with respect to controls set to 1.0, ± SD. * (p ≤ 0.01), # (p ≤ 0.05) (single treatments compared to untreated spheroids). (b) Cell viability was measured by trypan blue exclusion assay in RKO and HCT116 cells treated with RuCUR (100 μM) for 24 h and expressed as cell death percentage ± S.D. * (p ≤ 0.01) (single treatments compared to untreated cells). (c) Western blot analysis of the indicated protein was performed in RKO and HCT116 cells untreated or 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. (d) Total mRNA was extracted from RKO and HCT116 cells treated with 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)