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).