Modfit lt 5

We performed cell cycle analysis as previously described [18 (link)]. First, we harvested each A549 stably transduced cell group individually. Following PBS washing, the cells were fixed in 3.7% paraformaldehyde and fixed in 70% ethanol. Subsequently, we stained the cells with propidium iodide (MP Biomedicals, LLC, Santa Ana, CA, USA) and ribonuclease-A (Sigma), and analyzed them using a BD FACS Calibur Flow Cytometer, with Cellquest Pro v. 6.0 software (BD Biosciences, Franklin Lakes, NJ, USA). We processed the data we obtained using ModFit LT 5.0 (Verity Software House, Topsham, ME, USA; Supplementary Figure S5). The cell cycle data are presented as the percentage of cell distributions in the different phases (G0/G1, S, and G2).

For cell cycle analysis, cells were harvested, washed with PBS, fixed with prechilled 70% ethanol, and maintained overnight at 4 °C. The fixed cells were then collected, washed and resuspended in PBS. The cells were incubated with 1 mg/mL RNase and 50 µg/mL PI for 30 min at 37 °C and subjected to flow cytometry analysis (Beckman Coulter, CA). The cell cycle results were analysed with ModFit LT 5.0 (Verity Software House). The histograms of each cell cycle phases were represented and compared by GraphPad Prism 8 software.

Cell cycle assays were performed at 48 h post-transfection. The cells were spun at 1,000 g for 5 min, and cell pellets were washed once in PBS at 4°C. Add 1 ml of 70% ethanol to each sample and incubate at 4°C for 2 h. The cells were spun at 1,000 g for 5 min, and cell pellets were washed once in PBS at 4°C. Remove the supernatant, add 1 mL propidium iodide (PI) working solution to each tube to re-suspend the pellets. Incubate for 30 min protected from light at 37°C. Cell cycle distribution was performed using an Accuri C6 flow cytometer (BD Biosciences). Data were analyzed using ModFit LT 5.0 (Verity Software House).

The ploidy levels of both parents and progeny were determined by fluorescence-activated cell sorting (FACS), similar to the protocol used by Skosireva et al. [43 (link)]. Briefly, cells grown overnight were harvested from YEPD agar medium (~10⁷ cells/mL), washed once in phosphate-buffered saline (PBS), and then fixed in 1mL of 70% ethanol at 4 °C for at least 6 h. Fixed cells were washed once in NS buffer, then stained with 5 µL of propidium iodide (10 mg/mL) in 180 µL NS buffer adding 20 µL of RNaseA (10 mg/mL) and incubated with agitation for 3 h at room temperature or overnight at 4 °C 50 µL of stained cells were diluted into 2 mL of 50 mM Tris-HCl (pH 8.0) and sonicated for 10s. Flow cytometry was performed on a Becton–Dickinson LSR II model with ~10⁴ cells. Data were analyzed and visualized by ModFit LT 5.0 (Verity Software House). Parental strain JEC21 was used as a haploid control, and D15 (RAS strain) was used as a diploid control [44 (link)].

Cells were detached by incubation with trypsin and fixed with 70% ethanol at 4°C overnight, washed with PBS, and reacted with propidium iodide (PI, 20 μg/mL) for 30 min in the dark. The cells were then subjected to cell cycle distribution analysis using a flow cytometer (BD Biosciences, San Jose, CA, USA). Ten thousand cells were assessed using CellQuest Pro software (BD Biosciences, San Jose, CA, USA). The ratio of G2+M/G1 phase cells was calculated using ModFit LT 5.0 software (Verity Software House, Topsham, ME, USA).

For the cell cycle assay, pretreated cells were mixed with 75% ethanol and placed at −20°C overnight. On the next day, the cell suspension was brought to room temperature, and the cells were subsequently washed with PBS and centrifuged at 200 g for 5 min. Then they were mixed with a cell cycle staining reagent (Multisciences) and incubated in the dark for 30 min. The cell cycle distribution of the cells was detected using BD FACSCanto™ II and analyzed using Modfit LT 5.0 (Verity Software House). For the apoptosis assay, the cells were harvested and incubated with 200 μL of binding buffer containing 2 μL of FITC and 4 μL of PI fluorescence reagent (Multisciences) for 10 min in the dark. The proportion of apoptotic cells was detected using BD FACSCanto™ II and analyzed using FlowJo 10.8.1 (Becton, Dickinson and Company).

Cell proliferation was assessed by flow cytometry using carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Life Technologies), which covalently binds cell components to yield a fluorescence that is divided equally between daughter cells at each division. Briefly, starved DLD-1 cells or normal fibroblasts or CAFs were incubated with CFSE according to the manufacturer’s instructions. After 30 min, the medium was removed, and fresh medium was added for the indicated time points. At the end of the specific treatment, cells were collected, washed twice with PBS and then incubated with PI (5 mg/mL, Life Technologies) for 15 min at 4 °C in the dark. CFSE- and/or PI-positive cells were determined by flow cytometry (FACSVerse BD Biosciences, San Jose, CA, USA), and the data were analyzed using ModFit LT 5.0 (Verity Software House, Inc., Topsham, ME, USA) and expressed as relative proliferation index with respect to unstimulated conditions.
Cell growth was evaluated by using a commercially available 5-bromodeoxyuridine (BrdU) assay kit (Roche Diagnostics, Monza, Italy). Briefly, 3500 cells were cultured in 96-well microplates and allowed to adhere overnight. Five-bromodeoxyuridine was added to the cell cultures 6 h before the end of the experiments, and cell growth was evaluated by ELISA at 450 nm.