Mouse 3t3 l1 preadipocytes

3T3-L1 mouse pre-adipocytes were purchased from the ATCC (ATCC, Manassas, VA, USA). After thawing, 3T3-L1 cells were resuspended in an α-minimal essential medium (α-MEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (F.B.S.; Invitrogen) and 1% antibiotic/antimycotic solution (Invitrogen). The cultures were maintained at 37 °C under an air/5% CO₂ atmosphere and the medium was changed after 48 h and every 3–4 days thereafter. Upon reaching confluence, the cells were recovered by the addition of 0.25% trypsin/EDTA (Invitrogen) and sub-cultured [17 (link),27 (link),28 (link)].
To generate adipocytes overexpressing PGC1-α or deficient in PGC1-α, 1 × 10⁶ cells were seeded in 6-well plates one day before transduction. Adipo-ORF PGC1-α lentivirus and Adipo-sh PGC1-α (Vector builder, Shenandoah, TX, USA) were applied to adipocyte (3T3-L1) cells to establish stably transduced cell lines. The transduction medium consisted of 1 × 10⁶ transducing units (T.U.) of lentiviral particles in 0.5 mL α-MEM growth medium, and this was applied to each well and incubated for 3 h to maximize the contact between the cells and lentiviral particles. The cells were also treated with the transduction medium without the lentiviral particles as described [17 (link),25 (link),26 (link)].

3T3-L1 mouse pre-adipocytes were purchased from ATCC (Manassas, VA, USA). The cells were maintained in Dulbecco modified Eagle medium (DMEM) with 10% FBS and 1% penicillin/streptomycin and incubated at 37 °C in 5% CO₂. Pre-adipocytes were differentiated as previously described [25 (link)]. Briefly, two days after 3T3-L1 pre-adipocytes reached 100% confluency, cells were treated with differentiation media containing insulin, isobutylxanthine, dexamethasone, and rosiglitazone. To examine the effects of METRNL, mature 3T3-L1 adipocytes were treated with 100 ng/mL recombinant METRNL (AG-40B-0149) on day 8 for 1 h, followed by 10 ng/mL tumor necrosis factor (TNF)-α (T7539, Sigma) for 24 h.

3T3-L1 mouse preadipocytes were purchased from ATCC (Manassas, VA, United States). For maintenance of preadipocytes, 100 mm culture plates were used. For gene expression analysis and Western blotting the cells were seeded in 6 well plates, whereas 96 well plates were used for cell viability assay and Oil Red O staining. The cells were maintained in Dulbecco modified Eagle medium (DMEM) with 10% FBS (HyClone, Australia) and 1% penicillin/streptomycin (Carlsbad, CA, United States) and incubated at 37°C in 5% CO₂. 3T3-L1 cells were treated with differentiation media (MDI; methylisobutylxanthine, dexamethasone, insulin) to induce adipogenesis as previously described (Huh et al., 2012 (link)). To examine the effect of irisin on adipogenesis, 100 ng/mL Recombinant irisin was treated every other day for 6 days, starting from 2 days before changing to MDI. Recombinant irisin was purchased from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, United States). Plasmid encoding FNDC5 gene and small interfering RNA were also used to examine the effect of genetic manipulation of irisin on adipocyte differentiation, as described below.