Fig. 2.

Telomere clusters are a common feature of ALT cells. (A) Telomere clusters, detected by colocalization of anti-TRF2 antibodies (green) with CFP-ICP0* (blue), are present in all ALT lines tested (GM847, U2OS, SAOS2), and in VA13 cells constitutively expressing telomerase (VA13+telom.), but are rarely observed in an atypical ALT line without APBs (VA13-C3-cl6). (Scale bars, 2 μm.) (B and C) Quantification of results from (A) based on 20 nuclei corrected for the level of random co-localization of TRF2 with ICP0* as observed in HT1080 cells (2 = 0.65 ± 0.21, 3 = 0.50 ± 0.17, 4 = 0, 5 = 0). Error bars, SEM. (D–G) Telomere clusters are present in G1, S, and G2 phase of the cell cycle but not in M. VA13 cells were transfected with CFP-ICP0* (blue) for 48 h, labeled with BrdU before fixation and stained with anti-TRF2 antibodies (green) and different markers for the cell cycle (red) as follows: G1 cells (D) were identified as negative after a double staining with markers for the S and G2 phases; S phase nuclei (E) were identified with anti-BrdU antibodies; G2 nuclei (F) were identified with anti-CENP-F antibodies and chromosome condensation allowed to distinguish CENP-F positive cells that were in M (G). (H) Quantification of results from (D–G). Error bars, SEM.