Figure 2.

Levels of telomeric G4s and R-loops are higher in ALT+ cells and are broadly associated with telomere length and coupled with DNA damage signals. (A andB) Top; (A) G4-ChIP assay or (B) R-loop-ChIP assay in ALT+ cell lines (blue shades) versus TERT+ cell lines (red shades). Data shown are the means from three biological repeat experiments. T-test (two-tailed) was performed for the combined ALT+ versus TERT+ datasets. Bottom; linear regression plot of Tel/Alu ChIP signal ratio against telomere length (as measured by telomere restriction fragment (TRF) analysis). Line of best fit is plotted along with R² values with HELA1.3 (red) or without HELA1.3 (black). (C) ChIP-Re-ChIP (γH2AX-G4) and quantified for telomeric DNA (Tel DNA) in ALT+ cells, GM847 and SKLU1. The ReChIP signal for each sample was then normalized to its respective beads only control. Two-way ANOVA with Sidak multiple correction was performed. Data shown are the means from three biological repeat experiments. Error bars represent the SEM. (D) ChIP-ReChIP (first ChIP for R-loop followed by second ChIP for G4) and quantified for Tel DNA in ALT+ cells, GM847 and SKLU1. ReChIP signal for each sample was then normalized to its respective beads only control. Two-way ANOVA with Sidak multiple correction was performed. Data shown are the means from three biological repeat experiments. Error bars represent the SEM.