Sp600125

Cells were seeded in 12-well plates for cell viability or in P100 plates (Corning, New York, NY, USA) for RNA and protein purification at a density of 100.000 cells/well or 1.2 × 10⁶ cells/plate, respectively. Then, cells were allowed to attach and reach 100% confluency for 24 h before removing the medium and replacing it with serum-free medium for 24 h. Treatments were then added and maintained for 24 h, with the exception of ELISAs that were maintained for 48 h, and some protein extracts that were collected at shorter times. Cells were treated with 8–20 μM 7KCh (Sigma-Aldrich, Madrid, Spain) alone or with 10 μM SA (PPQF, University of Alcalá, Madrid, Spain), 10 μM of the TLR4 inhibitor CLI-095 (Invivogen Inc., San Diego, CA, USA), 10–50 μM of the JNK inhibitor SP600125 (StressMarq Biosciences Inc., Victoria, BC, Canada) or 5–40 μM of the p38 inhibitor SB203580 (Sigma-Aldrich, Madrid, Spain). 7KCh was prepared in β-cyclodextrin (Sigma-Aldrich, Madrid, Spain) as previously described [15 (link),20 (link)], CLI-095 was dissolved in culture medium and SA, SP600125 and SB203580 were dissolved in DMSO (Sigma-Aldrich, Madrid, Spain).

All reagents were obtained from Sigma Aldrich/Merck (Darmstadt, Germany) unless stated otherwise. The c-Jun N-terminal kinase inhibitor SP600125 was obtained from StressMarq Biosciences (Victoria, Canada), and tanshinone IIA (TIIA) 2 was obtained from Generon (Slough, UK). All synthetic compounds were synthesised using chemistry as outlined in the previous section. All compounds were dissolved in dimethyl sulfoxide (DMSO).