Adipocyte Isolation and Genetic Manipulation

Primary adipocytes were isolated and cultured as per a previously described method [56 (link)]. In experiments involving the administration of Ad-Col15a1 or sh-Col15a1 (24 h or 48 h at the titer of 1 × 10⁹ IFU/mL), AMPK inhibitor (Compound C, Selleck, Houston, Texas, USA), mTORC1 inhibitor (rapamycin, Selleck), MMP agonist (4-Aminophenylmercuric acetate, Sigma–Aldrich, St. Louis, Missouri, USA), FGFR1 inhibitor (PD173074, Selleck), LOX inhibitor (β-APN, Sigma–Aldrich), or FGF2 recombinant protein (50037-M07E, Sino Biological, Beijing, China), cells were allowed to adhere for 48 h before treatment. The medium was subsequently replaced, and adipocyte petri dishes were treated with the indicated reagents. Cells were assessed by RT-PCR and western blot or fixed with 4% paraformaldehyde and stained for markers of interest.