Fig. 2: PCS^LT cells display a subset of ALT features.

A Fragile telomeres assessed by either FISH (blue) or CO-FISH (black) on metaphase spreads. FISH data are from Fig. 1D. For CO-FISH, fragility was the average fragility from both the leading and lagging strands. U2OS/ALT⁺ and IMRB/ ALT⁺ cells were used as controls for ALT⁺ cells. A minimum of 3800 chromosome ends were analyzed, based on n metaphase spreads collected from three independent biological replicates. Boxplot: Min, 1st quartile, Median, 3rd quartile, and Max. Two-tailed unpaired t-tests with Welch’s correction if needed. B Left: Representative CO-FISH pictures on metaphase spreads from PCS^LT cells showing lagging (green) and leading (red) telomere fragility (arrowheads). Right: Lagging (green) and leading (black) telomere fragility. A minimum of 5000 chromosome ends were analyzed, based on n metaphase spreads collected from three independent biological replicates. Boxplot: Min, 1st quartile, Median, 3rd quartile, and Max. Two-tailed Mann–Whitney tests. C Representative blots (above) and quantification (below) for CCA in siPOLD3-treated PCS^LT cells, using U2OS/ALT⁺ cells as control. Signals were normalized to siLuc. n = 5 (U2OS/ALT⁺) or n = 6 (PCS^LT) independent biological replicates. Mean ± SEM, Two-tailed ratio paired t-tests. D Left: Representative images for Telo-FISH (red)/IF targeting BLM (cyan) and PML (green) in PCS^LT cells, with U2OS/ALT⁺ cells as positive control. Arrowheads indicate PML-associated telomeres bound by BLM. Right: Percentage of PML-associated telomeres co-localizing with BLM per nucleus. n nuclei were analyzed from three independent biological replicates. Median in red, quartiles in orange. Two-tailed Mann–Whitney test. E Above: Representative CO-FISH pictures on metaphase spreads from IMRB/ALT⁺ cells showing T-SCE events (arrowheads). Below: T-SCEs in PCS^ST and PCS^LT, with U2OS/ALT⁺ and IMRB/ALT⁺ as positive controls. n metaphases were analyzed from three independent biological replicates. Median in red, quartiles in orange. Kruskal–Wallis test. F Above: Representative images from native Telo-FISH targeting ssC-rich telomeric DNA (red) in U2OS/ALT⁺ and IMRB/ALT⁺ cells. Below: ssTeloC signals in PCS^ST and PCS^LT, with U2OS/ALT⁺ and IMRB/ALT⁺ cells as positive controls. n nuclei were analyzed from three independent biological replicates. Median in red, quartiles in orange. Kruskal–Wallis test. Source data are provided as a Source Data file.