The RASFs were plated in 6-well dishes and grown to confluence. The culture medium was then exchanged with serum-free RPMI medium. Cells were pretreated for 30 min with pharmacological activators Angiotensin II (a FAK activator), 740-YP (a PI3K activator), SC-79 (an AKT activator) and prostratin (an NF-κB activator) to stimulate the RSAFs, before 24 h of Antcin K (0, 0.3, 1, 3, or 10 μM) treatment. The conditioned medium was collected and stored at –20°C until use. Secreted expression of TNF-α, IL-1β and IL-8 was examined using ELISA kits for TNF-α, IL-1β and IL-8 (R&D Systems, MN, USA), according to the manufacturer’s protocols.
Serum levels of TNF-α, IL-1β, IL-8 and IL-6 in CIA mice were examined using ELISA kits for TNF-α and IL-1β (R&D Systems, MN, USA), IL-8 (MyBioSource, Inc, San Diego, USA) and IL-6 (Max™ Set Deluxe Kits, BioLegend, USA) according to the manufacturers’ protocols.
Serum levels of TNF-α, IL-1β, IL-8 and IL-6 in CIA mice were examined using ELISA kits for TNF-α and IL-1β (R&D Systems, MN, USA), IL-8 (MyBioSource, Inc, San Diego, USA) and IL-6 (Max™ Set Deluxe Kits, BioLegend, USA) according to the manufacturers’ protocols.