Fig. 1: The exposure of ALT-positive osteosarcoma cells to G4Ls elicited DNA damage associated to telomere dysfunctions.

A Representative western immunoblotting showing the amount of proteins involved in the signalling of DNA damage assessed in untreated U-2 OS and Saos-2 cells and after a 2-day exposure to an equitoxic amount (IC₅₀) of NMe2 (green) or QN-302 (red). Vinculin was used to ensure equal protein loading. Cropped images of selected proteins are shown. The graphs show the quantification of the relative protein abundance in treated vs. untreated cells upon normalization to Vinculin. Data have been reported as mean values ± s.d. (N = 3); *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed unpaired t-test); B Representative photomicrographs showing the immunofluorescence analysis of γ-H2AX (green) and TRF1 (red) in untreated (UNT) U-2 OS and Saos-2 cells and after a 2-day exposure to an equitoxic amount (IC₅₀) of each G4L. Nuclei were counterstained with DAPI (blue). Arrows indicate the fluorescence foci (yellow) arising from the co-localization between the two proteins. Scale bar: 10 μm; magnification: × 60; C Quantification of DNA damage foci in untreated (UNT) and NMe2- or QN-302-treated U-2 OS and Saos-2 cells. Data have been reported as percentage of nuclei that stained positive for γ-H2AX (N = 5); *p < 0.05; **p < 0.01 (two-tailed Mann Whitney test); D Quantification of telomere dysfunction induced foci (TIF) in U-2 OS and Saos-2 cells after a 2-day exposure to an equitoxic amount (IC₅₀) of NMe2 or QN-302. Cells with one or more γ-H2AX co-localized with TRF1 (yellow dots in B) were scored as TIF-positive cells. Data have been reported as the percentage of TIF-positive nuclei within the overall population of γ-H2AX–positive nuclei in treated and untreated (UNT) cells. Bars represent mean values ± s.d. (N = 5); *p < 0.05 (two-tailed Mann–Whitney test) (E) Representative photomicrographs showing anaphase bridges (AB) and micronuclei (MN, white arrows) in NMe2- and QN-302-treated compared to untreated U-2 OS and Saos-2 cells stained with DAPI. Scale bar: 10 μm; magnification: × 40. The graphs on the right reports the percentage of anaphase bridges and of micronuclei within the overall cell population in untreated (UNT) and NMe2- or QN-302 treated U-2 OS and Saos-2 cells. Data represent mean values ± s.d. (N = 3); **p < 0.01: ***p < 0.001; ****p < 0.001 (two-tailed unpaired t-test).