Modfit lt software

For cell cycle analysis, cells stained with 50 μg/mL of propidium iodide (Sigma-Aldrich) were analyzed using BD FACSCalibur flow cytometer and the ModFit LT software (Becton Dickinson).

The Huh7-luc/neo cells were seeded at a density of 1 × 10⁵ cells/well into 6-well culture plates (without antibiotic G418), cultured for 24 h, and treated with different concentrations of the test substances for an appropriate time. The cells were scraped off in 0.5 mL PBS and fixed by a gradual addition of 0.5 mL of cold ethanol with gentle vortexing. After incubation for 20 min, the cells were precipitated by centrifugation at 4000 rpm for 5 min. The supernatant was removed, and the cells were resuspended in 0.5 mL of PI-RNAse buffer (10 µg/mL PI and 0.1 mg/mL RNAse A in PBS) and incubated in the dark for 20 min. The DNA content in the cells was analyzed using an LSRFortessa Cytometer (BD Biosciences, San Jose, CA, USA). The data were analyzed with the ModFit LT software (Becton Dickinson, Franklin Lakes, NJ, USA).

Flow cytometry was used to analyze mitochondrial membrane potential (MMP). Briefly, GC2 cells were harvested after transfection of miR-574 mimics or inhibitors for 24 h, and then stained with JC-1 according to the manufacturer’s instructions. The mitochondrial membrane potential detection kit (Beyotime) was used for cell staining. The stained cells were detected by a FACS Caliber (Becton Dickinson, Mountain View, NJ, USA) and analyzed using Modfit LT software (Becton Dickinson). All experiments were performed in three replicates.

Cell death was quantified by FACS analysis, staining cells with the nonvital dye PI (Immunological Sciences, Rome, Italy), following the manufacturer's instruction. Briefly, cells floating were collected by centrifugation and pooled with adherent cells recovered from the plates, fixed in 80% ethanol, and stained in a PBS solution containing PI (62.5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and RNase A (1.125 mg/ml; Sigma-Aldrich). Samples were acquired with a FACScan instrument (Becton Dickinson Europe Holdings SAS, Le Pont De Claix, France) and the percentage of cells in sub-G1 compartment was calculated using ModFit LT software (Becton Dickinson). Approximately 30 000 events were acquired and gated using forward scatter and side scatter to exclude cell debris.

Cell death was quantified by Fluorescence Activated Cell Sorting (FACS) analysis, staining cells with the nonvital dye propidium iodide (PI) (Immunological Sciences, Rome, Italy), following the manufacturer’s instruction [32 (link)]. Briefly, floating cells were collected by centrifugation and pooled with adherent cells recovered from the plates, fixed in 80% ethanol and stained in a PBS solution containing PI (62.5 mg/ml; Sigma-Aldrich), and RNase A (1.125 mg/ml; Sigma-Aldrich). Samples were acquired with a FACScan instrument (Becton Dickinson Europe Holdings SAS - Le Pont De Claix, France) and the percentage of cells in sub-G1 compartment was calculated using ModFit LT software (Becton Dickinson). About 30.000 events were acquired and gated using forward scatter and side scatter to exclude cell debris.