Figure 1. DNA damage promotes formation of a pRB-E2F1 complex in proliferating cells.

(A, B) Asynchronous T98G cells untreated (−) or treated (+) with 2μM doxorubicin (doxo) for 24 hours were (A) screened by immunoprecipitation (IP) using an antibody against pRB followed by western blotting (WB) to assess levels of pRB and associated E2F1 and P/CAF or (B) assayed for cell cycle distribution by FACS analysis. Bars represent the mean of three independent experiments (± SD). (C) T98G cells were highly enriched for G₀/G₁ or S/G₂/M (prolif.) cells, as determined by FACS (right panel), by culturing in 0.1% FBS for 72 hours and then maintaining in 0.1% or replating in 20% FBS for 16hrs. The cells were collected (samples 1 and 3) or treated with 2μM doxo for additional 48 hours (samples 2 and 4) and then assayed in parallel for pRB-E2F1 complexes by IP-WB (left panel). (D, E) Enriched populations of G₀/G₁ or proliferating T98G cells were untreated (samples 1 and 4) or treated with I.R. (10 Gy) for 1 hour (samples 2 and 5) or 2μM doxo for 24 hours (samples 3 and 6) as indicated (D, left panel). These samples were analyzed for: (D, right panel) cell cycle phasing by FACS; (E, left panel) the presence of ppRB-E2F1 complexes by IP with either IgG control or ppRB antibodies and then WB for pRB or associated E2F1; or (E, right panel) the levels of total pRB and E2F1 by WB of the input lysates. (F) Asynchronous T98G cells, untreated (−) or treated (+) with 2μM doxo for 24 hours, were screened for the presence of ppRB-E2F1 complexes by IP with either IgG control or E2F1 antibodies and then WB, first for pRB and subsequently (after stripping the blot) for ppRB. A bubble in the blot yielded the non-specific signal (*).