Monocyte and HLMVEC Gene Expression Analysis

Monocytes and HLMVECs were incubated with medium alone or in medium containing TNF-α (10 ng/mL) for 4 hours, as previously described (64 (link)). Total RNA of monocytes and HLMVECs was isolated using the Direct-Zol RNA Miniprep kit after treatment with DNAse (Zymo Research) (65 (link)). Aliquots of RNA (1 μg) were reverse-transcribed using the Transcriptor First Strand cDNA Synthesis kit (Roche). PCR assays for CD36 and β-actin cDNA were performed using TB Green Premix Ex Taq II (Tli RNase H Plus, TaKaRa) on a 7500 Fast Real-Time PCR System (Applied Biosystems). The cycling program consisted of 1 denaturation cycle of 30 seconds at 95°C, followed by 40 cycles of 5 seconds at 95°C and 34 seconds at 60°C. The primer was 5′-TGTAACCCAGGACGCTGAGG-3′, and the reverse primer was 5′-GAAGGTTCGAAGATGGCACC-3′ for CD36; the primer and reverse primer were 5′-CCTCACCCTGAAGTACCCCA-3′ and 5′-TGCCAGATTTTCTCCATGTCG-3′, respectively, for β-actin.