All procedures were approved by the Animal Care and Use Committees of Yonsei University Medical School (2015–0267) and were performed in accordance with the relevant guidelines. A single dose of 75 or 90 Gy was delivered using an X-RAD 320 platform (Precision x-ray, North Branford, CT) as described previously . Lung tissues (n ≥ 3 per group) were collected at each time point after IR. The generation of HSP25 TG mice and establishment of the orthotopic lung tumor model were described previously . Mice were administered i.p. J2 (15 mg/kg) on alternate days for indicated periods after 75 Gy IR.
C57BL/6 N (male, 6 weeks old) were purchased from Central Lab. Animal Inc. (Seoul, Korea) and housed in a pathogen-free and light-controlled room (12 h light and 12 h dark) with free access to food and water. Bleomycin (BLM) sulfate from Santacruz Biotechnology (Dallas, TX, USA) was used to induce fibrosis. The mice were anesthetized and treated with either saline (n = 3–4 mice) or BLM (n = 3–4 mice) (2.5 U/kg of body weight) in saline solution through an intratracheal injection. At 14 days post-BLM administration, the mice were sacrificed, and the lungs were removed for histological staining. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Ewha Womans University.
Generation of HSP25 TG mice
HSP25 Mice was generated by Macrogen, Inc and mice were interbred and maintained in pathogen-free conditions at Macrogen, Inc (Seoul, Korea). All animal experiments were conducted in accordance with the Macrogen Institutional Animal Care and Use Committee approval. Briefly PMSG and hCG were used to treat C57BL/6 N female mice for superovulation. PMSG (7.5 IU) and hCG were IP injected at 48-h intervals (5 IU) into the female mice at 5–8 weeks. After hCG Injection, the female mice were mated with C57BL/6 N stud male mice. Next day, the vaginal plug was examined and female mice were sacrificed, to harvest the fertilized embryos. HSP25 DNA was co-microinjected into a single-cell embryo. Standard microinjection procedures were used to generate transgenic mice (Macrogen, Seoul, Korea). DNA 4 ng/µL was microinjected directly into the male pronucleus of zygote using micromanipulator and the microinjected embryos were incubated at 37 ℃ for 1–2 h. Fourteen to 16 one-cell stage embryos were transplanted surgically into oviducts of pseudo-pregnant recipient mice (ICR). After the birth of F0 generation, genotypic testing of tail-cut samples for the presence of the transgene was performed and confirmed by PCR analysis of their genomic DNA. PCR screening was carried out using phenol-extraction method.
Human tissues analysis
The study of tissue specimens of 6 patients with RIPF was approved by Severance Hospital, Yonsei University. The tissue of each patient contained an irradiated fibrotic and a non-irradiated normal area. A human IPF tissue array (catalogue no. LC561) comprising 28 cases was purchased from US Biomax (Derwood, USA).
Cell culture and treatment
The human normal lung epithelial cell line (L132) was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Gibco) in a 37 °C incubator with 5% CO2. Human primary pulmonary fibroblasts (HPFs) and human primary small airway epithelial cells (HSAEpCs) were obtained from PromoCell. All cells were used within nine passages. Cell lines were tested by BioMycoX Mycoplasma PCR Detection Kit (JCBIO Co., Ltd) to ensure that they were mycoplasma-free.
siRNA and plasmids
Mammalian expression vectors such as p3xflag-myc-HSP27S15A/S78A/S82A and p3xflag-myc-HSP27S15D/S78D/S82D were prepared and pCMV5B-Flag-Smurf2-WT was from Addgene (plasmid #11746). siRNAs against Smurf2 (sc-41675) and a control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). For transient transfection of siRNA or plasmids, L132 cells were plated and incubated for 24 h to reach 70% confluency. Cells were then transfected with the designated plasmids in each experiment using Lipofectamine 2000 (Invitrogen) according to the manufacturers protocol.
Total RNA from the mouse lung tissues was extracted using the Easy-SpinTM total RNA extraction kit according to the manufacturer’s instructions (iNtRON Biotechnology, Seoul, Republic of Korea). Isolated total RNA was amplified and labeled using the Low RNA Input Linear Amplification kit PLUS (Agilent Technologies) and hybridized to a microarray containing approximately 44,000 probes (~ 21,600 unique genes), in accordance with the manufacturer's instructions (Agilent Mouse whole genome 44 K, Agilent Technologies). The arrays were scanned using an Agilent DNA Microarray Scanner (Agilent Technologies).
GSE18800, GSE161322, GSE150910 and GSE24206, publicly available gene expression datasets used for therapeutic response rate analysis, were obtained from the National Center for Biotechnology Information (NCBI) gene expression omnibus database (GEO).
