Antibodies specific for Pref-1, HIF-1α, and phospho-serine were purchased from Abcam (Cambridge, MA, USA). Secondary antibodies against IP detection reagent, Alexa Fluor-488 and Alexa Fluor-555 were purchased from Abcam (Cambridge, MA, USA). An antibody specific for c-Jun Ser63 was purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies specific for PEA3, c-Jun, horseradish peroxidase (HRP)-linked antibodies, including anti-goat immunoglobulin G (IgG), anti-rabbit IgG, and anti-mouse IgG antibodies, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A luciferase assay kit was purchased from Promega (Madison, WI, USA). The human PEA3 luciferase reporter was a obtained from Peter Hollenhorst (Watertown, MA, USA). Furthermore, α-tubulin antibody, fetal bovine serum, control small interfering RNA (siRNA) (scrambled), and PEA3 siRNA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine 3000 reagent, minimum essential medium (MEM), penicillin, and streptomycin were acquired from Invitrogen Life Technologies (Carlsbad, CA, USA). A Novolink Max Polymer Detection System was purchased from Leica (Wetzlar, Germany)
2.2 Cell culture
Human lung fibroblast (WI-38) cells, purchased from American Type Culture Collection (Manassas, VA, USA), were grown in MEM supplemented with 10% fetal calf serum, penicillin G (100 U/mL), streptomycin (100 µg/mL), and MEM nonessential amino acids. WI-38 cells were maintained in a humidified 37°C incubator with 5% CO2. After reaching confluence, cells were seeded onto 12-well plates for the transfection and luciferase reporter assay, onto 6-cm dishes for Western blot analysis, and onto 10-cm dishes for the immunoprecipitation assay.
2.3 Ovalbumin-induced animal model of airway fibrosis
For sensitization, on days 1, 7, and 14, C57BL/6 mice were intraperitoneally injected with 200 µL of 50 µg ovalbumin (OVA) emulsified in 2 mg of aluminum hydroxide. From day 21, 8- to 10-week-old C57BL/6 mice were challenged with aerosolized 5% OVA in phosphate-buffered saline (PBS) or PBS alone for 9 weeks. The frequency of the OVA challenge was twice weekly. After the final OVA aerosol challenge, C57BL/6 mice were sacrificed, and the lung tissue of mice was analyzed with further experiments.
2.4 Western blot analysis
Protein extraction and Western blot analysis were performed as previously described (33). In brief, cells were lysed with lysis buffer containing 20 mM Tris (pH 7.5), 1 mM MgCl2, 125 mM NaCl2, 1% Triton X-100, 1 mM PMSF, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 25 mM β-glycerophosphate, 50 mM NaF, and 100 µM Na3VO4. Whole cell lysates (30 µg) were electrophoresed through SDS-PAGE, and the gels were transferred onto PVDF membranes. The whole membranes were blocked through incubation with 5% bovine serum albumin for 1 hour. Subsequently, proteins were incubated with specific primary antibodies for 20 hours at 4°C. Then, the membranes were incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Immunoreactivity was analyzed using enhanced chemiluminescence following the manufacturer’s protocol.
2.5 Cell transfection
For transient transfection of siRNA into WI-38 cells, the transfection reagent/siRNA mixture was incubated at room temperature for 10 minutes and then added dropwise to MEM supplemented with 10% fetal bovine serum; it was then incubated in a humidified 37°C incubator for 24 hours.
2.6 PEA3-luciferase activity assay
In brief, WI-38 cells were transfected with PEA3-Luc (0.8 µg) and pBK-CMV-Lac Z (0.1 µg) by using transfection reagent for 24 hours. Cells were stimulated with hypoxia (1% O2) for 18 hours. Luciferase activity was measured using the luciferase assay kit (Promega, Madison, WI, USA).
