Mimcd3 cells

HEK293 and mIMCD3 cells (ATCC) were cultured in DMEM/F12 (Life Technologies) with 10% FBS and 100 units/ml penicillin/100 μg/ml streptomycin.

mIMCD-3 cells (ATCC CRL-2123) cultured in DMEM/F12 medium (Thermo Fisher Scientific) supplemented with 10% FBS, at 37°C in 95% air and 5% CO2. For subculture of cells in 75 cm2 flasks, monolayers were washed in 5 ml Dulbecco’s phosphate-buffered saline (DPBS). Cells were detached by incubation with 2 ml 0.25% (w/v) trypsin-EDTA at 37°C for 5–15 min. Cells were dispersed in 6 ml medium and pelleted by 5 min centrifugation at 200 g. Trypsin-containing medium was aspirated and cells re-suspended in fresh medium and seeded in a new 75 cm2 flask in a 1:10 ratio. Reagent volumes were scaled for subculture in vessels of varying sizes.

mIMCD-3 cells (ATCC) grown to 90%–95% confluence in 24-well plates were transfected using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, Cat. No. 11668030). The transfection efficiency was normalized using the Renilla pRL plasmid (15 ng), and the total quantity of transfected DNA was kept constant using pcDNA3.1 (+) Mammalian Expression Vector (Thermo Fisher Scientific, Cat. No. V79020). Between 48 and 72 h after transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Cat. No. E1910) according to the manufacturer’s instructions. The luminometer was programmed to perform a 2-s premeasurement delay, followed by a 10-s measurement period for each reporter assay. After measurement of the firefly luciferase activity, Stop & Glo Reagent was added to each sample and enzymatic activity of Renilla luciferase was measured.

The hICD construct containing the intracellular domain of human FPC (192 aa) was described previously [9] (link). The hTMC construct containing the human FPC C-terminal 225 aa was made by RT-PCR and cloned into pcDNA4 at Hind III/Xba I sites (Figure S1). For the establishment of stable cell lines, hICD construct or empty vector was transfected with FuGENE6 (Roche, Madison, WI, USA) into mIMCD-3 cells (ATCC, Bethesda, MD, USA) cultured in DMEM/F12 media (Cellgro, Manassas, VA, USA), supplemented with 10% FBS (Invitrogen), and screened for cells resistant to Zeocin (Invitrogen, 750 µg/ml). For amino acid deprivation condition, cells were serum-starved for 16 h and then cultured in DMEM/F12 media without amino acids for 30 min. For drug treatments, stable cells were incubated with culture media containing appropriate chemical reagents for a period of time as specified in the Results. FPC knockdown cells were described previously [13] (link). Different amounts of hICD expression construct DNA were transiently transfected into LLC-PK1 cells stably expressing full-length FPC or empty vector control cells P4L [12] (link). Two days after transfection, cells were harvested for western blotting analyses.