RNA isolation, qRT-PCR
Total RNA was isolated from the sample using TRIzol® reagent (Qiazen, Valencia, CA, USA). RNA purity and concentration were measured with a Nanodrop. RNA was reverse transcribed using a ReverTra Ace® qPCR RT Kit (TOYOBO, kita-ku, Osaka, Japan) following the manufacturer's protocol. The mRNA expression was assessed by real-time PCR using SensiFAST sybr Hi-Rox Mix (Bioline USA Inc, Taunton, MA, USA) with CFX96 Touch™ Real-Time PCR Detection System (Biorad, USA), equipped at Ewha Drug Development Research Core Center. The 2-ΔΔCt method was used to analyze the relative changes in gene expression based on real-time quantitative PCR. Gapdh was used as an internal control gene. Primer sequences for qRT-PCR are listed in Additional file 1: Table S1.
Tissue histology and immunohistochemical and immunofluorescence staining
Mice were euthanized and lung tissues were harvested and fixed in 10% (v/v) neutral buffered formalin before preparation of paraffin sections. Paraffin-embedded sections were deparaffinized and stained with hematoxylin and eosin (H&E), using a Masson's trichrome stain kit or Sirius red stain kit (Sigma-Aldrich) to detect collagen.
Before immunohistochemistry, deparaffinized sections were in antigen-retrieval buffer (Abcam) for 30 min and next incubated with 0.3% (v/v) hydrogen peroxide in methanol for 10 min. Sections were blocked in normal horse serum at 37 °C incubator for 1 h and immunostained overnight at 4 °C with primary antibodies. The target proteins were visualized using ABC and DAB kits (Vector Laboratories) and counterstained with hematoxylin.
For immunofluorescence staining, cell or sections were stained with primary antibodies and incubated with Alexa 488- labeled anti-mouse, Alexa 568- labeled anti-rabbit, Alexa 647- labeled anti-goat labeled secondary antibodies (1:1000; Thermofisher) and counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; 3 mmol/L). Images were obtained using a Zeiss microscope Apotome (Cal Zeiss), equipped at Ewha Drug Development Research Core Center. The detailed protocol and antibodies for immunohistochemical and immunofluorescence staining were provided in Additional file 1: Table S2.
In situ proximity ligation assay (PLA)
PLA was performed to detect the interaction between AAA/DDD (Flag) and Smurf2. To visualize the bound antibody pairs, the Duolink Detection Kit (Duo92008) with PLA PLUS and MINUS probes for mouse and rabbit (Sigma-Aldrich) was used, according to the manufacturer’s instructions.
Experiments of miRNA
The putative miR-15a, miR-128, miR-195 and miR-203a target sequences of Smurf2 mRNA were determined using TargetScan, miRDB and miRSystem. Total RNA was extracted from cells using Qizol Reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. To analyze miRNAs expression, cDNA was synthesized by a Mir-X miRNA First-Strand Synthesis Kit (Takara, Dalian, China). The quantitative real-time PCR (qRT-PCR) was performed using Mir-X miRNA (Takara, Dalian, China).
Expression levels of miRNAs in mouse tissues were quantified by quantitative polymerase chain reaction (miRCURY LNA microRNA PCR kit; Exiqon, Vedbaek, Denmark) according to the manufacturer’s protocol.
The miRNA inhibitors and mimics of each miRNAs were purchased from Genolution (Seoul, Korea). Upon reaching 60–70% confluence, the L132 cells were transfected with 100 nM of inhibitors or mimics of miRNAs using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The expression levels of miRNAs were quantified 24 h after transfection and the cells were used for a western blot and qPCR analysis.
Twist (GeneTex); p50, p65, Hsp27, Smurf2, Ubiquitin, and β-actin (Santa Cruz Biotechnology); phospho-Hsp27 (Ser78), phospho-Hsp27 (Ser82), phospho-IkBα (Ser32/36), and Lamin A/C, Flag (Cell Signaling Technology); IkBα, IL6, IL1β, pro-SPC, and phospho-Hsp27 (Ser15) (Abcam); phospho-Hsp27 (Ser86) (Thermo Fisher Scientific); α-SMA and Flag (Sigma); and Alexa488-conjugated phalloidin (Invitrogen). The detailed antibodies for immunoblotting, immunohistochemical and immunofluorescence staining were provided in Additional file 1: Table S2.
The histology score was scored according to Gallet et al. in 2011, and a score of severity for inflammatory infiltrates was established, from 0 (no alteration) to 3 (severe alterations), using the visual scale presented in this journal. This visual scale is 0 grade = no inflammatory infiltrate; Grade 1 = Some inflammatory elements and mast cells; Grade 2 = Frequent inflammatory infiltrates; Grade 3 = Ubiquiary inflammatory infiltrates .
Comparisons of all results were performed by t.test, one- or two-way ANOVA, and Newman-Keuls test where indicated. The difference was considered statistically significant at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001. All statistical analyses were performed using GraphPad Prism 8.