Twist

Western blot assays were performed using the following primary antibodies: anti-human MMP2 (Abcam, Cambridge, MA, USA; 1:500), Twist (Abcam; 1:500), fibronectin1 (FN1, Abcam; 1:500), MMP9 (cell signaling, Danvers, MA, USA; 1:500), Bcl2 (cell signaling; 1:500), Bax (cell signaling; 1:500), PCNA (cell signaling; 1:500), E-cadherin (Santa Cruz Biotech., Santa Cruz, CA, USA; 1:500), β-catenin (cell signaling; 1:1000), ERK1/2 (cell signaling; 1:1000), p-ERK1/2 (cell signaling; 1:1000), AKT (cell signaling; 1:1000), p-AKT(cell signaling; 1:1000) and GAPDH (Millipore; 1:1,000). Briefly, stimulated cells were lysed with RIPA buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate) containing protease inhibitors; 20–30 μg samples of the lysates were separated on 8%–12% SDS PAGE gels and transferred to PVDF membranes. The membranes were incubated with primary antibodies overnight at 4°C. The primary antibody incubation was followed by incubation with an HRP-conjugated secondary antibody. Finally, the bound antibodies were detected using an ECL substrate.

Antibodies to p‐PDGFR‐β, p‐VEGFR2, p‐Src, Src, p‐STAT3, STAT3, p‐Smad3, Smad3, TGF‐β1, p‐NF‐κBp65, p‐Akt, Akt and β‐Actin were purchased from Cell Signaling Technology. TGF‐β1 and antibodies to type I collagen and fibronectin were purchased from Santa Cruz Biotechnology. p‐FGFR1 antibody was purchased from Life Span Biosciences. NF‐κBp65 antibody was purchased from Prosci Inc. Antibodies to CD68, CD31, MMP‐2, TIMP‐2, E‐cadherin, vimentin, Snail, Twist, and MCP‐1, TNF‐α, IL‐1β and IL‐6 ELISA assay kits were purchased from Abcam Inc. Nintedanib was purchased from Cayman. α‐SMA antibody, CG, Cell Counting Kit‐8 (CCK‐8) proliferation assay kit and all other chemicals were purchased from Sigma.

Cells or EVs were lysed in RIPA buffer (CWBIO, Beijing, China) with protease and phosphatase inhibitors (CWBIO). Identical quantities of proteins were electrophoresed by SDS‐PAGE, transferred onto PVDF membranes and incubated with primary antibodies specific for TSG101 (Abcam, Shanghai, China), CD63 (Abcam), TET1 (Abcam), Twist (Abcam) and GAPDH (Abcam) at 4°C overnight, followed by incubation with appropriate HRP‐conjugated secondary antibodies at room temperature for 1 hour. Signals were detected by Immobilon ECL substrate (Millipore, Germany), and the images were acquired using an Optimax X‐ray Film Processor (Protec, Germany).

Cells and murine lung tissue specimens were homogenized in extraction buffer (50 mM HEPES, 250 mM NaCl, 5 mM EDTA, 0.1% NP-40, 1 mM PMSF,1 mM DTT supplemented with Protease inhibitor cocktail). Whole proteins were extracted by centrifugation (12,000 × g) for 10 min at 4°C. Protein concentration was determined using the BCA kit (Pierce). Equal amounts of protein (20 μg/lane) from the cell lysates were electrophoresed under nonreducing conditions on 10% acrylamide gels. After SDS-PAGE, proteins were transferred to a polyvinylidene difluoride membrane. The membrane was incubated for 2 h in PBS plus 0.1% Tween-20 and 5% nonfat skim milk to block nonspecific binding. Subsequently, the membrane was incubated for 2 h with an antibody against E-cadherin (1:500, Invitrigen), Vimentin (1:500, Sigma) and Twist (1:400, Abcam), followed by incubation with fluorescent secondary antibodies (1:5000, IRDye 800 anti-mouse Molecular Probes, Rockland). Blots were stripped and reprobed by using anti-actin antibody (Santa Cruz Biotechnology). Images were acquired with the Odyssey infrared imaging system and analyzed by the software program as specified by Odyssey.

Western blot analysis was performed using specific primary antibodies against CD133 (Proteintech, USA), CD44 (CST, USA), Lgr5 (Abcam, USA), EpCAM (Abcam, USA), ALDH1 (Abcam, USA), β-catenin (Proteintech, USA), Nanog (CST, USA), E-cadherin (CST, USA), N-cadherin (Epitomics, USA), vimentin (CST, USA), Snail (Abcam, USA), Twist (Abcam, USA), Slug (Abcam, USA), ZEB1 (Abcam, USA), fibronectin (Abcam, USA), Sox2 (CST, USA), Oct4 (CST, USA), PRDX2 (Abcam, USA), Cyclin D1 (Abcam, USA), c-Myc (Abcam, USA), MMP-2 (Abcam, USA), MMP-9 (Abcam, USA), VEGF (Abcam, USA) and GAPDH (Goodhere, China).

Cells were lysed in lysis buffer (1% NP-40, 50 mM Tris, 0.1% SDS, 1 mM PMSF, 10 mM EDTA, 150 mM NaCl, and 0.5% sodium deoxycholate) as instructed (Beyotime, China). Supernatants of lysates were collected after centrifugation. A BCA protein detection kit (Beyotime, China) was used to determine the protein concentration. SDS/PAGE was used for the separation of the indicated amounts and then transferred on to PVDF membranes (Millipore, U.S.A.). Using nonfat milk, the membranes were blocked for 1 h. Next, primary antibodies were incubated overnight at 4°C. Then, the membranes were washed using TBST for 15 min and incubated with secondary antibodies (1:5000) at 37°C for 1 h. After washing with TBST for 15 min, the detection was performed with Fusion FX (Vilber, France) using an enhanced chemiluminescence kit (Millipore, U.S.A.). Specific bands were quantified using Fusion software. Each experiment was performed in triplicate. The following antibodies were used: CD133 (Proteintech, U.S.A.), CD44 (CST, U.S.A.), Lgr5 (Abcam, U.S.A.), EpCAM (Abcam, U.S.A.), ALDH1 (Abcam, U.S.A.), β-catenin (Proteintech, U.S.A.), CD166 (CST, U.S.A.), E-cadherin (CST, U.S.A.), N-cadherin (Epitomics, U.S.A.), vimentin (CST, U.S.A.), Snail (Abcam, U.S.A.), Twist (Abcam, U.S.A.), Slug (Abcam, U.S.A.), ZEB1 (Abcam, U.S.A.), fibronectin (Abcam, U.S.A.), and GAPDH (Goodhere, China).