Sp600125

BMSCs (2.0x10⁴) were seeded into HyCyte™ rat BMSC culture medium (Cas9X Biotech Co. Ltd.) and were allowed to attach for 24 h, after which the medium was replaced with HyCyte™ osteogenic differentiation medium (Cas9X) and changed every 3 days. After 14 days, cells were fixed with 4% polyformaldehyde for 15 min and then stained with 0.1% sodium alizarin sulfonate-Tris-HCL staining solution (pH=8.3) for 30 min at 37˚C. Stained cells were then washed with PBS and imaged through a light microscopy. For inhibitor treatments, both U0126 (cat. no. T21332; TargetMol Chemicals Inc.) and SP600125 (cat. no. T3109; TargetMol Chemicals Inc.) were used at a concentration of 10 µM.

U937 cells, with a density of 10⁶ cells/ml, were put in each well of the 48 well plates. In the experiment of gingival fibroblasts, a density of2x10⁴ (link) cells was selected. Before studies, the starvation was accomplished in a serum-free medium for 24hr. After being treated with different concentrations of S. aureus LTA and PGN for 48hr, the supernatants were collected, centrifuged, and stored at −80 °C for later analysis. The ranges of 0–10 μg/ml and 0–100 μg/ml were selected for LTA and PGN, respectively, according the previous report.²⁷ (link) In the experiment treated with NF-κB inhibitor (100 μM PDTC) (Sigma–Aldrich) and MAPK inhibitors (10 μM SCH7 72984 for extracellular signal-regulated kinase 1 and 2, ERK 1/2; SB203580 for P38 MAPK; and SP600125 for c- Jun N-terminal kinase, JNK) (TargetMol, Boston, MA, USA), cells were pretreated with inhibitor 1–2 h prior to stimulation. Then, S. aureus LTA or PGN (10 μg/ml in each) was added for 48 h. The supernatants were collected, centrifuged and stored at −80 °C for later zymography.