Figure 6.

BER is also impaired in U2OS cells that are siRNA-depleted of lamin A/C. (A) A representative western blot showing effective siRNA knockdown of lamin A/C in U2OS cells at the indicated days after transfection. (B) Cell proliferation assays were performed by seeding 25 000 cells per well in 6-well plates and counting the cells each day on a hemocytometer (n = 3, mean ± SD). (C) Cell survival was assessed by the WST-1 assay at the indicated stressor concentrations. All data points are the mean WST-1 absorbance (as percent of the value from untreated cells) from six wells (in 96-well plates) ± SD. Cells were seeded at 20 000 cells per well, 24 h prior to treatment. Treatment durations for the stressors were as follows: H₂O₂, 4 h; Menadione, 2 h; MMS; 1 h. (D) FPG-comet assay. 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). (E) Western blotting was performed on lysates prepared from untreated siScr and siLamin A/C U2OS cells in triplicate. Fold change in protein levels from siLamin A/C cells relative to siScr cells was quantitated using ImageJ (n = 3, mean ± SD). (F) Western blotting was also performed at the indicated time points, pre- and post- treatment (100 μM H₂O₂ for 30 min). P values from the growth curves and survival assays were determined by two-way ANOVA Sidak’s multiple comparisons test. The other P values were determined by Student’s t-test. ***P < 0.0001; **P < 0.005; *P < 0.05.