Pkf118 310

To investigate the impact of Wnt/β-catenin-mediated pathways on IoN pathophysiology, PKF 118-310 (a Frizzled-independent Wnt pathway antagonist that prevents the nuclear translocation of β-catenin) was injected in the close vicinity of the right IoN using an intraoral transmucosal injection technique as previously described.⁵ Each injection consisted of 50 µL of either PKF 118-310 (50 μM; Sigma-Aldrich, Saint-Quentin Fallavier, France) or vehicle (NaCl 0.9%). Rats were pretreated 24 h before surgery (three injections spaced 6 h apart), with the last injection occurring during the surgical procedure (one half of injection volume deposited on the exposed nerve and the other half injected transmucosally after suture of the oral mucosa). Evaluation of resulting molecular alterations and/or BNB permeability was performed 3 h after last perineural injection. After sacrifice, IoN samples were harvested and immediately frozen in liquid nitrogen then stored at −80℃, except for BNB permeability assessment (see “Evaluation of BNB permeability” section).
All the procedures described above were performed by the same investigator (NM) so as to ensure the homogeneity and comparability of resulting data.

Suberoylanilide hydroxamic acid (SAHA) or Vorinostat (Cayman, Germany), Lysotracker Red DND-99 (Invitrogen, Germany), B27 supplement 1:50 (Invitrogen, UK), Anti-E-cadherin (Cell signaling, Danvers, MA, USA), Anti-Snail (Cell signaling, Danvers, MA, USA), Anti-β-actin (Santa Cruz Biotech, CA, USA) primary antibodies and Tetrachloroauric acid (HAuCl4‚3H2O)(Acros organics, USA) were purchased. Sodium borohydride (NaBH4), L-ascorbic acid, Cetyltrimethylammonium bromide (CTAB), Sodium thiosulfate, Silver nitrate (AgNO3), RPMI-Medium (Biosera, France), Fetal bovine serum (FBS)(Gibco, USA), Penicillin/streptomycin (Biowest, France). TWEEN20, Dimethyl sulfoxide (DMSO), and formaldehyde were all purchased from Merck (Merck, Germany). PKF118-310, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole (DAPI), Fluorescein isothiocyanate (FITC), and Human Epidermal Growth Factor were purchased from Sigma Aldrich. Human plasma was obtained from the Blood Transfusion Organization (IRAN) and then aliquoted and stored at −80°C. SAHA was dissolved in deionized water (pH 12, 5 mg/mL) and PKF was dissolved in deionized water (5mg/mL) as stock solutions and were used for further experiments.