Figure 3.

RHPS4 impairs telomere replication and alters the dynamics of telomeric proteins. BJ-HELT fibroblasts were synchronized at the G₁/S boundary with HU, treated with RHPS4 or released in drug-free medium and harvested every 2 h. Prior to harvest of each individual time point, the cells were incubated with BrdU for 1 h. (A) FACS analysis of untreated and RHPS4-treated BJ-HELT fibroblasts released from G₁/S block at the indicated times. (B) ChIP experiments on synchronized BJ-HELT fibroblasts incubated with BrdU. Precipitations were performed with antibodies against TRF1 and TRF2. The precipitated DNA was analyzed by western blotting with anti-BrdU antibody. The total DNA (input) represents 10% of genomic DNA (C) The graphs show the densitometric evaluation of three independent experiments, with error bars indicating the SD. (D) Protein extracts from cells at the indicated times were subjected to ChIP experiments using antibodies against TRF1, TRF2 and POT1. IgG antibody was used as negative control. The total DNA (input) represents 10 and 1% of genomic DNA. Southern blot analysis was performed by using telomeric or ALU repeat-specific probes. (E) The signals obtained were quantified by densitometry, and the percentage of precipitated DNA was calculated as a ratio of input signals and plotted. Four independent experiments were evaluated and error bars indicate the SD.