Fig. 2. Correlation between DNA damage and WRN acetylation.

a) 8-D cells were incubated with or without 40 μM O⁶-benzylguanine (O6-BG) for 4 h followed by incubation with 1 mM MMS for an additional 4 h. For treatment with O6-BG alone, cells were treated with 40 μM O6-BG for 8 h. After harvesting and processing, cell lysates were subjected to IP with anti-acetylated lysine antibody and analysis of IP products with anti-WRN antibody (upper panel). In parallel, cell lysates (40 ug each) were analyzed by Western blotting with anti-WRN antibody (lower panel). b) Bar graph for WRN acetylation from experiments performed as in A (mean ± SEM of 3 independent experiments; * = p < 0.05 when compared with untreated cells). c) 8-D cells were incubated in growth medium with or without 5 nM olaparib for 38 h followed by incubation with 1 mM MMS for an additional 4 h. For treatment with olaparib alone, cells were treated with 5 nM olaparib for 42 h. Cell lysates from each treatment were analyzed by IP as in A, showing (upper panel) IP products and (lower panel) cell lysates (60 ug each). d) Bar graph for WRN acetylation for experiments performed as in C (mean ± SEM for two independent experiments; * = p < 0.05 when compared with control and # = p < 0.05 when compared with cells treated with MMS alone). Lanes 1 in A and C are purified acetylated WRN (upper panel) or unmodified WRN (lower panel) used as markers (MKR)