Multicycle av program

Cells were seeded at 1 × 10⁵ cells per mL into 6 well plates (1 mL per well) and were allowed to adhere for 24 h. The medium was replaced with fresh medium containing SOCLAC (5 μM final concentration) and incubated for 1 h. After this time, the medium was replaced with fresh medium containing RBM5-177, RBM5-019 or RBM2-37 (5 μM final concentration) ± SOCLAC and incubated for 20 min. In the experiments with DM120, the compound, SOCLAC and RBM5-177 were incubated together for 4 h. After incubation with the probes, the medium was replaced with fresh medium containing CO-1 (1 μM final concentration), and cells were further incubated for 10 min. Then cells were washed 3 times with DMEM for 5 min at 37 °C, and finally washed with 400 μL PBS and harvested with 400 μL trypsin–EDTA and 600 μL of medium. Pellets were resuspended with 200 μL of PBS and stained cells were analyzed by using a Guava EasyCyte™ flow cytometer (Merck Millipore, Billerica, MA) measuring green fluorescence (488/525 nm). Data analysis was performed using the Multicycle AV program (Phoenix Flow Systems, San Diego, CA).