Quantitative real-time PCR (qRT-PCR) and Enzyme-linked immunosorbent assay (ELISA)
Total RNA was isolated from cultured cells using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. RNA was reverse-transcribed using a ReverTra Ace qPCR RT Kit (Vazyme, China) and qRT-PCR was performed using Light-Cycler 480 (Basel Roche, Switzerland). All primers were synthesized by BGI (Beijing, China) and the sequences of all primers used in this study are listed in Online Supplementary Table 2. Relative mRNA levels are measured using the 2-ΔCycle Threshold (2-ΔCT) method. Three independent experiments were performed, and each reaction was repeated three times.
Following treatment with AZM or ETC, cell culture supernatants were collected for the measurement of IL-1α, IL-1β, IL-6, IL-8, TNF-α, MMP-1, MMP-3, CXCL9, CXCL10 and VEGF using ELISA kits (Hangzhou Multi-Science Company of China). Fresh blood was extracted from 20-week-old mice treated with indicated drugs, and the serum was collected for measuring the release of IL-6, IL-1β, COMP, IL-10, IL-13, RANKL and OPG by ELISA kits (Hangzhou Multi-Science Company of China).