Kn 93

The PDLtms were randomly assigned into three groups, i.e., the control group, the compression group (Cg) and the compression+KN-93 (an inhibitor of the CAMK II pathway) group (CKg). To simulate the pressured periodontium in OTM, a modified “weight” method was used. Briefly, a cover glass and a bottle of granules were placed on the PDLtm to produce a compression of 25 g/cm², which has been proved to be the optimal force level for this model¹⁸. In the compression+KN-93 group, 0.01 mM KN-93 (Sigma) was added to the media to suppress the CAMK II pathway.

Astrocyte-enriched cultures were generated from newborn CD1 Swiss mice and maintained in culture as previously described [28 (link)]. Generation/maintenance of U251 cell lines expressing wild type (MLC1-WT) or mutated MLC1 (MLC1-S280L) has been previously described [20 (link)]. Cell treatments were performed in serum-free (SF) medium, as follows: 5 min with adenosine triphosphate (ATP, 100 µM, Sigma-Aldrich, Saint Louis, MO, USA), 30 min with isotonic solution (140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 20 mM D-glucose, 5 mM HEPES, pH 7.4) or hypotonic solution prepared by adding 30% distilled water to the isotonic solution (Hypo) [29 (link)]. For high K⁺ stimulation, cells were treated for 30 min with the same isotonic buffer, with the KCl concentration increased up to 60 mM (HK), as previously described [30 (link)]. For CaMKII inhibition U251 MLC1-WT cells and mouse astrocytes were treated for 15 min with 10 µM of KN93 (10 µM, Sigma-Aldrich, Saint Louis, MO, USA) in isotonic solution or co-stimulated for 15 min with 10 µM KN93 in hypotonic solution. Cycloheximide (CHX) treatment (100 µg/mL, Sigma-Aldrich) was maintained for 1, 3, 4, or 5 h.