Optimem glutamax media

Cells were seeded in six-well plates and transfected with siRNA or plasmid or both with Lipofectamine reagent 2000 (Invitrogen) for at least 6 hr in antibiotic-free culture media mixed with OptiMEM glutaMax media (Gibco). After transfection, the medium was changed to DMEM or RPMI (as described above) supplemented with FBS and antibiotics. At 72 hr post-transfection, the cells were harvested for RNA or protein analysis. To check for apoptosis, the media from the culture vessels were also collected and the dead cells were collected by centrifugation. For survival assays, the cells were trypsinized and counted at least 4 hr after changing the transfection medium. Depending on the cell line, between 2,000 and 3,000 cells were plated in a 96-well plate and cultured for 96–120 hr. Cells were then washed with PBS once and fixed in 3.7% formaldehyde (Thermo Fisher Scientific, Waltham, MA) at room temperature for 12 min. Next, cells were stained with Syto-60 Red fluorescent nuclein acid stain (Thermo Fisher Scientific) and scanned with ODYSSEY infrared imager (LI-COR, Lincoln, NE) at 700 nm.

CAD5 cells were a generous gift of Dr. S. Mahal (The Scripps Research Institute Florida)^{29 (link),30 (link)}. Cells were grown in Opti-MEM glutamax media (GIBCO, USA) containing 10% (v/v) bovine growth serum (Hyclone, USA). Immortalized mouse embryonic fibroblasts (MEF) were obtained from Dr. N. Mizushima (Tokyo Medical and Dental University, Tokyo, Japan) and grown in DMEM glutaMax media (GIBCO, USA) containing 10% fetal bovine serum (Sigma, USA)^{31 (link)}. Human embryonic kidney (HEK-293FT) cells for lentiviral transduction were purchased from Invitrogen (Karlsruhe, Germany) and maintained in DMEM glutaMax media (GIBCO, USA) containing 10% fetal bovine serum. All cells were grown at 37 °C in a 5% CO₂ atmosphere.