Extended Data Fig. 1: Overexpression of Rad51 prevents MMC-mediated DNA damage, and MMC does not induce apoptosis.
(a) PMVECs (5 × 103 cells) transfected with empty vector (mock) or Rad51 expression plasmid (Rad51) were subjected to the comet assay. The bar graph indicates the levels of DNA damage as a percentage of nuclei with damaged DNA out of total nuclei as mean ± SEM. Approximately one hundred nuclei per condition were examined. n = 5 independent samples. (b) PMVECs (1 × 106 cells) were transfected with empty vector (mock) or Rad51 expression plasmid (+Rad51), followed by vehicle or MMC treatment for 14 h. Total cell lysates were subjected to immunoblot of VE-Cad, Rad51, γH2AX, and β-actin (control). (c) PMVECs were treated with vehicle (Veh), MMC for 14 h or 0.4 mM hydrogen peroxide (H2O2) for 2 h, followed by fluorescence staining of Annexin V (green) and DAPI (blue). Annexin V-positive cells’ fraction (%) is shown as mean ± SEM. Scale bar=10 mm Approximately one hundred cells per condition were examined. A similar result was obtained by flow cytometric analysis of FITC-Annexin V staining. The number of Annexin V-positive cells is shown as mean ± SEM. About 300 cells and 1.5 × 106 cells per condition were analyzed by fluorescent staining and flow cytometry. Because of the autofluorescence of PMVECs, the signals of unstained PMVECs were used to set the gate for FITC-Annexin V-positive cells. A green rectangle indicates Annexin V-positive cells. Data are analyzed by one-way ANOVA with Tukey’s post-hoc test (c, d); two-way ANOVA With Tukey’s post-hoc test (a).
