Mccoy s 5a modified media

HCT116 cells were purchased from American Type Culture Collection (authenticated by STR DNA profiling) and were tested negative for Mycoplasma contamination.
HCT116 cells were cultured in McCoy’s 5A (modified) Media (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and Pen/Strep at 37 °C in humidified incubator with 5% CO₂. Cells were passaged every 2–3 days.
HCT116 cells in 6-well plates were transfected by Lipofectamine LTX (15338100, Thermo Fisher) with 2.5 μg of pX330-puro plasmid containing gRNA (5′-TCCAGTAAGTACAGAAAATG-3′) and Cas9 for RHBDL4 knockout. Twenty-four hours after transfection, cells were trypsinized and plated in a 10 cm petri dish. After 24 h, the cells were treated with puromycin (1 μg ml⁻¹). The puromycin selection stopped after 48 h. Cells were trypsinized and limited dilution was performed to generate single clones, which were expanded and analysed by western blot (anti-RHBDL4) and genotyped by sequencing the genomic DNA region targeted by the gRNA.
To detect the proteolytic fragments from endogenous substrates generated by endogenous RHBDL4, 10 million wild-type or RHBDL4 knockout HCT116 cells were cultured in hybridoma serum free medium for 40 h. The medium was collected, filtered and concentrated by a 30 kDa cut-off concentrator. Proteins were precipitated by TCA and dissolved in 1× LDS loading buffer for immunoblotting analysis.

HT29 colon carcinoma cells containing a ZnCl₂-inducible wt-APC or β-galactosidase (control) expression vector were provided by Dr. Bert Vogelstein’s lab (Johns Hopkins University) [9 (link)]. We refer to the modified HT29 cells as HT29-APC or HT29-GAL cells. HT29 cells were chosen for most of the study because they contain a homozygous APC mutation and do not have any known retinoic acid receptor gene mutations. Modified cells were cultivated in McCoy’s 5A modified media (Gibco/Thermo Fisher Scientific, Waltham, MA USA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals/R&D Systems, Minneapolis, MN, USA), 1% penicillin:streptomycin (pen-strep; VWR), and 600 μg/mL hygromycin B (Thermo Fisher Scientific, Waltham, MA, USA). All variations of HT29 cells grew equally well in terms of cell density. Respective expression plasmids were induced with 120 μM ZnCl₂ (Sigma-Aldrich, St. Louis, MO, USA) for the times indicated. HT29-APC cells treated with ZnCl₂ are referred to as wt-APC and those not treated with ZnCl₂ are referred to as mt-APC. All cells were maintained in a 37 °C incubator with 5% CO₂.

Two colorectal cancer cell lines (DLD-1 p53+/+ and HCT116 p53+/+), both a gift from Prof. Vogelstein (John-Hopkins-University, Baltimore, MD) were maintained in McCoy´s 5A (modified) Media (Gibco) supplemented with 10% foetal bovine serum (FBS) (Sigma Aldrich), 1% penicillin-streptomycin (Biochrom), 1% Sodium pyruvate and 1% Non-Essential Amino acids (100X) (Gibco). For lentiviral particle production, HEK293T cells (Thermo Fisher) were maintained in Hyclone- Dulbecco’s Minimum Essential Media (DMEM, Gibco), supplemented with 10% FBS and 200 μM L-glutamine. All cell lines were kept in an incubator at 37°C in 5% CO2.