Fig. 4: Assessment of ROS and mitochondrial dysfunctions as well as of the antioxidant response in osteosarcoma cells upon exposure to G4Ls.
A Representative photomicrographs showing the detection of ROS upon H2DCFDA staining of living U-2 OS and Saos-2 cells both untreated (UNT) and after a 1-day exposure to NMe2 or QN-302. Hydrogen peroxide (H2O2) in the absence or presence of N-acetylcysteine (NAC) was used as positive and negative controls for ROS detection, respectively. Nuclei were counterstained with Hoechst 3342. Scale bars: 100 μm; magnification: × 10; B Representative photomicrographs showing mitochondrial morphological alterations in NMe2-treated vs. untreated U-2 OS and Saos-2 cells probed with an anti-COX IV antibody (green). Nuclei were counterstained with DAPI. Merged images are shown; scale bar: 10 μm; magnification: × 60; C Representative western immunoblotting showing p21Waf1 protein amounts in untreated U-2 OS cells and after a 2-day exposure to either G4L (IC50). β-tubulin was used to ensure equal protein loading. Cropped images of selected proteins are shown. The graph on the bottom reports the quantification of p21Waf1 protein amounts in the indicated samples. Data have been reported as relative protein amounts with respect to β-tubulin and represents mean values ± s.d. (N = 3). ***p < 0.001; ****p < 0.0001 (two tailed unpaired t-test); D Quantification of the total antioxidant capacity (TAC) in untreated (UNT) U-2 OS and Saos-2 cells and after a 2-day exposure to equitoxic (IC50) amounts of NMe2 or QN-302. Data have been reported as the total antioxidant capacity, expressed as μM copper-reducing equivalents (μM CRE), according to the manufacturer’s instructions. Bars represent mean values ± s.d. (N = 4); *p < 0.05 (two-tailed Mann-Whitney test); E Quantification of the Nrf2 binding activity to ARE sequences in untreated U-2 OS cells and after a 2-day exposure to either G4L (IC50). Data have been reported as OD read at 450 nm in tested samples in the presence of wild-type (wt) or mutated (mut) ARE-containing consensus sequence used to test for binding competition. C + : internal positive control for Nrf2 binding activity provided with the kit. Bars represent mean values ± s.d. (N = 4); *p < 0.05 (two-tailed Mann–Whitney test); F Assessment of NQO1 mRNA levels in untreated U-2 OS and Saos-2 cells and after a 2-day exposure to either G4L (IC50). RT-qPCR data were reported as 2−ΔCt and represents mean values ± s.d. (N = 3). **p < 0.01; ***p < 0.001; ****p < 0.0001 (two tailed unpaired t-test). GAPDH was used as normalizer; G Representative western immunoblotting showing the amounts of the Nrf2 downstream target NQO1 in untreated U-2 OS and Saos-2 cells and after a 2-day exposure to either G4L (IC50). β-tubulin was used to ensure equal protein loading. Cropped images of selected proteins are shown. The graph at the bottom reports the quantification of NQO1 protein amounts in the indicated samples. Data have been reported as relative protein amounts with respect to β-tubulin and represents mean values ± s.d. (N = 3). *p < 0.05; **p < 0.01 (two tailed unpaired t-test); H Assessment of cell growth kinetics in siCTR (•)- and siNrf2 (▲)-transfected U-2 OS cells either untreated (blue) or after a 2-h exposure (pulse) to subtoxic amounts of NMe2 (green) or QN-302 (red). Data have been reported as the percentage of phase image confluency (determined by Incucyte® SX5 Live-Cell Imaging and Analysis System) normalized to the first time point (T0) using the normalization function in GraphPad and represent mean values ± s.d. (N = 4); ***p < 0.001 (2-way ANOVA).
