Plasmid transfections were performed using Lipofectamine 2000 (Thermo Fisher) according to the manufacturer’s protocol but using Transfectagro (Corning) instead of Opti-MEM. Cells were incubated with transfection mix in Transfectagro supplemented with 10 % FBS for 6–8 h, following by a change of media to regular growth media and analysis after 18–20 h.
DsiRNA duplexes were obtained from Integrated DNA Technologies. Transfections with siRNA were performed with the appropriate duplexes (seeKey Resources Table ) using Lipofectamine RNAiMAX (Thermo Fisher) following the manufacturer’s protocol except using Transfectagro in place of Opti-MEM. Cells were incubated wit transfection mix in Transfectagro supplemented with 10% FBS for 12–16 h, followed by exchange with fresh media. NC1 (negative control 1, IDT) was used as the control siRNA duplex for all experiments. Forty-eight h post transfection, cells were subjected to analysis via western blot, microscopy or flow cytometry.
DsiRNA duplexes were obtained from Integrated DNA Technologies. Transfections with siRNA were performed with the appropriate duplexes (see