Figure 3.

ATR repression by POT1b is limited by its interaction with CST. A, schematic of the role of POT1b in recruiting CST and polymerase α/primase to telomeres (top). Schematic of POT1b domains and the mutations that disrupt the POT1b-CST interaction (bottom). B, immunoblot for POT1b in POT1a^F/F MEFs expressing the indicated proteins before and after treatment with Cre (96 h). ctrl, nonspecific band used as loading control. C, representative IF-FISH images showing the DNA damage response at telomeres in Cre-treated POT1a^F/F MEFs expressing POT1a, POT1b, or POT1bΔCST (96 h). γH2AX foci that co-localize with telomeres (TIFs) were detected using α-γH2AX antibody (red) and a telomeric FISH probe (green). D, quantification of the TIF response as shown in C of POT1a^F/F MEFs before and after Cre treatment. Bar graphs represent averages from three experiments and S.D.s. p values were derived from a two-tailed Student's t test. p values: **, p ≤ 0.01; *, p ≤ 0.05. E, immunoblot for POT1a, POT1b, and Stn1 in POT1a^F/F MEFs (with and without 96 h Cre treatment) expressing exogenous POT1b and/or Stn1 shRNA (indicated with +) or a scrambled sh (indicated with −). Asterisks indicate nonspecific bands. ctrl, nonspecific band used as loading control. F, representative images of IF-FISH to monitor the DNA damage response at telomeres in the indicated Cre-treated (96 h) POT1a^F/F MEFs with and without POT1b and expressing Stn1sh or a scrambled control (ctrl sh). γH2AX foci that co-localize with telomeres (TIFs) were detected with IF with an α-γH2AX antibody (red) and FISH with a telomeric PNA probe (green). G, quantification of TIF response in the indicated POT1a^F/F MEFs (as in F). Bar graphs represent averages from three independent experiments and S.D.s. p values: **, p < 0.001; *, p < 0.05; ns, not significant.