Sasi mm02 00354635

U2OS, U2OS-EB1 (a gift from Dr. Pavel Draber (Vinopal et al. 2012 (link))), and HaCaT (a gift from Dr. Paul Higgins) cells were cultured in DMEM (Invitrogen, 11965092) supplemented with 10% fetal bovine serum and 1% Glutamax at 37°C in the presence 5% CO2. L929s (a gift from Dr. Bin Zhou) were cultured in EMEM (ATCC, 30–3000) and 10% FBS, under the same conditions.
For in vitro FL2 knock down studies, cells were transfected using 2.5 µl Lipofectamine RNAiMAX (Invitrogen, 13778075) or 7.5 Lipofectamine 3000 with 40 or 75 pmol, respectively, of either FL2 siRNA (Dharmacon, ON-TARGETplus set of four), mouse FL2 (Sigma-Aldrich, SASI_Mm02_00354635), or a negative control siRNA (Sigma-Aldrich, SIC001).
For FL2 rescue experiments, 24 h after siRNA treatment, U2OS cells were transfected with 1.5 µg of either pEGFP-C1 or pEGFP-FL2 using 4 µl of Lipofectamine 2000. Media was replaced 5 h later and cells were processed the next day depending on the experiments (see below).
Unless otherwise noted, experiments in U2OS cells were repeated 3 times and experiments in HaCaT and L929 cells were repeated twice, independently.