*4.1. Mice*

All animal procedures conformed to the Japanese regulations for animal care and use, following the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science. Male and female C57BL/6J mice were purchased from Clea Japan (Tokyo, Japan). Using the 3.7SP-C/SV40

vector kindly provided by Dr. Je ffrey A. Whitsett (Children's Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, Ohio), we generated the following transgenic mice: C57BL/6J-hSP-C-M2 flag-p38 α dominant-negative (d.n., dual mutations in wild mouse p38 α: Thr180 to Ala; Tyr182 to Phe) TG (p38-DN) mice [62] and C57BL/6J-hSP-C-3HA-tag-MKK6 constitutive-active (c.a., dual mutations in wild human MKK6: Ser207 to Asp; Tyr211 to Asp) TG (MKK6-CA) mice [63]. We confirmed that each transgene-derived product was expressed at least in surfactant protein C (SP-C)-positive AEC II in the lung by using anti-M2-Flag or anti-HA tag antibody. Male heterozygous TG mice and WT littermates aged 10–12 weeks were used for the experiments. The animals were housed in standard laboratory cages and allowed food and water throughout the experiments. The studies were performed according to a protocol approved by the Committee of Animal Welfare of Chiba University.

#### *4.2. BLM-Induced Pulmonary Fibrosis Model*

Mice were anesthetized and the neck skin of each was cut longitudinally to expose the trachea. After a single intratracheal instillation of BLM hydrochloride (3 mg/kg; Nippon Kayaku, Tokyo, Japan) dissolved in PBS using a repeating syringe dispenser (Hamilton, Reno, NV, USA), the skin was sutured. Control mice were administered a sham treatment with PBS. Then, changes in body weight were measured daily. To evaluate the histopathological changes in the lung samples at 8 and 15 dpi of BLM, freshly cut lung sections (5 μm thick) were placed on adhesive glass slides (Matsunami Glass Ltd., Osaka, Japan) and stained with Masson's trichrome. The changes in the fibrotic lung samples were evaluated semi-quantitatively according to the modified Ashcroft method with a scoring grade of 0 to 8 [64]. In addition, the collagen content of the left lung was measured using the Sicol Soluble Collagen Assay Kit (Biocolor Life Science Assays, Carrickfergus, United Kingdom) according to the manufacturer's protocol.

#### *4.3. Evaluation of Inflammatory Cells in BALF*

At 8 dpi, the trachea was exposed and lavaged three times with 1 mL ice-cold PBS using a 20-gauge catheter. The BALF was centrifuged at 400× *g* for 10 min and the resulting supernatants were stored at −80 ◦C for protein array analysis. The resulting cell pellets were resuspended in PBS and subjected to cell counting using a hemocytometer in combination with Di ff-Quick (Sysmex Corporation, Kobe, Japan) staining.

#### *4.4. Measurement of Left Lung Compliance*

As described previously [65], the lung compliance of the mice was measured by drawing static air pressure–volume relationships in a mixture of medetomidine, midazolam, and butorphanol (M/M/B: 0.3/4/5 mg/kg)-anesthetized mice tracheotomized with polyethylene tubing (O.D. = 0.8 mm). Total lung capacity was defined as the lung volume of full inflation judged by visual inspection of the lung that fully occupied the chest cavity. Functional residual volume was defined as deflation at 0 cm H2O. Lung volumes at an airway pressure of 20 cm H2O were estimated between mice at 8 dpi with BLM and PBS in the three genotypes (WT, p38-DN, and MKK6-CA mice).

## *4.5. Immunofluorescence Staining*

The lung sections were pretreated with 1:10 FcR blocking agen<sup>t</sup> (Miltenyi Biotech, Gladbach, Germany) for 10 min. They were then treated with primary antibodies (1:100 dilution) as follows: goa<sup>t</sup> anti-proSP-C polyclonal antibody (sc-7706; Santa Cruz Biotech, Dallas, TX, USA), rabbit anti-p38 polyclonal antibody (original production [66]), rabbit anti-proSP-C antibody (customized production [60]; Sigma-Aldrich Japan Genosys, Ishikari, Japan), and mouse anti-phospho-p38 MAPK (pT180/pY182) (clone30, 612281; BD Biosciences, NJ, USA), followed by staining with appropriate fluorescein-conjugated secondary antibodies (1:200 dilution), and <sup>4</sup>,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. The stained sections were observed under a fluorescence microscope (Axio Imager A2; Zeiss, Oberkochen, Germany).

