Bleomycin

The animal maintenance and handling procedures followed the Henan Normal University Institutional Animal Care and Use Committee (IACUC, SMKX-2118BS1018) guidelines, which coordinate with the Association of Animal Behavior and National Regulations. Eight-to-ten-week-old C57BL/6 N male mice were purchased from Beijing Charles River Laboratory Animal Technology Co., Ltd. (Beijing, China) and maintained in a specific pathogen-free environment. For bleomycin-induced PF, a single 50 μl injection containing 1.5 U/kg of bleomycin (Nippon Kayaku Co., Tokyo, Japan) diluted in PBS or PBS only (vehicle) was intratracheally administered. At designated time points, mice were sacrificed by intraperitoneal injection of ethyl carbamate, and samples were collected for further analysis.
IPF lung tissues and control non-IPF lung tissue samples were recruited based on the ATS/ERS/JRS/ALAT Clinical Practice Guidelines at Henan Provincial Chest Hospital. The study was approved by the Henan Provincial Chest Hospital Medical Research Ethics Committee (No. 2019-05-07), and informed consent was obtained from all the patients before surgery. The work was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.

IRI (CAS No. 100286-90-6, in the form of hydrochloride trihydrate salt) was the product of LC Laboratories (Woburn, MA, USA). Prior to use for the treatments, it was dissolved in 0.9% NaCl solution (Croatian Institute for Transfusion Medicine, Zagreb, Croatia).
Bleomycin (CAS No. 11056-06-7, in the form of Bleomycin sulfate) was the product of Nippon Kayaku Co., Ltd., Tokyo, Japan. It served as a positive control, and was also dissolved in 0.9% NaCl solution before use.
AH was composed of fructose (Kemika, Zagreb, Croatia), glucose (Sigma-Aldrich, Steinheim, Germany), maltose (Torlak, Belgrade, Serbia), and sucrose (Fluka, St. Gallen, Switzerland). It was freshly prepared by dissolving sugars in 10 mL of distilled water, as follows: 40 mg of fructose + 30 mg of glucose + 8 mg of maltose + 2 mg of sucrose [3 (link)].
Unless otherwise specified, other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

All procedures involving animals were conducted in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experimentation of the Institutional Animal Care and Use Committee of the College of Medicine of the Catholic University of Korea. The study protocol was approved by the Institutional Review Board of the Catholic University of Korea (CUMC-2017-0128-05). To generate mice with bleomycin-induced SSc, C57BL/6 mice were injected subcutaneously with 1 μg bleomycin (NIPPON KAYAKU) every day for 3 weeks. The model was generated by daily subcutaneous bleomycin injections. Starting 3 days after the bleomycin injections began, the mice were also injected subcutaneously with 10 mg/kg raloxifene (Takeda). Starting three days later, the mice were also treated with daily subcutaneous injections of raloxifene. The mice were killed. The double injections were continued for the remaining 21 days of the experiment. The skin was subjected to histology and western blot analysis of fibrosis markers expression.

bleomycin treatment was performed as previously reported [14] (link). In brief, bleomycin (Nippon Kayaku) was dissolved in phosphate buffered saline (PBS) at a concentration of 1 mg/ml and sterilized by filtration. bleomycin (100 µl) was injected intradermally into the shaved back of the 8-week-old mice daily for 4 weeks. The back skin was removed on day after final bleomycin injection.

Bleomycin (BLM) was purchased from Nippon Kayaku (Tokyo, Japan). The P-FAK inhibitor defactinib (a specific FAK phosphorylation inhibitor) were purchased from Medchemexpress (no. HY-12289). Mouse and human recombinant OPN were purchased from R&D Systems (no. 441-OP-050, no. 1433-OP-050). Mouse biotin-OPN was synthesized by Medchemexpress. The LV-OPN-siRNA was synthesized by GENECHEM (Shanghai, China). Antibodies used in this study were listed in Additional file 3: Table S1.

Female C57BL/6 J mice (6–7 weeks) were obtained from Beijing Vital Laboratory Animal Technology (Beijing, China). They were maintained in a specific pathogen-free laboratory with food and water ad libitum. The study protocol was approved (20162032) by the Animal Ethical and Welfare Committee of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. Animal procedures were in accordance with the Institutional Animal Care and Use Committee guidelines.
The Bleomycin-induced model of skin fibrosis was described by Yamamoto and colleagues [32 (link)]. Bleomycin (Nippon Kayaku, Tokyo, Japan) was dissolved in physiologic 0.9% NaCl (1 mg/mL) and sterilized by filtration. Mice were divided randomly into six groups with ≥ 9 mice in each group. After shaving of the back, Bleomycin (100 μL) or 0.9% NaCl (100 μL) was injected (s.c.) into well-defined areas (1 cm²) once daily, along with oral gavage with vehicle control (0.5% carboxymethylcellulose sodium), D-penicillamine (125 mg/kg; positive control) or baicalein (25, 50, or 100 mg/kg) once daily for 4 weeks.