Cell Cycle Analysis of Bleomycin-Treated ECs

Flow cytometry analysis was performed as previously reported^{61 (link)}. In brief, human ECs cultured in 12-well plates were infected with lentivirus(es) for 48 h. Cells were then incubated for 24 h in fresh medium with or without 0.5 μg ml^–1 bleomycin and collected by trypsin. After washing in FACS buffer (PBS containing 5% bovine serum albumin (BSA)), cells were permeabilized with the FoxP3 permeabilization kit (eBioscience). Cell cycle analysis was performed with anti-Ki67 antibody and propidium iodide (eBioscience) staining. Data were acquired and analyzed using a Fortessa Flow Cytometer (Becton Dickinson) and FlowJo software (Tree Star). Data for flow cytometry analysis represent n = 3 independent biological samples.