Ad pparγ

HPASMC monolayers were grown at 37°C in a 5% CO₂ atmosphere in culture media (SmGM-2, Lonza) containing 5% fetal calf serum, growth factors, and antibiotics. Human PPARγ in adenovirus (Ad-hPPARγ) or Ad-GFP (Vector Biolabs, Philadelphia, PA) were applied to cells at 3-28 MOI (Ad-GFP was applied at 3 MOI and Ad-PPARγ was applied at 28 MOI) for 4 hours in 2% FBS media. Media were replaced with fresh SmGM-2 media, and HPASMC were cultured for 72 hrs.

The plasmid that contained the cDNA of the human ACSL1 (pBS-hACS) was purchased from Open Biosystems. The hACSL1 cDNA was isolated with double digestion using BamHI and XbaI restriction enzymes. The 5′ and 3′ ends of hACSL1 were blunted with DNA polymerase I, Large (Klenow) Fragment. Accordingly, pAd-TrackCMV plasmid was digested with SalI restriction enzyme and ends were blunted with Klenow fragment. The cDNA of hACSL1 was then cloned in pAd-TrackCMV. The pAd-TrackCMV-hACSL1 plasmid was used to produce adenoviral particles as previously described [24 (link)] using the Ad-Easy-1 system [25 (link)]. The recombinant adenoviral vectors were linearized with PacI and used to infect human embryonic kidney 293 cells. The recombinant adenoviruses were purified by two consecutive cesium chloride ultracentrifugation steps, dialyzed, and titrated. Usually, titers of −5 × 10¹⁰ plaque-forming units (pfu)/ml were obtained. The adenovirus expressing the human PPARγ cDNA (Ad-PPARγ) was purchased from Vector Biolabs (Philadelphia, PA, USA). The adenovirus expressing ATGL was constructed as described previously [26 (link)]. Recombinant adenoviruses expressing green fluorescent protein (Ad-GFP) served as control.