Chemicals and animals
Eperisone hydrochloride, tolperisone hydrochloride, isoflurane, Dulbecco’s modified Eagle’s medium (DMEM), L-hydroxyproline, sodium acetate, trichloroacetic acid (TCA), perchloric acid, and formalin neutral buffer solution were obtained from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). Tizanidine hydrochloride, baclofen, methocarbamol and pirfenidone were from Tokyo Chemical Industry (Tokyo, Japan), and nintedanib was from Cayman Chemical (Ann Arbor, MI). Inaperizone hydrochloride and lanperizone hydrochloride were synthesized in our laboratory. Details of the synthesis method are described in the Supplementary Materials and Methods. BLM was from Nippon Kayaku (Tokyo, Japan). Chloramine T and 4-dimethylaminobenzaldehyde (DMBA) were obtained from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was purchased from BioWest (Nuaille, France), and Ham’s F-12K (Kaighn’s modification) medium was from Thermo Fischer Scientific (Waltham, MA). An antibody against α-smooth muscle actin (α-SMA) was from Abcam (Cambridge, Cambridgeshire), and Alexa Fluor 594 goat anti-rabbit immunoglobulin G were from Invitrogen (Carlsbad, CA). Recombinant human TGF-β1 were from R&D Systems (Minneapolis, MN), and mounting medium for immunohistochemical analysis (VECTASHIELD) was purchased from Vector Laboratories (Burlingame, CA). Mayer’s haematoxylin, 1% eosin alcohol solution, primary mordant agent, secondary mordant agent, 0.75% Orange G solution, Masson’s staining solution B, 2.5% phosphotungstic acid solution, aniline blue solution, mounting medium for histological examination (malinol) and Weigert’s iron haematoxylin were from MUTO Pure Chemicals (Tokyo, Japan). 4,6-diamidino-2-phenylindole (DAPI) was purchased from Dojindo (Kumamoto, Japan). The RNeasy kit was obtained from Qiagen (Valencia, CA), PrimeScript™ II 1st strand cDNA Synthesis kit was from TAKARA Bio (Ohtsu, Japan), and THUNDERBIRD® SYBR qPCR Mix was from Bio-Rad (Hercules, CA). ICR mice (6-7 weeks old, male) were purchased from Charles River (Yokohama, Japan). The experiments and procedures described here were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health, and were approved by the Animal Care Committee of Musashino University.