Fig. 2: KSHV⁺ cell lines exhibit hallmarks of telomere maintenance by ALT.

a Representative image of metaphases from KSHV-infected cells. Small panels show chromosomes with normal signal pattern (left column) and T-SCE (right), respectively. X symbol within image denotes T-SCE events on the metaphase depicted. b Blinded quantification of T-SCE/metaphase. Experiment was performed in biological triplicate (a). Statistical significance was tested with unpaired, two-tailed t-test (all ****p < 0.0001). c Representative C-circle telomere slot blot. Circular telomeric DNA was analysed upon amplification with Φ29 ( + ) as well as in its absence (−) as background control. Slot blot was probed for telomeric DNA (upper panel) and Alu probe as loading control (bottom panel). d Quantification of signal obtained from biological quadruplicate. Signal quantification was carried out by subtraction of Φ29-negative controls. Dotted line represents threshold, which determined positive samples in line with published reports (>5). Error bars represent SEM. Significance tested with two-tailed Mann–Whitney test (EA.hy926 ^nsp = 0.1714, SLK *p = 0.0006). e Representative interphase telomere FISH. Telomere clustering is marked by white arrows in image. f Quantification of mean telomere signal intensity. Data from biological triplicate and statistical significance determined as in b. g Count of telomere FISH foci used for intensity measurement (f). Significance inferred in a manner equivalent to b, f. h Analysis of relative telomere length by metaphase qFISH. AU denotes relative telomere signal intensity, see associated Methods section. Experiment was performed in biological triplicate for each cell line, whiskers represent 1–99 percentile and box is median with two adjacent quartiles. Statistical significance was tested with Student’s t test (all ****p < 0.0001). i Representative TRAP products. SYBR Gold stained gel image shown for each cell line, representative of biological triplicate, along with control conditions (HeLa, telomerase positive cell line, ΔHeLa heat inactivated lysate). j Expression of hTERT. RT-qPCR measurement is plotted as relative mean, normalised to uninfected control cell lines, respectively. Data obtained from three independent biological replicate cell lines, performed in technical triplicate. Statistical significance was tested with unpaired, two-tailed Student’s t test (EA.hy926 *p = 0.0351, SLK *p = 0.0442). Error bars represent SEM.