Taq green master mix

S-nitroso-N-acetyl-penicillamine (SNAP), 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT),N^ω-nitro-L-arginine methyl ester (L-NAME), Griess reagent, and interleukin-1α(IL-1α) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A microchemoattraction chamber (No. 3415; Transwell, Sigma-Aldrich) was purchased from Corning (Acton, MA, USA). Fetal bovine serum (FBS), Dulbecco's modified eagle's medium (DMEM), penicillin/streptomycin, and trypsin were purchased from Gibco (Invitrogen, Carlsbad, CA, USA). Primers for mRNA were obtained from Genet bio (Seoul, Korea). Trizol was purchased from Invitrogen and Taq Green Master Mix was purchased from Promega (Fitchburg, WI, USA). All other reagents and general lab chemicals were purchased from Sigma-Aldrich Chemical.

Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA). Northern blot analysis to measure the mRNA expression of lipoprotein lipase (LPL) and PPARγ2 was performed before and after the growth arrest to evaluate the effects of PGs on early differentiation. Briefly, an RNA denaturation mix composed of isolated RNA, oligo dT primer, and nuclease-free water was used to denature the RNA. The polymerase chain reaction (PCR) primer pairs for cDNA amplification were as follows: LPL (forward) 5'-TGCCGCTGTTTTGTTTTACC-3' and (reverse) 5'-TCACAGTTTCTGCTCCCAGC-3', PPARγ2 (forward) 5'-TGCCGCTGTTTTGTTTTACC-3' and (reverse) 5'-AATCAGCAACCATTGGGTCA-3'. cDNA was synthesized by adding prime RT premix (Genet Bio). Taq Green Master Mix (Promega, Madison, WI, USA) and 10 pmol of forward primer and reverse primer were added to the synthesized cDNA, and amplified with annealing for 30 cycles with DNAEngine cycler (Bio-Rad, Hercules, CA, USA). The DNA band of amplified PCR products was analyzed by multi-Gauze (Fujifilm, Tokyo, Japan) after electrophoresis. The level of β-actin was used as an internal standard.