Fig 2: PML-α3 and PML-IE1 colocalization. IFA was performed at 4 h in non-infected, infected, and infected-compound-treated cells. Co-staining was performed with mouse anti-CMV IE1/2 and rabbit anti-PML (A), rabbit anti-PML, and mouse anti-α3 (B). The experiment was repeated thrice, and the best images are shown. Quantification of colocalization of PML and IE1 (A, upper) and PML and α3 (B, lower) is shown using Pearson’s correlation coefficients. Data represent an average of nine measurements with SD. Significance levels were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test (**** p <0.0001, ** p <0.01, ns not significant).

Fig 3: Evaluation of DNA damage, APBs formation, and clonogenic potential following treatment with RIBO‐ISCH‐1. A) Representative flow cytometry histograms showing γH2AX fluorescence in U2OS cells treated with Scr (50 nM), ISCH (100 nM), RIBO‐ISCH‐1 (100 nM), or ASO (50 nM) for 48 h (n = 3). B) Overlay of γH2AX fluorescence intensity histograms comparing all treatment conditions. C) Immunofluorescence images of ALT‐associated PML bodies (APBs) in U2OS cells stained with anti‐PML antibody (green) and DAPI (blue) after treatment with ISCH (100 nM) or RIBO‐ISCH‐1 (100 nM) for 48 h. Scale bar = 10 nm. D) Quantification of APBs number per nucleus in treated U2OS cells. (n = 120 cells for each treatment over three different experiments), statistical comparison was performed by a two‐tailed unpaired t‐test (**** p < 0.0001). E) Representative images of crystal violet–stained U2OS cell colonies after treatment with ISCH (1 µm) or RIBO‐ISCH‐1 (1 µm). F) Quantification of colony numbers over 4 different experiments. Statistical analysis was performed by unpaired t‐test (* p < 0.05).

Fig 4: Alternative lengthening of telomeres-positive glioma cell lines exhibit high levels of DNA replication stress relative to telomerase+ glioma cell lines. (A) Western blot detection of ATRX protein expression in patient-derived glioma cell lines. Cell lines labeled in blue text represent ALT-positive cell lines, while red-labels indicate telomerase-positive cell lines. β-actin is used as a loading control for total protein. Data are representative of 3 independent experiments. (B) Confocal microscopy images of ALT-positive (TB096 and 08-0537) and telomerase-positive (12-0160 and TS603) cell lines stained with a TelC PNA probe, anti-PML antibody, and a CHK1 pSer317 antibody. (C) Flow cytometry data showing the extent of chromatin-bound γH2AX, a marker of DNA DSBs, between ALT-positive (TB096 and 08-0537) and telomerase-positive (12-0160 and 13-0302) cell lines. (D) Quantification of data shown in Figure 1C and analyzed using a one-way ANOVA and Tukey’s multiple comparisons test for differences between geometric means between conditions.