DMEM–Dulbecco's Modified Eagle Medium

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	supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin (Mediatech/Corning Life Sciences, Manassas, VA, USA), and 10% FBS (Gemini Bio-Products, West Sacramento, CA, USA)

Protocol tips
supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin (Mediatech/Corning Life Sciences, Manassas, VA, USA), and 10% FBS (Gemini Bio-Products, West Sacramento, CA, USA)

Publication protocol

Human breast cancer cell lines T47D (mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection (www.atcc.org; Manassas, VA, USA). Cells were maintained at 5% CO2 in DMEM (Life Technologies, Carlsbad, CA, USA) with 50 U/ml penicillin, 50 μg/ml streptomycin (Mediatech/Corning Life Sciences, Manassas, VA, USA), and supplemented with 10% FBS (Gemini Bio-Products, West Sacramento, CA, USA) in a 37 °C humidified incubator. T47D cells generated with inducible MDM2 knockdown were described previously [6]. Constitutive MDM2 or MDMX knockdown cell lines were generated by retroviral infection with MLP.GFP vector (a generous gift from Scott Lowe) containing mir30 short hairpin RNA (shRNA)-expressing vector, mdm2 151656 shRNA, or mdmx 13023 shRNA. The mir30 shRNA inducible expressing vector has been used as a control for numerous previous high-impact studies [23, 24], and the only difference for the stable knockdown cell lines was a constitutively active promoter. Cell lines were generated and selected as previously described. All stable knockdown cell lines were used as selected pools.

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