Modfit lt software version 5.0

HCT116 cells were trypsinized after being treated with 0.5 mM HU for 0, 3, and 6 h. At 4 °C overnight, 10⁶ cells were suspended and fixed as single-cell dispersions in 70% ethanol. After washing two times with PBS, cells were resuspended in propidium iodide buffer (CF0031; Beijing Ding-guo, Beijing, China) and incubated for 30 min at 37 °C. EPICXLTM cytometers were used to obtain data (Beckman Coulter, Brea, CA, USA). MODFIT LT software (Version 5.0) was used to evaluate the collected data (Verity Software House, Topsham, ME, USA).

HGFs were seeded in 6-well plates and treated for 6 h and 24 h, as previously described. For each time point, the cells were trypsinized, collected by centrifugation, and fixed overnight at 4 °C in cold ethanol 70% v/v. After fixation, cells were washed in cold phosphate buffer saline (PBS) and centrifuged at 4000 rpm for 10 min: the supernatants were discarded and each sample was stained with 300 µL of a staining solution containing PBS, RNase 100 µg/mL (stock solution 10 mg/mL in 10 mM sodium acetate buffer, pH 7.40), and propidium iodide (PI) 10 µg/mL (stock solution 1 mg/mL in water) (all purchased from Merck Life Science, Milan, Italy) and kept overnight at 4 °C in the dark. The PI fluorescence was detected by a flow cytometer equipped with a 488 nm laser (CytoFlex flow cytometer, Beckman Coulter, CA, USA) in the FL-3 channel. At least 10,000 events/sample were collected and analyzed with the CytExpert Software, version 2.3 (Beckman Coulter, CA, USA), and the percentages of cells in the G1, S, or G2 phase of the cell cycle were calculated using the ModFit LT™ Software, version 5.0 (Verity Software House, Topsham, ME, USA).

Cell cycle analysis was performed as we previously reported [12 (link)]. Briefly, cells were fixed overnight in 70% precooled ethanol at 4 °C and stained with 0.5 mL of propidium iodide (PI)/RNase Staining Buffer (BD Biosciences, Cat. No. 550825) for 15 min at room temperature. The DNA content was assessed by flow cytometry (Attune NxT Flow Cytometer, Thermo Fisher Scientific Co., Ltd., USA) at an excitation wavelength of 488 nm and an emission wavelength of 585 ± 21 nm. ModfitLT software version 5.0 (Verity Software House, Inc., Topsham, ME, USA) was used for the analysis of DNA distributions.