Fibronectin

Whole-cell lysates were separated in 12% SDS-PAGE gels and blotted on nitrocellulose membranes, and probed with antibodies against β-Actin (Santa Cruz Biotechnology, Inc.), RhoGDI1, Fibronectin, Snail (Proteintech), N-cadherin (Epitomics), E-cadherin, and β-catenin (Cell Signaling Technology). After incubation with primary antibodies, the membranes were washed with TBS/0.05% Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. Signals were detected using enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA).

As reported previously [55 (link)], proteins in the lung or cells were extracted by RIPA lysis buffer containing protease and phosphatase inhibitors (Servicebio, China), and the protein concentration was determined using the BCA protein kit (Thermo, United States). After denaturation, proteins were separated by SDS-PAGE (Epizyme, China) and blotted onto activated PVDF membranes (Millipore, United States). After blocking with protein free rapid blocking buffer (Epizyme, China) for 10 min, the membranes were incubated with primary antibodies against GAPDH (1:50000, Proteintech), β-actin (1:50000, Proteintech), CDA1 (1:2000, Proteintech), α-SMA (1:1000, Abcam), collagen I (1:1000, Abcam), fibronectin (1:1000, Proteintech), TGF-β1 (1:1000, Proteintech), Smad3 (1:2000, Abcam), phosphorylated Smad3 (P-Smad3) (1:2000, Abcam), GFP (1:2000, Proteintech) at 4 °C overnight. After washing, the membranes were incubated with the appropriate secondary antibodies and visualized using an ECL Assay Kit (Epizyme, China). GAPDH/β-actin served as an internal control. The intensities of the target bands were quantified using ImageJ software.

The following commercial antibodies and reagents were used: ZEB2 (ab138222, 1:500; Abcam, Cambridge, UK); TWIST1 (46702S, 1:500; Cell Signaling Technology, Danvers, MA, USA); E-cadherin (610181, 1:1000; BD Biosciences, San Diego, CA, USA); HDAC2 (5113T, 1:1000; Cell Signaling Technology); GAPDH (60004-1-Ig, 1:2000; Proteintech, Wuhan, China); ACTIN (66009-1-Ig, 1:2000; Proteintech); Tublin (11224-1-AP, 1:2000; Proteintech); GST (10000-0-Ig, 1:1000; Proteintech); HIS (66005-1-Ig, 1:1000; Proteintech); Flag (20543-1-AP, 1:1000; Proteintech); PRMT5 (18436-1-AP, 1:1000; Proteintech); RbAp48 (20364-1-AP, 1:1000; Proteintech); MTA2 (66195-1-Ig, 1:1000; Proteintech); Vimentin (10366-1-AP, 1:1000; Proteintech); Fibronectin (15613-1-AP, 1:1000; Proteintech); H3K56ac (39281, 5 μg/ChIP; Active Motif, Carlsbad, CA, USA); H4R3me2S (61187, 5 μg/ChIP; Active Motif). siRNAs and shRNAs were synthesized by GenePharma Co., Ltd. (Shanghai, China). The targeted sequences are listed in Supplementary Materials (Supplemental Table S1).

Collagen I and α-SMA antibodies were from Abcam (Cambridge, MA); p-Smad2/3 and Smad2/3 antibodies were obtained from Cell Signaling Technology (Beverly, MA, United States); Fibronectin and ERK1/2 antibodies were purchased from Proteintech (Wuhan, China); IL-11 and p-ERK1/2 antibodies were obtained from ABclonal Technology (Wuhan, China); IL-11RA antibody was from Santa Cruz Biotechnology (Santa Cruz, CA); GAPDH antibody was obtained from Wuhan Gugeshengwu Technology Co., Ltd (Wuhan, China); all secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, United States).
Osthole (Ost, Figure 1A) was obtained from Sigma-Aldrich (St. Louis, MO); Fetal bovine serum (FBS) was purchased from Gibco (Thermo Fisher Scientific, Waltham, MA); Dulbecco’s modified Eagle medium/Ham’s F-12 medium (DMEM/F12) was purchased from Hyclone Laboratories Inc (Logan, UT, United States); Recombinant human TGF-β1 and IL-11 were obtained from Peprotech (Rocky Hill, NJ, United States); U0126 was provided by Selleck Chemicals (Shanghai, China); Trizol, Hifair^™ II 1st Strand cDNA Synthesis Super Mix and Hieff^®qPCR SYBR^®Green Master Mix were purchased from Yeasen (Shanghai, China); The immunohistochemistry kit was from Wuhan Gugeshengwu Technology Co., Ltd. (Wuhan, China). All other regular reagents were obtained from Wuhan Gugeshengwu Technology Co., Ltd. unless otherwise specified.

Total protein was extracted from the thoracic aorta tissues with a Pro-Prep Protein Extraction Solution (Intron Biotechnology, Gyeonggi-do, Republic of Korea) according to the manufacturer's instructions. Western blot analysis was performed using the following antibodies: transforming growth factor-β (TGF-β, R&D Systems, MN, USA), collagen IV (Abcam, Cambridge, UK), fibronectin (Proteintech Group Inc., IL, USA), Ang II (Novus Biologicals, CO, USA), ACE (Santa Cruz Biotechnology, TX, USA), ACE2 (R&D Systems, MN, USA), AT1R (Santa Cruz Biotechnology, TX, USA), AT2R (Novus Biologicals, CO, USA), PRR (Sigma Life Science, MO, USA), MasR (Novus Biologicals, CO, USA), endothelial nitric oxide synthase (eNOS, Cell Signaling Technology Inc., MA, USA), NADPH oxidase 2 (Nox2, BD Biosciences, MD, USA) and NADPH oxidase 4 (Nox4, Santa Cruz Biotechnology, TX, USA), superoxide dismutase 1 (SOD1, Enzo Life Sciences, NY, USA), superoxide dismutase 2 (SOD2, Abcam, Cambridge, UK), and β-actin (Sigma Life Science, MO, USA).

Protein samples derived from mouse skins and cells were extracted by RIPA lysis buffer containing phosphatases and proteases inhibitor cocktails (Roche, USA). Protein concentration was determined by BCA protein assay kit (Pierce, USA). For immunoblotting analysis, protein samples were subjected with SDS-polyacrylamide gel electrophoresis and proteins were transferred to PVDF membranes (Millipore). After being blocked, and incubated with primary antibodies and indicated HRP-coupled secondary antibody, membranes were visualized with ECL and images were captured using the Bio-Rad system. Band intensities were detected, normalized, and quantified with the Image Lab 5.0 software (Bio-Rad). The following antibodies were used in this research: NAT10 (Abcam, ab194297, 1:1000 dilution), STAT3 (CST, 30835, 1:1000 dilution), p-STAT3 (Abcam, ab76315, 1:1000 dilution), p-p65 (CST, 3033, 1:1000 dilution), p65 (CST, 8242, 1:1000 dilution), p-FAK(CST, 8556, 1:1000 dilution), FAK (Abcam, ab40794, 1:1000 dilution), Lamin B1 (Abcam, ab133741, 1:1000 dilution), poly-Ubiquitin (CST, 3936, 1:1000 dilution), Fibronectin(CST, 26836, 1:1000 dilution), α-SMA(Proteintech, 14395-1-AP, 1:1000 dilution), α-Tubulin (Beyotime, AT819, 1:5000 dilution), and GAPDH (Beyotime, AG019, 1:5000 dilution).