Adipocyte Differentiation and Manipulation

The SVF was isolated from iWAT, C3H10T1/2 (CCL-226, American Type Culture Collection) cells, and 293T (CRL-3216, American Type Culture Collection) cells were maintained in DMEM supplemented with 10% FBS. To induce adipocyte differentiation, confluent SVF or C3H10T1/2 cells were cultured with media containing an adipogenic cocktail consisting of 2.5 μM or 1 μM dexamethasone (D4902, Sigma), 5 µg ml⁻¹ insulin (I0516, Sigma), 500 μM isobutylmethylxanthine (I5879, Sigma) and 1 μM rosiglitazone (R2408, Sigma) for 2–3 days. Subsequently, the cells were switched to maintenance medium (culture medium supplemented with 5 µg ml⁻¹ insulin). For Lats1/Lats2 deletion in mature adipocytes differentiated from iAKO SVF, cells were treated with 1 µM 4-hydroxytamoxifen (H7904, Sigma) for 10 days, starting at 7 days after adipocyte differentiation. C3H10T1/2 cells transduced with TAZ4SA-ER^T2 retroviruses containing a puromycin-resistance cassette were subjected to puromycin (2 µg ml⁻¹) selection to establish tamoxifen-inducible TAZ4SA cells. Next, 1 µM 4-hydroxytamoxifen (H6278, Sigma) was treated for 24 h to activate TAZ4SA-ER^T2. For adenoviral expression, cells were infected with adenoviruses encoding TAZ4SA or GFP as described previously^{51 (link)}. For pharmacological inhibition or activation of YAP/TAZ, C3H10T1/2 day-10 adipocytes were treated with 5 μM verteporfin (SML0534, Sigma) for 12 h or C3H10T1/2 day-8 adipocytes were treated with 2 μM lysophosphatidic acid (L7260, Sigma) for 2 h after 12 h of serum starvation. To modulate YAP/TAZ pharmacologically, C3H10T1/2 cells at 8 or 10 days after adipocyte differentiation were treated with either 2 μM lysophosphatidic acid (L7260, Sigma) for 2 h following 12 h of serum starvation or 5 μM verteporfin (SML0534, Sigma) for 12 h, respectively, with ethanol as the vehicle for verteporfin and DMSO for lysophosphatidic acid.