Anti α sma

Cells or lung tissues were harvested and lysed in radio immunoprecipitation assay buffer and phenylmethanesulfonyl fluoride (100:1). The protein concentration was measured using bicinchoninic acid protein assay kit (Coolaber, China). After separation in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were transferred to a polyvinylidene fluoride membrane, and the protein bands were blocked with 5% nonfat dry milk for 2 h, and mixed with anti-collagen III (1:1,000, Affinity, China), anti-collagen I (1:1,000, Affinity, China), anti-α-SMA (1:1,000, Affinity, China), anti-Vimentin (1:1,000, Affinity, China), Anti-GAPDH (1:10,000, Affinity, China), anti-ULK1 (1:1,000, Affinity, China), anti-FAP1 (1:1,000, Cell Signaling, United States), anti-TGF-β1, anti-ATG5, anti-DRAM2 (1:1,000, Cell Signaling, United States) 1,000, Bioss, China), anti-GABARAP (1:1,000, Affinity, China), anti-EZH2, anti-FOXK1, anti-STAT1 (1:1,000, Abcam, United Kingdom), anti-P62, anti-HuR (1:1,000, Proteintech, China)), anti-ATF3 (1:1,000, Proteintech, China), anti-LC3 polyclonal antibody and incubated overnight. Membranes were washed three times with 1× tris buffered saline tween and then incubated with goat anti-rabbit/mouse secondary antibody for 1 h. Finally, protein expression was detected by enhanced chemiluminescence kit (Spark Jade, China).

Tissues or cells were collected and lysed in radioimmunoprecipitation assay (RIPA, Solarbio, R0020) buffer and phenylmethylsulfonyl fluoride (PMSF, Solarbio, P0100) (RIPA buffer: PMSF = 100:1). The protein concentration was measured by bicinchoninic acid protein assay kit (Coolaber, SK1070). After separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was transferred to polyvinylidenefluoride membranes. The protein was sealed for 2 h with 5% skim milk and incubated overnight at 4°C with antibodies: anti-collagen I (Affinity, AF7001), anti-collagen III (Affinity, AF0136), anti-vimentin (Affinity, AF7013), anti-α-SMA (Affinity, AF1032), anti-FAP1 (Cell Signaling Technology, 66562S), anti-S100A4 (Cell Signaling Technology, 13018S), anti-Myo1c (Abcam, ab194828), anti-YAP1 (Cell Signaling Technology, 14074S), anti-phospho-YAP1 (Cell Signaling Technology, 53749S), anti-F-actin (Abcam, ab130935), anti-GAPDH (Affinity, AF7021). 1×Tris buffered saline Tween was used to wash the membranes for three times. Then membranes were incubated for 1 h with secondary antibodies at room temperature. The expression of proteins was detected by enhanced chemiluminescence reagent kit (SparkJade, ED0015-B).

Western blotting was performed as described previously [54 (link)]. Briefly, cells and liver tissue samples were collected and lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Solarbio, Beijing, China). Protein concentration was measured using a BCA protein assay kit (LABLEAD, Beijing, China). Proteins were separated by 10% SDS-PAGE gel at 115 V for 1.2 h, then were transferred to a PVDF membrane at 200 mA for 1 h. The membranes were blocked with 5% milk in TBST for 1 h and incubated overnight at 4 °C with primary antibodies. Primary antibodies included anti-Smad4, anti-α-SMA, anti-GAPDH, anti-E-cadherin, anti-ID1, anti-CTGF, anti-p65, anti-p-p65, anti-p38, and anti-p-p38 (Affinity Biosciences, Cincinnati, OH, USA). Followed by HRP-conjugated goat, anti-mouse and goat anti-rabbit IgG (Solarbio, Beijing, China) were used as secondary antibodies. Protein bands were scanned using a Clinx Science Instrument and quantified with Image J software.

MRC-5 cells were inoculated into 24-well plates, and paraffin sections of mouse or human lung tissues were baked for dewaxing, fixed with 4% paraformaldehyde fixative, punched with TritonX-100 and then washed 2–3 times with PBS, and then closed with goat serum for 1 h. Primary antibodies were added, and the plates were incubated at 4°C overnight with antibodies: anti-α-SMA (1:200, Affinity, AF1032), anti-S100A4 (1:250, Affinity, DF6515), anti-F-actin (1:250, Abcam, ab130935), anti-SPC (1:250, Affinity, DF6647), anti-E-cadherin (1:250, Affinity, BF0219), P16 (1:250, Affinity, BF0580). On the next day, fluorescently labeled secondary antibody was added and incubated for 60 minutes at room temperature protected from light. Cell nuclei were stained by adding DAPI and then washed with 1 × PBS. Finally, a drop of anti-fluorescence quencher was applied to the cell slides. All images were collected under fluorescence microscopy (Invitrogen EVOS M7000, Thermo Fisher, USA).

Lung tissues were sectioned at 8 μm. Verhoeff–Van Gieson staining (VVG, HT25A; Sigma Aldrich, St. Louis, MO, USA) was conducted, and the ratio of staining area to artery area was calculated in both pulmonary arterioles (diameter < 100 μm and diameter ≥ 100 μm) [27 (link)]. The media of pulmonary arteries was measured using a primary antibody against smooth muscle-specific alpha-actin (anti α-SMA; Affinity, OH, USA) [28 (link)], as described previously [27 (link)]. Slides were incubated with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (sc-2005; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37 °C. After adding 3, 3′-diaminobenzidine chromogenic, slides were examined and assessed for the ratio of media/(media + lumen + intima). The immunostaining density of eNOS in the endothelium was measured using an eNOS antibody (ab5589; Abcam, Cambridge, UK) [26 ], following the manufacturer’s protocol [14 (link)]. Six slides (five arteriole within each section) were selected for taking pictures using Leica microscope (DM2500, Leica Camera AG, Solms, German) and quantification using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).