Macrophage Differentiation and Gene Expression

U937 cell line was differentiated in foam macrophages with phorbol 12-myristate 13-acetate (PMA) 10 ng/ml (Sigma) for 72 hours at 37 °C in RPMI 10% FBS. At day 3 T0901317 (10 μM) or different amount of PFM037 (50, 25 and 10 μM) were added for 16 hours. Total RNA was purified by TRIZOL (Invitrogen). Reverse transcription was performed by MLV-reverse transcriptase (Promega). QPCR was performed using SYBR Green Master Mix (Applied Biosystems) and real-time PCR (Viia 7 Real Time PCR System, Applied Biosystems). PCR reactions were done in triplicate. The comparative Ct method was used to quantify transcripts that were normalized for human GAPDH. We used the following primer pairs:
GAPDH-F 5’-ACA TCA TCC CTG CCT CTA CTG-3’; GAPDH-R 5’-ACC ACC TGG TGC TCA GTG TA-3’
ABCA1-F 5’-CCA GGC CAG TAC GGA ATT C-3’; ABCA1-R 5’-CCT CGC CAA ACC AGT AGG A-3’
SREBP-1c-F 5’-GGC GGG CGC AGA TC-3’; SREBP-1c-R 5’-TTG TTG ATA AGC TGA AGC ATG TCT-3’
FAS-F 5’-ACA GCG GGG AAT GGG TAC T-3’; FAS-R 5’-GAC TGG TAC AAC GAG CGG AT-3’
SCD1-F 5’-TTC AGA AAC ACA TGC TGA TCC TCA TAA TTC-3’; SCD1-R 5’-ATT AAG CAC CAC AGC ATA TCG CAA GAA AGT-3’