Modfit lt version 5.0 analysis software

A total of 5×10³ ITK-SYK⁺ or ITK-SYK⁻ Jurkat cells per well were seeded in 96-well plate with RPMI-1640 medium containing DMSO, or 0.5, 2.5 or 5 µM tofacitinib for 6, 18, 24 and 48 h at 37°C. DMSO exposure at a concentration of 0.1% in controls had no effect on the viability of examined cells lines, therefore 0.1% DMSO was used to treated the cells for 6, 18, 24 and 48 h at 37°C; these cells were used as the control cells. Cell viability was evaluated using a Cell Counting Kit-8 assay (CCK-8; Dojindo Molecular Technologies, Inc.) according to the manufacturer's protocol. Cells were measured at 450 nm using a Varioskan Flash multimode reader (Thermo Fisher Scientific, Inc.).
The apoptotic rate of transduced cells that were treated with tofacitinib was detected using an Annexin V-PE/7-ADD Apoptosis Detection kit (BD Pharmingen™). Separately, the transduced and tofacitinib-treated cells (1×10⁶) were stained with propidium iodide (Sigma-Aldrich; Merck KGaA) at room temperature in the dark for 30 min. The cells were analysed using a flow cytometer using ModFit LT version 5.0 analysis software (Verity Software House, Inc.). Experiments were performed in triplicate.

FTC-133, BCPAP, SW1736, C643 and TT cells were harvested following 24-h incubation with mitotane (25 and 50 μM), washed with cold PBS, and fixed in 100% ice-cold methanol for 1 h. The fixed cells (1×10⁶) were incubated with RNase A for 30 min at room temperature, and stained with 50 μg/ml propidium iodide (PI) solution (Thermo Fisher Scientific, Inc.) at room temperature in the dark for 30 min. Flow cytometry analysis was performed on a BD LSRII flow cytometer (Becton, Dickinson and Company). A 488-nm laser was used for the dye excitation; 595 long pass and 610/20 band pass filters were used for emission detection. Single cells were gated using forward scatter height and area parameters. The single cell population gate was confirmed by using area and width parameters of PI channel. The distribution of cells in the G₀/G₁, S and G₂/M phases of the cell cycle was estimated using ModFit LT version 5.0 analysis software (Verity Software House, Inc.). As an estimate the extent of apoptosis, the percentage of apoptotic cells was calculated in the DNA histograms. The measurements were performed in triplicate.