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. 2006 Oct 9;175(1):55–66. doi: 10.1083/jcb.200604009

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Copyright © 2006, The Rockefeller University Press

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Figure 5. — The increase in PML NB number in response to DSBs is independent of new protein translation and p53. NHDF cells (GM05757) in the presence or absence of 150 μM cycloheximide (CHX), Saos-2 human osteosarcoma cells, and isogenic HCT116 human colon carcinoma cells (+ or − p53) were treated with etoposide (20 μM VP16) for 30 min (*, P < 0.0001). (A) Western blot analysis of PML protein levels after etoposide treatment in the presence or absence of cycloheximide. NHDFs were treated with etoposide (20 μM VP16 for 30 min) and harvested at the indicated times for SDS-PAGE and Western blot analysis. Ratio of PML protein levels in the control lane to PML protein at the indicated time points after etoposide treatment are shown normalized against actin. (B) Comparison of mean PML NB number after VP16 treatment in NHDFs, NHDFs treated with cycloheximide (+CHX), and Saos-2 cells. (C) Comparison of mean PML NB number after VP16 treatment in isogenic HCT116 and HCT116 p53-null cells. (D) Comparison of DNA synthesis activity of NHDFs, NHDFs treated with cycloheximide (+CHX), and Saos-2 cells at 18 h after VP16 treatment. 18 h after VP16treatment, cells were incubated with BrdU, fixed, and processed for immunodetection of BrdU, and DNA was counterstained with DAPI. Asterisks represent BrdU-positive cells. Bars, 5 μm.