## *4.6. RNA Sequencing*

At 8 dpi, mice under anesthesia were intracardially perfused with ice-cold PBS to wash out blood cells in the lungs and sacrificed. The left lung lobes were homogenized in ISOGEN plus (TaKaRa Bio, Kusatsu, Japan), and total RNA was extracted. Thereafter, 500 ng of total RNA was ribosomal RNA-depleted using a NEBNext rRNA Depletion Kit (New England Biolabs) and was converted to Illumina sequencing library using NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs). The library was validated to determine the size distribution and concentration using a Bioanalyzer (Agilent Technologies). Sequencing was performed on a NextSeq 500 (Illumina) instrument with paired-end 36-base read options. Reads were mapped on the mm10 mouse reference genome and quantified using CLC Genomics Workbench version 12.0 (QIAGEN). All RNA-seq data sets were deposited in the Gene Expression Omnibus database at the National Center for Biotechnology Information with accession number GSE154074.

#### *4.7. Identification of Di*ff*erentially Expressed Genes (DEGs)*

To estimate the expression patterns of transcripts among the three genotypes (WT, p38α d.n.-TG and MKK6 c.a.-TG mice) with or without BLM instillation, the read counts were normalized by calculating the number of reads per kilobase per million for each transcript in individual samples using CLC Genomics Workbench version 12.0 (QIAGEN) [67]. Filtering characteristics of fold change −2 to 2 (FDR at *p* < 0.05) were used to identify the DEGs. Subsequently, the distinct gene expression patterns were analyzed comparatively through PCA and clustering heatmaps using CLC Genomics Workbench. GSEA for p38 MAPK pathways in the BLM-treated group among the three genotypes was also performed using GSEA\_4.0.3. [68]. K-means functional enrichment analysis of DEGs was analyzed using integrated differential expression and pathway analysis (iDEP) online tools [69]. A volcano plot was used to compare the gene expression levels in terms of the log2 fold change. The GO (molecular function and biological process) and KEGG pathway analyses of DEGs between BLMand PBS-treated groups were performed using ToppGene Suite (https://toppgene.cchmc.org) [70]. Finally, K-means cluster analysis was performed to identify BLM-upregulated genes that depended on the theoretical intrinsic activity of the p38 signal (p38-DN < WT < MKK6-CA) using CLC Genomics Workbench.

## *4.8. Statistical Analysis*

Data are expressed as means ± standard error of the mean (SEM). Statistical analysis was conducted using GraphPad Prism Version 6 (GraphPad Software, San Diego, CA, USA). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey's or Student's *t*-tests, and *p*-values < 0.05 were considered significant.

#### **Supplementary Materials:** Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/18/ 6746/s1.

**Author Contributions:** S.M., J.-D.K., and Y.K. developed the concept and designed the experiments. S.M., J.-D.K., F.S., Y.M., J.I., K.M., K.N. (Kanako Nakamura), K.N. (Kana Namiki), T.S., T.K., and Y.K. performed the experiments. M.H., K.T., and A.F. provided conceptual advice throughout the project. S.M., J.-D.K., and Y.K. wrote the paper. All authors discussed the results and implications and commented on the manuscript at all stages. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported in part by Grants-in-Aid for Scientific Research ((B), 24390137 to Yoshitoshi Kasuya) from the Ministry of Education, Science, Sports and Culture of Japan, a Therapeutics Research Initiative Grant from Chiba University, School of Medicine (2018-G7 to Yoshitoshi Kasuya and 2019-Y10 to Shuichi Matsuda) and the Cooperative Research Project Program of Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA Center), University of Tsukuba, Japan.

**Acknowledgments:** We would like to thank Ben-Shiang Deng and Hiromi Takahashi for guidance in lung compliance measuring, Tomoko Misawa for technical support of pathologically staining, Kento Yoshioka for assistance with mouse genotyping.

**Conflicts of Interest:** The authors declare no conflict of interest. *Int. J. Mol. Sci.* **2020**, *21*, 6746
