Figure 2.

mSSB1 and mSSB2 protect telomeres. (A) Endogenous mSSB1, Flag-mSSB1 and Flag-mSSB2 colocalized with the telomeric PNA probe (telomere: Cy3-OO-(CCCTAA)₄ (red)) or the telomere binding protein TRF1 on a subset of telomeres. Colocalized foci are displayed in greater detail in magnified images for Flag-mSSB1 and 2. (B) Immunoblot detection of TRF2, p-Chk2, mSSB1, mSSB2 and INTS3 in MEFs of the indicated genotypes untreated or treated with shRNA against TRF2 for 5 days. γ-tubulin is used as a loading control. (C) Examples of chromosome aberrations in metaphase spreads using Cy3-OO-(CCCTAA)₄ (red) and FAM-OO-(TTAGGG)₄ (green) telomere peptide nucleic acid probes. Arrows point to sites of chromosome fusions. Not all fusion sites are indicated. (D) Immunoblot detection of TPP1^ΔRD, TRF2, pChk2, mSSB1, mSSB2 and INTS3 in MEFs of the indicated genotypes untreated or treated with TPP1^ΔRD. γ-tubulin is used as a loading control. (E) Quantification of total telomere fusions in MEFs either untreated or treated with shRNA against TRF2. Two independent cell lines were analyzed, and a minimum of 35 metaphases and 1 500 chromosomes were scored per cell line. Mean values were derived from two independent cell lines. The one-tailed t-test was used to calculate statistical significance. Error bars: s.e.m. (F) Quantification of total telomere fusions in MEFs either untreated or treated with TPP1^ΔRD and analyzed as in D. Error bars: s.e.m. (G) Immunoblot detection of p-Chk1 in WT or mSSB1^Δ/Δ MEFs untreated or treated with shRNA against TRF2 or expressing TPP1^ΔRD. γ-tubulin was used as a loading control.