2.7 Immunohistochemistry and immunofluorescence
Lung tissues were fixed in 10% formaldehyde overnight, embedded in paraffin, and sectioned for immunohistochemistry (IHC) and immunofluorescence staining. For IHC staining, 2-µm sections were deparaffinized and then processed for antigen retrieval by using citrate buffer (pH 6.0). Endogenous peroxidase in tissue was neutralized through peroxidase blocking. The tissue was then blocked with a blocking buffer and incubated with Pref-1 and PEA3 antibodies for 40 minutes followed by polymer secondary antibody incubation for 30 minutes. Finally, the tissues were stained with hematoxylin and DAB solution. The integrated density of IHC staining was analyzed using ImageJ Fiji software. For immunofluorescence staining, 2-µm sections were deparaffinized and then processed for antigen retrieval by using EDTA buffer (pH 9.0). The tissue was blocked with a blocking buffer and incubated with Pref-1 and HIF-1α antibody for 24 hours at 4°C. Then, the tissue was incubated with Alexa Fluor 488-conjugated and Alexa Fluor 555-conjugated secondary antibodies for 1 hour at room temperature. Images were scanned using a ScanScope CS or fluorescence microscope.
WI-38 cells were cultured on slides. After reaching confluence, cells were subjected to hypoxia (1% O2) for 30 minutes and then fixed through incubation in 4% paraformaldehyde in PBS for 10 minutes at room temperature. Permeabilization with PBS containing either 0.5% Triton X-100, and washed slides in PBS three times for 5 minutes. The coverslips were blocked with 5% bovine serum albumin in PBST for 1 hour and incubated at 4°C overnight with antibodies specific to PEA3. Cells were incubated with Alexa Fluor 488-conjugated secondary antibody for an additional 1 hour. Counter staining was performed DAPI for 1 minute and the coverslip was mounted with a mounting medium. They were then observed under a fluorescence microscope.
WI-38 cells were seeded onto 10-cm dishes. After reaching confluence, cells were subjected to hypoxia (1% O2) for the indicated time intervals. Cells were then harvested, lysed in 100 µL of IP lysis buffer (Thermo Fisher Scientific, MA, USA), and centrifuged. The supernatant was then immunoprecipitated with a specific Ab against PEA3 (Santa Cruz, CA, USA) or c-Jun (Santa Cruz, CA, USA) in the presence of protein A/G beads at 4°C overnight. The immunoprecipitated beads were washed three times with IP lysis buffer. The immune complex was analyzed through 8% SDS-PAGE, transferred to PVDF membranes, and then subjected to immunoblot analysis with Abs specific for serine (Abcam, Cambridge, UK), PEA3 (Santa Cruz, CA, USA), or c-Jun (Santa Cruz, CA, USA).
2.10 Chromatin immunoprecipitation (ChIP)
WI-38 cells were subjected to hypoxia (1% O2) for 30 minutes and then fixed with 10% formaldehyde for 10 minutes. Cells were collected and subjected to sonication; then, anti-PEA3, or anti-c-Jun was used for immunoprecipitation, and mouse anti-IgG antibody was used as the control. The Pref-1 promoter region was amplified through the polymerase chain reaction, and the following primers were used: AP-1, 5′-ACCACGAGTCAGCTGGGTAT-3′ (sense) and 5′-TGCACACCCAAACACGCAAA-3′ (antisense) and PEA3, 5′-TTGTGTTTCAGCGCGGCTA-3′ (sense) and 5′-CAAGCGGACCTGCGGTTA-3′ (antisense). DNA was analyzed with 1% agarose gel containing ethidium bromide.
2.11 Study approval
All animal protocols were approved by the Animal Ethics Committee of Taipei Medical University (approval no. LAC-2016-0361 and LAC-2019-0042).
2.12 Statistical analysis
All experimental data are presented as the mean value ± standard error of the mean for at least three independent experiments. The results of comparisons were conducted using one-way analysis of variance (ANOVA) followed by Dunnett’s test analysis, unpaired t test, or Mann–Whitney U test. The results were considered statistically significant if p was <0.05.