Figure 4.

DNA incision activity of APE1 is reduced by Lmna knockout in MEFs in a PARylation-dependent manner. (A) APE1 DNA incision product formation from extracts of Lmna^+/+ and Lmna^−/− MEFs and the effects of NAD⁺ (2 mM, 2 h) and/or PARP1 inhibitor 3-AB (10 mM, 2 h) media supplementation on product formation. A representative gel image is shown, as well as the APE1 activity quantitation based on three separate experiments (mean ± SD). APE1 DNA incision activity was determined as the intensity of product band relative to the combined intensities of substrate and product bands. (B) A representative western blot showing the PAR bands (PARylation) from lysates that were made at the same time as the extracts used for the APE1 DNA incision assay. (C) FPG-comet assay on MEFs overexpressing wild-type APE1, empty vector (EV) or DNA incision mutant D201N. The MEFs were treated with oxidative stress (100 μM H₂O₂) for 30 min and then allowed to repair for 8 h in fresh medium. DNA repair efficiency was expressed as percent of FPG-sensitive sites remaining, after 8 h of repair, relative to 5 min of repair, after correction for the FPG-sensitive sites in untreated cells (n = 100 comet tails, mean ± SEM); Pos, positive control (purified APE1 instead of lysate); Neg, negative control (no lysate); n.s, not significant. P values were determined by Student’s t-test; ***P < 0.0001; **P < 0.005; *P < 0.05.