2.1 Reagents and antibodies
Recombinant human TGF-β1 was obtained from R&D systems (Minneapolis, MN, USA). Bleomycin sulfate was obtained from Hisun Pharm (Taizhou, China). Sodium orthovanadate was obtained from Sigma-Aldrich (Shanghai, China). Wiskostatin was obtained from Abcam (Shanghai, China). N-WASP was obtained from Proteintech (Wuhan, China). Goat anti-rabbit IgG of β-actin, α-SMA, pY256N-WASP and GAPDH, were purchased from Affinity Biosciences (Changzhou, China). Goat anti-mouse IgG of Vimentin and E-cadherin were purchased from Cell Signaling Technology (Shanghai, China), Goat anti-rabbit IgG of N-cadherin, Fibronectin and Collagen I were purchased from Cell Signaling Technology (Shanghai, China), TUFT1 was purchased from Thermo Fisher (Suzhou, China).
2.2 Human Lung tissues and the mouse lung fibrosis model induced by bleomycin
Human lung tissues were obtained by surgical lung biopsy (SLB) from patients in Xinxiang central hospital. Wild-type male C57BL/6N mice (9–10 weeks, 19–24 g) were obtained from the Charles River (Beijing, China). Mice were raised in a controlled environment with a cycle of 12-hour light/dark, auto-regulated temperature and humidity, and access to food and water unrestricted. To knock down the Tuft1 gene in vivo, Tuft1/shRNA-AAV(5 × 1012 Vg/mL) (HANBIO, Shanghai, China) was administered via intratracheal injection of adenoviral particles 7 days before the experiment. The control group received vector adenoviral particles (shNC). On day 0, the mice were anesthetized by isoflurane and bleomycin (1.5 U/kg.bw, i.t.) or 0.9% saline (50 µL) was instilled into the trachea. The mice were euthanized and lung samples were collected on day 14.
2.3 Cell culture
The human lung adenocarcinoma cell line A549 (ATCC ® CCL-185™, ATCC, Manassas, VA), negatively tested for mycoplasma, was cultured in DMEM/F12 medium with 10% fetal bovine serum and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin). The human lung normal fibroblast cell line (MRC-5) was purchased from Procell Life Science &Technology Co., Ltd. (Wuhan, China). MRC-5 was cultured in MEM medium (containing NEAA) (Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd., ZQ-300) with 10% fetal bovine serum and antibiotics (100units/ml penicillin and 100 µg/ml streptomycin) in an atmosphere of 5% CO2 and humidified at 37°C.
2.4 Small interfering RNA (siRNA) transfection
TUFT1/siRNA-1(5’-GGAGTCCCATGATGGACAT-3’), TUFT1/siRNA-2(5’-GCAGAGGCUGUGUGACAAATT-3’), TUFT1/siRNA-3(5’-GAGGAACTTCGGAGCAACA-3’) were purchased from RIBOBIO (Guangzhou, China). To transfect siRNA into A549 and MRC-5 cells, the cells were cultured on 6-wellplates at 50–70% coverage before transfection. Lipofectamine RNAi MAX (RIBOBIO, Guangzhou, China), Opti-MEM (Thermo Fisher) and Individual siRNA (50 nM) were mixed completely and incubated for 5–10 min at room temperature. The efficiency of transfection was testified by western blot in 48 h after transfection.
2.5 Adenovirus generation and infection
The specific shRNA of Tuft1 was obtained from HANBIO (Shanghai, China). Adenovirus generation and subsequent infection were conducted following previously described protocols[25]. Briefly, HEK293T cells were transfected with lipofectamine 3000 (Thermo Fisher Scientific, Shanghai, China) and plasmids to generate adenovirus. The adenovirus-containing medium was collected 48 hours post-transfection and add fresh medium immediately. After another 24 hours, collected the medium again and the adenovirus-containing medium was centrifugated immediately (25,000 rpm, 4°C, 2 hours; Beckman Ti70 rotor). The supernatant was divided into aliquots after aseptic filtration and frozen at -80°C for later use.
2.6 Hydroxyproline assay
Ahydroxyproline assay kit (MAK008, Sigma, St. Louis, MO, USA) was used to detect the content of hydroxyproline in mice right lung [26]. Briefly, the fresh lung tissue was homogenized in water (100 µL water of every 10 mg lung tissue). Hydrolyzed the samples for 3 hours at 120°C. Next, the samples were centrifuged (12,000rpm, 4°C, 3 mins; Beckman Microfuge 20R) and extracted 10 µL supernatant of every sample, the samples were transferred to a 96-well plate and dry them at 56℃. The samples in the plate were incubated for 5 minutes at 20℃ after added 100 µL chloramine T reagent into each well. Afterward, the samples were incubated for 90 min at 56°C after mixed with 100 µL p-dimethylaminobenzaldehyde reagent. Absorbance of samples was measured by BioTek ELx800 plate reader at λ = 560 nm (Winooski, VT, USA). The hydroxyproline content of samples was calculated by a standard rat tail collagen (Sigma, St. Louis, MO, USA).
