Journal: Cell Death & Disease

Article Title: Cellular adaptations impact the biological activity of naphthalene diimide G-quadruplex ligands in ALT-positive osteosarcoma cells

doi: 10.1038/s41419-025-07908-2

Figure Lengend Snippet: A Representative western immunoblotting showing the amount of the indicated proteins in U-2 OS ( top panel ) and Saos-2 ( bottom panel ) cells either untreated (UNT) and after a 2-day exposure to an equitoxic amount (IC 50 ) of NMe2 or QN-302. Vinculin or β-tubulin were used to ensure equal protein loading. Cropped images of selected proteins are shown. The graphs on the side of each panel report the quantification of the protein amounts in treated vs . untreated cells following normalization with respect to vinculin or β-tubulin and represents mean values ± s.d. (N = 3); * p < 0.05; ** p < 0.01; **** p < 0.0001 (two-tailed unpaired t -test); B Representative Western immunoblotting showing BLM protein amounts in the cytoplasmic (c) and nuclear (n) fractions of untreated U-2 OS cells and after a 2-day exposure to NMe2 (IC 50 ). Lamin B1 and vinculin were used to ensure equal protein loading and proper nucleus/cytoplasm fractionation. Cropped images of selected proteins are shown. The graph on the bottom reports the quantification of the protein amounts. Data have been reported as relative protein amounts with respect to vinculin (cytoplasmic fraction) or lamin B1 (nuclear fraction) and represents mean values ± s.d. ( N = 3); ** p < 0.01 (two tailed unpaired t -test); C Representative photomicrographs showing the co-localization between BLM (green) and TRF1 (red) assessed by immunofluorescence in untreated U-2 OS cells and after a 2-day exposure to NMe2 (IC 50 ). Nuclei were counterstained with DAPI. Arrows indicate the fluorescence foci (yellow) arising from the co-localization between the two proteins. Merged images are shown; scale bars: 10 μm; magnification: × 60; D Representative western immunoblotting showing the amount of BLM protein in non-transfected (UNT), siCTR- and siBLM-transfected as well as DMSO-exposed and ML216-treated U-2 OS cells. β-actin was used to ensure equal protein loading. Cropped images of selected proteins are shown. The graphs on the bottom reports the quantification of the protein amounts. Data have been reported as relative protein amounts with respect to β-actin and represents mean values ± s.d. ( N = 3); **** p < 0.0001 (two tailed unpaired t -test); n.s.: not statistically significant. E Quantification of telomeric C-circle DNA levels in untreated (UNT), siCTR- and siBLM-transfected U-2 OS cells and after a 2-day exposure to 30 μM ML216. Data have been reported as relative CCA score in the indicated samples with respect to UNT cells and represent mean values ± s.d. ( N = 4); * p < 0.05 (two-tailed Mann–Whitney test); F Assessment of cell growth kinetics in siCTR (•)- and siBLM (▲)-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 (T 0 ) using the normalization function in GraphPad. Data represent mean values ± s.d. ( N = 4); * p < 0.05; ** p < 0.01; *** p < 0.001 (2-way ANOVA). The panel on the right reports a representative western immunoblotting showing BLM protein amounts in siCTR- and siBLM-transfected U-2- OS cells at the indicated time points. The graphs report the quantification of BLM protein amounts. Data have been reported as relative protein amounts with respect to β-actin and represents mean values ± s.d. ( N = 3). G Quantification of cell growth inhibition in U-2 OS cells exposed for 2 days to NMe2 (0.25 μM) and ML216 (30 μM) alone or in combination. Data have been reported as the extent of cell growth inhibition (fraction affected) and represent mean values ± s.d. ( N = 4); * p < 0.05 (two-tailed Mann–Whitney test); H Synergistic pharmacological interaction observed in U-2 OS cells after a 2-day exposure to the combination NMe2:ML216 (1:120) as indicated by the values of the combination index (C.I.) as a function of the fraction affected (Fa) calculated as previously reported .

Article Snippet: Target-specific siRNAs designed to silence the expression of TP53 (sip53; sc-29435); CDKN1A (sip21; sc-44214), BLM (siBLM; sc-29808) and NFE2L2 (siNrf2; sc-44332) as well as a control siRNA (siCTR; sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX). siRNAs were dissolved in RNase-free water to make a 10 μM stock solution, stored at −20 °C and diluted to obtain a final concentration of 50 nM immediately before use.

Techniques: Western Blot, Two Tailed Test, Fractionation, Immunofluorescence, Fluorescence, Transfection, MANN-WHITNEY, Live Cell Imaging, Inhibition