4mm citrate buffer

The cell cycle arrest analysis was carried out as previously described.⁴⁸ Briefly, 1 × 10⁶ MDA-MB-468 cells were infected as described above (Cell Lines and Viral Transductions). Thirty six hours post-infection, cells were collected, washed in PBS (Invitrogen), and fixed in 70% cold ethanol at −20°C for 1 h. Fixed cells were then washed three times with PBS and suspended in a staining solution containing 10 % v/v of 0.5 mg/mL propidium iodide (Sigma-Aldrich, St. Louis, MO), 5% v/v of PBS, 84 % v/v of 4mM Citrate Buffer (Sigma-Aldrich), 0.62 %v/v of RNase A (Sigma-Aldrich), 1% v/v of Triton X-100 (Sigma-Aldrich), and 3% m/v of PEG 6000 (Fluka, St. Louis, MO) for 20 min at 37°C in the dark. An equal volume of salt solution containing 5% of propidium iodide, 1% of Triton X-100, 89% v/v of 0.4M NaCl (Thermo Fisher Scientific Inc, Pittsburgh, PA), and 3% m/v of PEG 6000 was added to the stained cells and incubated at 4°C for 1 h in the dark. Cells were gated at similar intensity level of ZsGreen1. ZsGreen1 and PI were excited at 488 nm and detected at 507 nm and 670 nm, respectively. The means from three separate experiments (n=3) were analyzed using one-way ANOVA with Bonferroni’s post hoc test.