*4.6. QRT-PCR*

RAW264.7 cells plated into a 6-well plate at a density of 3 × <sup>10</sup><sup>5</sup> cells/well were pre-treated with 150 μM of wistin for 30 min, followed by LPS (0.1 μg/mL) treatment. After 0, 2, 8, 12, and 24 h, cells were washed with DPBS and harvested. Total RNA was isolated using the TRIzol reagent (Invitrogen, Waltham, MA, USA). cDNA was synthesized from total RNA using a SimpliAmp Thermal Cycler (Applied Biosystems Co., Waltham, MA, USA), and then qRT-PCR was performed on a Step One Plus Real-time PCR System Cycler (Applied Biosystems Co.) using a Power SYBR Green PCR mix Cycler (Applied Biosystems Co.). The PCR primer sequences (forward and reverse) used are listed in Table 1, and β-actin was used as the reference gene.

**Table 1.** Primers used in the quantitative reverse transcription polymerase chain reaction.


