Psuperior retro puro

3T3-L1 pre-adipocyte cells and C3H10T1/2 mesenchymal cells were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% calf serum or 10% fetal calf serum (FCS). For adipogenic differentiation, 2 day post-confluent cells were differentiated with a standard adipogenic cocktail (1 µg/ml insulin, 0.25 µg/ml dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX) with 10% FCS) or with the indicated subset of components. Where indicated, forskolin (10 µM, Calbiochem) was added to serum-containing differentiation media lacking the differentiation cocktail components for the first two days of the differentiation process. 3T3-L1 cells were also pre-treated for 1 hour prior to administration of differentiation cocktail with either 10 µM H89 (Sigma) or 10 µM myr-PKI (Calbiochem). BOSC23 retroviral packaging cells were cultured in DMEM containing 10% FCS and were cycled through selective media every 1–2 months as described [31] (link). pSuperior-retro-puro (OligoEngine, Seattle, WA), pSuperior-retro-puro-PKAC1α, pSuperior-retro-puro-PKAC1β, and pSuperior-retro-puro-C/EBPβ [23] (link) viral packaging was achieved by transfection of the plasmid into BOSC23 cells using Fugene6 (Roche) as described [23] (link). Collection of viral supernatant and infection of cells was also described previously [23] (link).

Retrovirus expressing shRNA against SIRT3 (5′-GCGCCTATCAGTACACAAT-3′ for SIRT3-KD1, 5′-CTCCTCTGTTGCCTTGGTA-3′ for SIRT3-KD2) or scrambled (5′-CAACAAGATGAAGAGCACC-3′) was made by cotransfecting pSUPERIOR.retro.puro (Oligoengine) construct containing the above shRNA sequences with pVSV-G retroviral packaging vector into Phoenix cells (ATCC). Retrovirus was collected from supernatant of Phoenix cells and used to infect MSCs, which were subsequently selected with 0.5 μg/mL puromycin for 5 days. Puromycin was removed prior to differentiation experiments. For SIRT3 overexpression, SIRT3 cDNA was cloned into pcDNA3.1 vector [34 (link)] and transfected with Fugene (Promega). Efficiencies of knockdown and overexpression were assessed by western blots.