2.7 Histological and morphometric analysis
The lungs of mice were collected and fixed with 4% paraformaldehyde. The lung tissues were embedded in paraffin, and the lung tissues would be cut into 3 µm sections for further study on slides. The sections were stained with Masson's trichrome (Beyotime) and H&E (Beyotime) to assess the collagen content and evaluate the morphometric changes. The acquisition of images was performed using a BioTek Cytation C10 Confocal Imaging Reader. Images were analyzed by Image Pro Plus software (version 6.0, Media Cybernetics).
2.8 Immunohistochemical staining
The lung slides were prepared following previously described protocols [27]. The lung sections were incubated with the primary antibodies overnight at 4°C, then the secondary antibody biotinylated anti-rabbit IgG (1:100, Beyotime) was incubated for 60 min at 37°C. Followed by incubating with SABC (1:100, Beyotime), and then visualized by DAB stain (Beyotime).
2.9 Wound-healing assay
SiRNA control or TUFT1 siRNA was transfected into A549 and MRC-5 cells. After 24 hours, the cells were placed in a 6-well plate using a culture medium containing 1% FBS. Upon reaching confluence, a pipette tip was utilized to create a direct scratch across the cell monolayer. Pictures were taken at the predetermined time intervals to document the progression of wound healing. Wound healing is calculated as follows: [1-(Width of the wound at a given time/width of the wound at t = 0)] × 100%.
2.10 Transwell assay
Cell migration assay was performed using a transwell chamber. A549 cells that transfected with SiRNA control or TUFT1 siRNA were seeded into transwell plates with serum-free medium, then medium containing 10% FBS was added to the bottom of the chamber. After 24 hours, Migration cells were fixed and stained with 1% crystal violet and counted in three random fields under an optical microscope.
2.11 Collagen gel contraction assay
Collagen gel contraction assay was performed using a cell contraction assay kit (CBA-201, Cell Biolabs, Inc. (San Diego, CA)). MRC-5 cells were suspended in bovine type 1 collagen solution (chilled on ice) at the concentration of 1.85 mg/mL and seeded at a density of 1 × 106 cells/well in a Costar low attachment surface polystyrene 24-well plate (Corning; Kennebunk, ME). The plate was then placed in a 37°C incubator for 1 h to allow for collagen gel polymerization, and 1 mL of culture medium was added on top of the gel lattice, and cells were incubated overnight. Then cells were pre-incubated with Sodium orthovanadate (20 µM) for 24 hours or Wiskostatin (10 µM) for 1 h. Once the contraction agonists were added, the gels were meticulously detached from the sidewalls of each well by a sterile scalpel. Photographs were captured after 24 hours, and the analysis of gel disc area was conducted using Image J software for quantification.
2.12 Immunofluorescence staining
The 4% paraformaldehyde was used to fix the lung sections and cell slides and 0.3% Triton X-100 was used to elevate the permeable ability of cells. Then, the sections were block by 10% goat serum for 0.5 h at 37°Cand incubated with primary antibodies overnight at 4°C. Corresponding secondary antibodies were incubated for 1 h at 37°C. F-actin was stained with Phalloidin-iFluor 594 Reagent (ab176757, Abcam, USA) and nuclei were stained with DAPI. The images were captured by a fluorescence microscope (Zeiss LSM780, Carl Zeiss).
2.13 Western blotting
The steps of Western blot analyses were described as previously [28]. Initially, lung homogenates or cell lysates were prepared by RIPA lysis buffer (AR0102, Boster, Wuhan, China). The protein concentrations were detected by BCA kit (Solarbio, Beijing, China). The protein was loaded onto SDS-PAGE as equal amounts and electrotransferred onto PVDF membranes completely. The membranes were incubated with primary antibodies overnight at 4°C and incubated with corresponding secondary antibodies for 1 hour at room temperature. The images were captured by Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The densitometry band quantification was measured by the Image Studio software (LI-COR Biosciences, Lincoln, NE, USA).
2.14 Co-Immunoprecipitation (co-IP)
HEK293T cells were lysed in co-immunoprecipitation buffer (AR0107, Boster, Wuhan, China) and incubated for 30 min on ice. The lysate was then centrifuged at 10,000 × g for 10 min and the supernatant was collected. The supernatant was incubated with anti-HA beads (Bimake, B26202, Shanghai, China) or anti-Flag beads (B26102, Bimake, Shanghai, China) at 4°C overnight. The beads were washed three times with immunoprecipitation wash buffer (50 mM Tris, 150 mM NaCl, 0.5% Tween 20, pH 7.5) and subjected to further analysis.
2.15 Statistical Analysis
The statistical analysis involved using a two-tailed Student’s t-test for comparing two experimental groups. One-way or two-way analysis of variance was used for multi-group comparisons, followed by Tukey’s multiple comparison test. Differences were considered statistically significant in all cases when P < 0.05.