53BP1 deficiency in TRF1Δ/Δ-immortalized MEFs leads to stronger CHK1 activation. (A) Percentage of nuclei exhibiting ≥10 γ-H2AX foci and pannuclear γ-H2AX staining in Cre-infected LT-immortalized MEFs 6 d after selection. Error bars indicate standard deviation. (B) Number of TIF per nuclei determined by immunofluorescence detection of γ-H2AX and RAP1. The number of cells analyzed in each case is indicated. Statistical differences were calculated using the Student’s t test, and p-values are indicated. Red lines correspond to mean values. (C) Representative combined images of γ-H2AX foci, RAP1, and DAPI of the indicated genotypes. Insets represent larger magnification of the squared areas, and TIF are visualized as yellow spots. (D) Western blot detection of DDR factors in Cre-infected LT-immortalized MEFs 4 d after selection of the indicated genotypes. RAD21 and actin were used as loading controls. The black line indicates that intervening lanes have been spliced out. ACT, actin.
