Pam2csk4

N-acetyl-L-cystein (NAC), protease inhibitors, and sodium orthovanadate (Na₃VO₄) were from Sigma. S-LPS from Salmonella enterica serotype abortus equi was a gift from Dr. Brandenburg (Borstel, Germany). Phosphatase inhibitors, set III, was from Merck and 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA), flagellin, poly(I:C), and Pam₂CSK₄ were from Invitrogen. Goat anti-mouse was from Santa Cruz Biotechnology, anti-tubulin from NEB, anti-GFP from Abcam, anti-Myc from Sigma, and anti P-p44/42 MAP kinase was from Cell Signaling Technology. LPS was labeled with Cy5 bifunctional reactive dye (part of labeling kit PA35000; GE Healthcare).

LPS was purchased from Sigma-Aldrich; Pam₂CSK₄, Pam₃CSK₄, poly(A:U),
and poly(I:C) from Invitrogen; mouse IFN-γ from PeproTech; MRT68601,
bosutinib, dasatinib, and ponatinib from Tocris; losmapimod from Biorbyt;
and nilotinib, imatinib, NG-25, and the other compounds from Table S1 were provided by LifeArc. All compounds
are >95% pure by HPLC analysis.

Monolayer chondrocytes were used at passage 2. 150 000 cells were seeded in 6 well plates and were cultured in complete chondrocyte media to confluent. At confluence, cells were pre‐treated with 1/1000 dilution of Sparstolonin B, o‐Vanillin or PBS in serum‐free chondrocyte media for 1 hour before adding a final concentration of 1 μg/mL S100A8/9 (R&D Systems) or 2.5 µg/mL biglycan (R&D Systems). The synthetic agonist Pam2CSK4 (Invitrogen) was also used here as a positive control at a lower concentration of 1 ng/mL. The lower dose was chosen for physiological relevance to simulate the low‐grade inflammation found in OA. For gene expression analysis, cells were lysed in TRIzol 16 hours following alarmin treatment. Culture media were collected after 48 hours.