Research Paper Volume 16, Issue 20 pp 12977—13011

Figure 4. WRN, HDAC2, and HP1α associations are reduced in RAF oncogene-induced senescence. (A) 4HT-inducible expression of RAF oncogene in WI38hTERT leads to cessation of proliferation and onset of senescence marked by the formation of SAHF after three days. (B) Quantitations of H3K9me3 (left panel, three independent experiments), and WRN, HP1α, and HDAC2 (center and right panels, two independent experiments each) nuclear levels in WI38hTERT after 4 days in 4HT compared to the contemporaneous control without 4HT, determined by IF in situ. (C) Quantitations of nuclear area (in pixels) of WI38hTERT treated with 4HT as in (B). The graph represents three independent experiments. (D) Total levels of the WRN, HP1α, and HDAC2 proteins in WI38hTERT under the same conditions, determined by Western blotting. Quantitations below the image average three independent experiments. The values were normalized to NCL as loading control and shown relative to no-4HT conditions. (E) An example of HDAC2/WRN PLA data in WI38hTERT with and without 4HT. Scale, 10 µm. (F) Quantitations of HDAC2/HP1α, WRN/HP1α, and HDAC2/WRN PLA in WI38hTERT after 4 days in 4HT compared to the contemporaneous no-4HT control. The left and center panels represent two independent experiments each, and the right panel represents two biological replicas. Crossbars in graphs are distribution means.