F9423

Unless otherwise stated, cell culture reagents were purchased from Gibco (ThermoFisher Scientific). A549 cells were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM:F12) with HEPES, supplemented with 10% foetal bovine serum (JRH Biosciences #12003C or Sigma-Aldrich #F9423) and 2 mM GlutaMAX™ or 2 mM L-Glutamine. HEK-293 cells were cultured in DMEM supplemented with 10% FBS (Sigma-Aldrich #F9423) and 2mM L-glutamine. MCF10A cells were cultured in DMEM:F12 containing 5% horse serum (#16050-122), 20 ng/mL epidermal growth factor (EGF, Peprotech), 0.5 mg/mL hydrocortisone (Sigma-Aldrich #H-0888), 100 ng/mL cholera toxin (Sigma-Aldrich #C-8052) and 10 μg/mL insulin (available from pharmacy). The Flp-In™ T-Rex™ U2OS cells were cultured in DMEM supplemented with 10% FBS and 1 μg/mL Blasticidin (Melford). For expression of MDM2 protein, the cDNA for the human full-length MDM2 sequence was cloned into the pcDNA5 vector (ThermoFisher Scientific) to enable expression of the proteins with an N-terminal 2xFLAG-PreScission protease site-His6 (FLAG) tag. The plasmid was then transfected into the cells for stable integration into the host genome and selected using Hygromycin B (Formedium) according to the manufacturers’ instructions.

To generate MEFs, C57BL/6 Mdm2^C305F/C305F mice or their wild-type C57BL/6 littermates were plug mated. Mouse embryos were isolated at E13.5 and prepared as similarly described (Durkin et al., 2013 (link)), digesting individual embryos in 1 mL of 0.25% trypsin-EDTA (Gibco) containing 100U DNaseI (Sigma-Aldrich #D5025) at 37°C at 10% CO₂ for 15 minutes. To stop the digestion, 1 mL of DMEM (Gibco) supplemented with 10% FBS (Sigma-Aldrich #F9423), 1x Antibiotic-Antimycotic, 2 mM GlutaMAX™ and 1x MEM non-essential amino acids (NEAA) was added, and the cells were centrifuged at 300 x g for 5 minutes to pellet the cells. Cell pellets were then resuspended and cultured in fresh DMEM containing 10% FBS, 1x Antibiotic-Antimycotic, 2 mM GlutaMAX™ and 1x MEM NEAA at 37°C.