*4.9. Measurement of Cytokine Expression Levels by RT-PCR*

Total RNA was extracted using a Quick-RNA MiniPrep kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol. RNA concentrations were quantified using a spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific, Waltham, MA, USA). The mRNA expression levels of interleukin 1 beta (*IL-1*β), *TNF-*α, *IL-6*, *FAK*, *paxillin*, and *PAK1* were quantified by RT-PCR by a one-step RT-PCR kit (TaKaRa, Shiga, Japan) with an ABI Prism 7500 Fast Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) as previously described [30]. The relative expression levels of mRNA were defined by the following equation: (39—threshold cycle (Ct) after being adjusted by the expression of 18S ribosomal RNA).
