Bleomycin

All cytokines and growth factors including TGFβ1 were purchased from Peprotech (Rocky Hill, NJ, United States). Hydroxyproline assay kits, and CCl₄ were purchased from Sigma-Aldrich (St. Louis, MO, United States). Bleomycin and target specific pooled siRNAs siSTAT3 and siSTAT6 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). EPRS in pEXPR-103-Strep vector (IBA Lifesciences, Göettingen, Germany) were gifts from Dr. Myung Hee Kim at the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Daejeon, Korea). EPRS (1–1440 amino acids) consists of ERS (1–687 amino acids) and PRS (935–1,440 amino acids) linked via non-catalytic WHEP repeat domains (688–934 amino acids) (Ray and Fox, 2014 (link)). The PRS domain of EPRS was cloned into pEXPR-103-Strep vector (IBA Lifesciences). pRc/CMV-WT STAT3 was previously described (Choi et al., 2009 (link)) and pCMV-STAT6-IRES-Neo was a gift from Axel Nohturfft (Addgene plasmid # 35482). Adenovirus expressing SMAD2 or SMAD3 were described previously (Lee et al., 2005 (link)).

A549, HeLa, 293T and U2OS cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS), and 50 U ml⁻¹ penicillin and 50 ng ml⁻¹ streptomycin (Gibco). None of the cell lines used in this study are listed in the database of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee70 (link). The A549 cell line was a gift from the Bradner Lab at Dana-Farber Cancer Institute and was verified by short tandem repeat (STR) profiling. The HeLa and U2OS cells originated from the Jasin Lab at Memorial Sloan Kettering Cancer Center. The 293T cells were gifts from the Alt Lab at Boston Children’s Hospital. Cells were tested for mycoplasma contamination by the IDEXX Laboratories or using the Lonza Mycoplasma kit. Puromycin (Gibco) selection in A549 and 293T cells was at 1.0 μg ml⁻¹. Hygromycin B (Invitrogen) selection in A549 cells was at concentration (200 μg ml⁻¹). Cell synchronization of A549 cells was achieved by growing cells to confluence, reducing FBS to 0.1% for 72 h and releasing cells into DMEM with 20% FBS. A549 cells were treated at the indicated concentrations of pyridostatin (Sigma Aldrich), PhenDC3 (Polysciences), ME0328 (Selleck), KU0058948 (Axon MedChem) or Bleomycin (Santa Cruz Biotechnology) when indicated. All cells were cultured under normal oxygen conditions (21% O₂, 5% CO₂, 37 °C).

After exposure to room air or hyperoxia, or treatment with bleomycin (Santa Cruz, Dallas, TX), cells were trypsinized, pelleted and stored at −20 °C until analysis. Total DNA was isolated using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Ten micrograms of DNA was digested with BamHI and subjected to gel electrophoresis on a 0.6% agarose gel without ethidium bromide. DNA was transferred to a nylon membrane (MSI MagnaGraph, 0.45 µM) using standard southern blotting technique [33] . A complimentary DNA fragment encoding the 12S and 16S rRNA region of mtDNA (5′-GGTCACACGATTAACCCAAG-3′ and 5′-GTTGGTTGATTGTAGATATTGG-3′) was radiolabeled using the Prime-a-Gene kit (Promega, Madison, WI) with α³²P-dCTP and hybridized to the DNA on the blot overnight. Hybridization was visualized on blue x-ray film (Laboratory Products Sales, Rochester, NY).

Wildtype (WT) EPRS^+/+ (n = 4 for vehicle and n = 9 for bleomycin) and EPRS^−/+ hetero-knockout (n = 5 for vehicle and n = 7 for bleomycin) C57BL/6 mice were housed in a specific pathogen-free room with controlled temperature and humidity. Mouse protocol and animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-161201-1-3). For the lung fibrosis model, bleomycin (Santa Cruz Biotechnology) was dissolved in sterilized saline and intratracheal instillation was performed through surgically exposed trachea as a single dose of 1 mg/kg in 100 μl solution per animal. Mice were sacrificed 4 weeks post-intratracheal instillation. Lung tissue samples were snap frozen in liquid nitrogen for western blot, qPCR, and hydroxyproline analysis, or fixed in 4% formaldehyde in PBS for histological analysis.

Wild-type C57BL/6 mice (4 weeks old) were purchased from Orient Bio Inc. (Seongnam, South Korea). Global TM4SF5-knockout C57BL/6 mice (Tm4sf5^−/−) were generated by Macrogen (Seoul, South Korea). Briefly, base pairs of genomic TM4SF5 surrounding exon 1 were deleted using the RGEN/Cas9 genetic scissor. The genotypes of both wild-type and Tm4sf5^−/− mice were determined by PCR analysis of genomic DNA obtained from tail clippings from 3-week-old animals. Male wild-type and Tm4sf5^−/− mice at 8 weeks old were treated with either saline vehicle for the control or bleomycin (1 mg/kg, Santa Cruz Biotechnology) by intratracheal injection on day 0. Mice were sacrificed on day 28 for histological analysis and hydroxyproline assay.