Fig. 3: EXD2-deficient cells exhibit hyperactivation of ALT-associated phenotypes and poor telomere maintenance. | Nature Communications

Fig. 3: EXD2-deficient cells exhibit hyperactivation of ALT-associated phenotypes and poor telomere maintenance.

From: Pathway choice in the alternative telomere lengthening in neoplasia is dictated by replication fork processing mediated by EXD2’s nuclease activity

Fig. 3

a Quantification of extrachromosomal telomeric C-circles by rolling circle PCR amplification and radio-labelled slot blotting using a probe complimentary to the telomeric repeat sequences in U2OS control and EXD2−/− cells. HeLa samples act as a non-ALT control. Samples processed without Phi129 polymerase act as a polymerase-negative control (n = 5 biologically independent samples examined over 3 independent experiments, statistical analysis by two-sided student’s t-test, bars represent +/− SEM). b Analysis of C-circle abundance as in (a) in a panel of ALT-reliant cell lines in the presence or absence of control siRNA or siRNA targeting EXD2 as indicated. VA-13+hTel cells act as a telomerase-revertant control (n = 2 independent experiments, statistical analysis by two-sided student’s t-test, bars represent +/− SDM). c Western blotting depicting EXD2 expression in a panel of ALT-reliant cell lines. HeLa and VA-13+hTel cell lines act as a non-ALT and Telomerase-revertant control, respectively. α-Tubulin acts as a loading control. Verification of expression was carried out three times independently. d Quantification of the incidence of ALT-associated PML bodies in EXD2−/− U2OS compared to parental control cells as assayed by immunofluorescence staining using antibodies against TRF2 (red) and PML (green), DAPI (blue) acts as a nuclear stain (n = 316, 330 and 307 cells examined over 3 independent experiments, statistical significance confirmed by two-sided Mann–Whitney analysis, bars represent +/− SEM, scale bar = 10 μm). e Quantification of telomeric sister chromatid exchanges (T-SCEs) per chromosome per cell in U2OS WT and EXD2−/− cells (n = 75 metaphase spreads from 3 independent experiments, statistical significance confirmed by two-sided Mann–Whitney analysis). f Quantitative FISH staining using strand-specific SpO and FITC-labelled PNA FISH probes specific to telomeric repeat sequences showing average telomere length (n = 4187 measurements per condition examined over 2 independent experiments, statistical significance confirmed by two-sided Mann–Whitney test, error bars represent +/−SEM). g Quantification of telomere length in U2OS WT and EXD2−/− cells by TeloSizer® analysis employing DNA combing and immuno-FISH staining to measure discreet telomere lengths (n = 433, 493 and 577 measurements examined over 3 independent experiments, statistical significance confirmed by two-sided Mann–Whitney analysis, bars represent +/− SEM). Source data are provided as a source data file.

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