Pi solution

Cells were washed with PBS, dissociated with 200 μl trypsin/EDTA and centrifuged for 6 min at 1000 × g. The pellet was resuspended in 100 μl Annexin V-APC binding buffer 100 mM HEPES, 140 mM NaCl, 25 mM CaCl2 and pH 7.4 in water. One microliter Annexin V-APC staining solution (Biolegend, San Diego, CA, USA) was added and the cells incubated for 15 min at room temperature in the dark. Immediately before measuring with the LSRII SORP flow cytometer (Becton Dickinson), 400 μl Annexin V binding buffer and 5 μl PI solution (1 mg/ml in PBS, Sigma-Aldrich) were added. The Annexin V-APC and PI signals were collected on a logarithmic scale using a 640 nm laser in combination with a 660 nm filter (20 nm bandwidth) and a 488 nm laser in combination with a 695 nm filter (40 nm bandwidth), respectively. Analysis was performed with FlowJo software (Version 10.0.7, Treestar Inc. Ashland, OR, USA).

Cells were washed with PBS and fixed overnight at − 20 °C with 70% ethanol. Fixed cells were stained with PI solution (Sigma-Aldrich St. Louis, MO, USA) solution containing RNase A (Qiagen, Hilden, Germany). Fluorescence was measured with FACS Canto II (BD Biosciences, Heidelberg, Germany) flow cytometer and data was analyzed with FACSDiva software.

For both cell lines, HT-29 and ZR-75-1, cells were harvested 7 days after transfection and stained with anti-Annexin V FITC-conjugated antibody (BD Bioscience, 556420) at 20 μl/1 X 10⁶ cells in Binding Buffer 1X (BD Bioscience), for 15 min RT protected from light. Cells were finally resuspended in 400 μl of Binding Buffer 1X and 100 μl of propidium iodide (PI) solution (250 nM) (Sigma-Aldrich) was added to the cells and incubated for 1 min before analysing with the flow cytometer (FACSCalibur, BD). Data was analysed using FlowJo software (FlowJo, BD).

After transfection, the cells were washed with PBS, and fixed with ethanol at 4° C overnight. Thereafter, 150 μL PI solution (Sigma, USA, #25535-16-4) was added at 4° C for 30 min in the dark. The distribution of the cells in the different cell cycle phases was analyzed using a flow cytometer (Beckman, USA, #A00-1-1102). PI was activated using 488 nm argon lasers and received using a 630 nm filter.

After treating with BTFS (0, 1.5, 2.0, 2.5 μg/mL) for 24 h, the cells were washed with cold PBS twice and then fixed with ice-cold 70% ethanol at 4 °C overnight. Afterwards, the cells were washed twice with PBS and incubated with 180 μg/mL RNase (Invitrogen) for 15 min at 37 °C, then incubating with 50 μg/mL propidium iodide (PI) solution (Sigma) for 15 min in the dark at 37 °C. The cells were then analyzed by FACSCalibur flow cytometry (BD Biosciences, San Jose, CA, USA) and data were analyzed by FCS software (De Novo Software, CA, USA).