Propidium iodide (PI) staining and cell cycle analysis

Huh-7.5 cells were seeded in 10 cm dishes at 1 × 10⁶ cells/dish and infected on the next day at an MOI of 1. The PI staining protocol used here was adapted with minor modifications from the standard method reported by Riccardi and Nicoletti³⁸. Briefly, cells were harvested and fixed with 70% cold ethanol and then stored at −20 °C until all cells were ready to be stained. To prepare for PI staining, the cells were centrifuged at 400 × g for 5 minutes and supernatants discarded. Cells were washed and resuspended in 0.5 ml of PBS, 0.5 ml of DNA extraction buffer was added, and incubated for 5 minutes. Next, cells were pelleted as above, supernatants were removed, and the cells were resuspended in 1 ml of DNA staining solution and incubated for 30 minutes at room temperature. The DNA extraction buffer and the DNA staining solution were prepared as described in ref. ³⁸. PI intensity in the stained cells was measured using a Becton Dickinson FACS Calibur. Cellular debris and doublets were gated out during the analysis. Following that, hypodiploid cells with an intensity lower than that of the diploid cells (G1) were counted.