Trizol assay

RT-PCR analysis was performed as mentioned previously [46 (link)]. Briefly, THP-1 cells were incubated with vehicle control or α-MMC (40 μg/mL) for 24 h at 37 °C and the total RNA was then extracted using TRIzol assay (Invitrogen), followed by conducting the reverse transcription using the Prime Script RT Kit (Takara, Shiga, Japan) according to the manufacturer’s protocol. cDNA products were analyzed by Human Inflammatory Response and Autoimmunity RT² Profiler™ PCR Array following the manufacturer’s instructions. LPS at sub-lethal dose of 1 µg/mL was used as positive control. Alternatively, real-time PCR on cDNA was carried out in an Applied Biosystems ViiA 7 real-time PCR machine (Thermo Fisher Scientific, Carlsbad, CA, USA) using SYBR Green assays (Bio-Rad, Hercules, CA, USA). The PCR primers were as follows: 5′-TTCGACACATGGGATAACGAGG-3′ and 5′-TTTTTGCTGTGAGTCCCGGAG-3′ for human IL-1β; 5′-ACTGAGAGTGATTGAGAGTGGAC-3′ and 5′-AACCCTCTGCACCCAGTTTTC-3′ for human IL-8; 5′-GAGGCCAAGCCCTGGTATG-3′ and 5′-CGGGCCGATTGATCTCAGC-3′ for human TNF-α; 5′-CAGCCAGATGCAATCAATGCC-3′ and 5′-TGGAATCCTGAACCCACTTCT-3′ for human MCP-1.

TRIzol assay (Invitrogen, Carlsbad, CA) was used to extract the total RNA according to the instructions. The quality and quantity of RNA were monitored by OD_260/280. The RNA OD_260/280 (1.8–2.0) was used for the experiments.