Version 7

All data was acquired using FACSDiVa data acquisition software (BD Biosciences, San Jose, CA) and analyzed using FlowJo version 7.6.5 (FlowJo LLC, Ashland, OR). The level of FP fluorescence was expressed as a staining index (SI), using the median and slope distributions of labeled and background cell or bead populations to calculate a value proportional to the level of fluorochrome fluorescence, as described previously [23 (link)]. Fluorescent compensation was calculated in FlowJo using the software package’s compensation algorithm.

The drug treatment was carried out as previously described with either 100 nM of NVP-BGJ398 or 0.01% DMSO. 72 h post drug treatment, cells were trypsinized and fixed with 80% cold ethanol. After PBS wash, cells were stained for 20 min with PE-conjugated anti-Ki67 (BD Biosciences, New Jersey, United States) or the corresponding isotype control (BD Biosciences, New Jersey, United States) at recommended dilutions, followed by PBS wash and Hoechst 33342 (BD Biosciences, New Jersey, United States) staining at a concentration of 2 μg/ml for 15 minutes. Staining was measured using LSR II Flow Cytometer (BD Biosciences, New Jersey, United States). For Hoechst, 355 nm UV laser with 540/50 BP filter and for PE-conjugated Ki67, 488 nm blue laser with 585/42 BP and 550 LP filters were used. The results were analyzed with FlowJo, version 7.6.5 (FlowJo, Oregon, United States). After cell doublet discrimination, the Ki67 negative cell population was gated as G₀. The isotype control was used to confirm the gating strategy with Ki67. Cell cycle analysis was done using the built in software Cell Cycle Analysis module. Statistical analysis was performed using two-tailed, unpaired Student's t-Test. p-values < 0.05 were considered significant.

TiHo-0906 cells (P7 and P77) were seeded in 6-well plates (2 × 10⁵ cells/well) and incubated for 18 h at 37 °C in a humidified atmosphere of 5% CO₂. Afterwards, the cells were exposed to different dilutions of doxorubicin (1 nM, 10 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1000 nM, and 2000 nM [3 mL/well]) and then re-incubated for 72 h. After this period, media containing non-adherent and dead cells were collected. Adherent cells were dissociated with 1 mL TrypLE^TM and centrifuged (1000 rpm for 6 min) together with the previously collected media. The supernatant was discarded, and cell pellets were resuspended in 500 μL of 1X Binding Buffer. Cell suspensions were incubated (10 min at room temperature in the dark) with 5 μL Annexin V-FITC and 1 μL SYTOX Green Dye (Annexin V-FITC Detection Kit plus, PromoCell). Flow cytometry was performed using a MACSQuant® Analyzer 10 (Miltenyl Biotec). Dead cells treated with 1 mL 0.2% Triton^TM X-100 (Sigma-Aldrich) and non-treated viable cells were used to set the gates. Annexin V-FITC and SYTOX Green Dye were measured in the FL-1 channel, and results were analysed with FlowJo Version 7.6.5 (FlowJo, Ashland, OR, USA). For data analysis, cell debris on the left-side of the plot were set as negative gate. Afterwards, intact cells were gated for viability on three different populations: viable, apoptotic, and dead.