Synchronizing Cell Cycle for TGF-β Signaling

The 786-O and A498 RCC cell lines were bought from ATCC in 2012. Prior to experiments, both cell lines were authenticated by STR profiling (Identicell, Denmark) on 12-06-2014, and were cultured in RPMI media supplemented with 10% FBS.
One day prior to transfection, 1 × 10⁶ cells (786-O and A498) were seeded in a 10 cm plate. To synchronize the cell cycle, cells were starved by adding Opti-MEM and incubating at 37°C for 15 min. Transient transfection of the cell lines with 20 μg C-terminally hemagglutinin (HA)-tagged ALK5 (ALK5-HA) [17 (link)] or 20 μg pcDNA 3.1(+) (control) was done using Lipofectamine 3000 reagent (Life Technologies) according to manufacturer's protocol. Cells were collected the next day after stimulation with 10 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN, USA) for 30 minutes. For the kinetic experiment, cells (786-O and A498) stimulated with 10 ng/ml TGF-β1 were collected at 0 min, 30 min, 1 h, 2 h, and 6 h. For immunoblot experiments, the cells were treated with 10 ng/ml TGF-β or 10 ng/ml TGF-β and 20 μM TAPI-2 (Enzo life sciences, Farmingdale, NY, USA) or untreated cells were collected after 6 h.