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.