Retro x tet on advanced system

First, 3T3-L1 cells were differentiated into adipocytes using differentiation medium containing 3-isobutyl-1-methylxanthine (0.5 mmol/l), dexamethasone (1 μmol/l), insulin (1 μmol/l), and pioglitazone (10 μmol/l). The cells were used in experiments 7 days after differentiation. Treatment with recombinant human TGF-β1 (R&D Systems) was performed after changing the media to serum-free Dulbecco's modified Eagle's medium to avoid unknown serum effects. PlatE cells were used to produce retrovirus, followed by transfection in 3T3-L1 cells. Stable 3T3-L1 cells expressing tetracycline (tet)-inducible TGF-β1 (3T3-L1–tet–TGF-β1) or control cells (3T3-L1–tet–empty cell) were produced using the Retro-X Tet-On Advanced system according to the protocol supplied by the manufacturer (Clontech). The coding region of mouse TGF-β1 was subcloned into the expression vector pRetroX-Tight-Pur. Retroviral particles were generated using pRetroX-Tight-Pur-TGF-β1 or pRetroX-Tight-hygro-empty and pRetroX-tet-on advanced vectors. Infected 3T3-L1 cells were selected in 400 mg/ml G418 and 5 mg/ml puromycin. Other materials were as described: SB431542 (Selleck Chemicals), SIS3 HCl (Selleck), PF-573228 (Selleck), MK2206 (Selleck), collagenase (Sigma–Aldrich), rapamycin (Selleck), and U0126 (Selleck).

The Retro-X™ Tet-On^® Advanced system (Clontech) was used to establish 143B cells with doxycycline-inducible expression of LONP1-FLAG, OXA1L-FLAG and mtHSP70-HA. Cells were incubated with doxycycline (0.1 μg/ml for OXA1L-FLAG and mtHSP70-HA; as indicated concentration for LONP1-FLAG) to induce protein expression.