3T3-L1 fibroblast culture and differentiation into adipocytes

Mycoplasma-free 3T3-L1 fibroblasts obtained from 3T3-L1 Howard Green (Harvard Medical School, Boston, MA) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS) (Thermo Fisher Scientific), 1% GlutaMAX (Thermo Fisher Scientific) in a humidified atmosphere with 10% CO₂. HA-GLUT4 overexpressing 3T3-L1 fibroblasts were generated by retroviral transduction as previously described (Govers et al., 2004). Confluent 3T3-L1 cells were differentiated into adipocytes by the addition of DMEM containing 0.22 µM dexamethasone, 100 ng/mL biotin, 2 µg/mL insulin, 500 µM IBMX (day 0). After 72 hr, medium was replaced with DMEM/10% FCS/GlutaMAX containing 2 µg/mL insulin (day three post differentiation). After a further 72 hr (day six post differentiation), cells were cultured in DMEM/10% FCS/GlutaMAX. Medium was subsequently replaced every 48 hr. Cells were used between days 10 and 15 after the initiation of differentiation.

For stable isotope labelling of amino acids in cell culture (SILAC)-based proteomics, 3T3-L1 fibroblasts were passaged for six cell divisions in DMEM (Sigma Alrich)/10% dialysed FCS (Thermo Fisher Scientific) containing L-arginine (Arg 0) and L-lysine (Lys 0) (‘light’), L-arginine-U-¹³C₆¹⁴N₄ (Arg 6) and L-lysine-²H₄ (Lys 4) (‘medium’) or L-arginine-U-¹³C₆¹⁵N₄ (Arg 10) and L-lysine-U-¹³C₆¹⁵N₂ (Lys 8) (‘heavy’). Final concentrations of arginine and lysine were 33 µg/mL and 76 µg/mL, respectively. This strategy generated three distinct SILAC populations. We periodically tested labelling efficiency by mass spectrometry analysis. SILAC-labelled fibroblasts were differentiated into adipocytes as above.