Gentamicin reagent solution

Human osteosarcoma U2OS cells (ATCC) with MDM2-knockout and doxycycline-inducible p53 shRNA (U2OS^mod)^{44 (link)} and unmodified U2OS cells were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, 0.1 mg ml⁻¹ streptomycin, 100 U ml⁻¹ penicillin, 20 mM l-glutamine, and 6 mg l⁻¹ gentamicin reagent solution (Invitrogen, USA). For U2OS^mod cells, the medium was further supplemented with 50 μg ml⁻¹ doxycycline to keep p53 knocked down. The cells were cultured in monolayer at 37 °C in 5% CO₂ and transfected using jetPRIME transfection reagent (Polyplus transfection) following the manufacturer’s protocol. For cycloheximide chase experiments (Fig. 6a, d), U2OS^mod cells were transfected with 2.5 μg MDM2 variants. Cell-based ubiquitination assays (Fig. 6b, f) were performed by transfecting U2OS^mod cells with 5 μg MDM2 variant or EV as indicated in the figures, along with 1 μg 12× His-Ub. In Fig. 6c, U2OS cells were co-transfected with 1 μg Myc-p53 and 5 μg of the indicated MDM2 variant or EV, while in Fig. 6e, U2OS^mod cells were transfected with 5 μg MDM2 variant or EV as indicated. Cells were harvested 36 h post transfection.

The human CC cell lines SiHa and C-33A (ATCC, Manassas, VA) were maintained in Eagle's Minimum Essential Medium supplemented with 10% heat-inactivated fetal bovine serum (Bioserum, Japan). The normal human lung fibroblast cell line NHLF (Clonetics, San Diego, CA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Bioserum, Japan). Vero cells were used to produce measles virus and maintained in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated fetal bovine serum (Bioserum, Japan). All media used in this study contained 100 U/ml of penicillin - streptomycin. Primary human CC tissues were established using surgical specimens immediately after resection from the First Affiliated Hospital of China Medical University after institutional review board approval and informed patient consent. Briefly, tissues were treated with collagenase (GIBCO, Invitrogen, Carlsbad, CA) at 37°C for 2 hours on a shaker, and then filtered through a nylon mesh (100-μm diameter) to obtain single cell suspensions. Harvested cells were cultured in Minimum Essential Medium-α medium (GIBCO, Invitrogen) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and 4 μg/ml of Gentamicin Reagent Solution (GIBCO, Invitrogen). All cell lines used in this study were cultured in a humidified atmosphere containing 5% CO₂ at 37°C.