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Protocol: Adipogenic Differentiation of Mouse Embryonic Fibroblasts (MEFs) Strains: Wild type (Tsc2 +/+) and Tsc2 -/- (p53−/−) MEFs Source: DJ Kwiatkowski (Brigham and Women's Hospital, Boston, MA) Papers: • Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, et al. (2002) A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in TSC1 null cells. Hum Mol Genet 11: 525–534. • Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, et al. (2003) Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 112: 1223–1233.

Materials • DMEM (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092) • FBS (we test several batches from several sources for differentiation and insulin stimulated glucose uptake then order large quantities of that particular lot. Currently we are using Sigma cat# F2442, lot# 072K8403. Aliquot and store in 50 mL tubes at -20) • NCS (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20) • Penicillin/Streptomycin (100X Invitrogen Cat# 100378-016) • Insulin (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid (28.7μl/50ml) as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20 • Dexamethasone (Sigma D-1756). Dissolve in ethanol as a 2.5 mM stock (0.049g/50ml). Aliquot into 1.5 tubes and store at -20. • MIX (Sigma I-5879). Add 2.78g MIX and 0.98g KOH and bring up to 50 mL. Aliquot into 1.5 mL tubes and store at -20 • Fibroblast Media (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle) • Adipocyte Media (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle) • DMI Media (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions • Insulin Media (Adipocyte Media + Insulin; Sterile filter into new bottle after adding insulin)

Induction of MEF differentiation: 1. Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0). 2. On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM). 3. At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off). • Note1 Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well) 4. Protein lysate and RNA extraction is taken at day 4 (D3). • Note2: In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection. • Note3: A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.