Fig. 3
Cell death assays in PARP-1 stable knockdown fibroblast cells compared to PARP-1 positive fibroblasts following oxidative stress. (A) The GMU6, GMSiP, and GMRSiP were treated with 0.5 mM of H2O2, followed by nuclear and cytoplasmic fractionation and immunoblotting using mouse monoclonal anti-AIF antibody to detect AIF translocation. H3 and actin were used as loading controls for nuclear and cytoplasmic fractionation, respectively. (B) The three types of cells were treated with 0.5 mM and 2 mM of H2O2, followed by immunoblotting of the total cell lysates using rabbit anti-Bcl2, rabbit anti-Bcl2XL, and rabbit anti-cleaved caspase-3 antibodies. (C) The plot depicted actin normalized expression of Bcl2 and Bcl2XL in GMU6, GMSiP, and GMRSiP after H2O2 treatment. (D) The plot showed actin normalized cleaved caspase-3 expression in three types of cells after H2O2 treatment with 0.5 and 2 mM. (E) Cells treated with 0.5 mM of H2O2 for 1 h were stained with both Annexin V-FITC and DAPI. Images were acquired in a Zeiss Epifluorescence Microscope with 45X magnification. The scale bar denotes 5 μm. (F) The plot showed %Annexin V positive cells after 0.5 mM H2O2 treatment. The data shown was the mean ± SD of three independent experiments. The significance values were denoted as ‘**’ (0.001 < p ≤ 0.01) and ‘***’ (p ≤ 0.001)