Tgf β1

CSCs were sorted and grown in serum‐free DMEM/F12 medium in glass‐coverslips for 16 hours before fixed with 2% paraformaldehyde at 37°C for 15 minutes. The fixed cells were then permeabilized with 0.1% Triton X‐100 at the room temperature for 15 minutes followed by incubation at 37°C for 2 hours with primary antibody against KLF4 (Santa Cruz Biotechnology) or TGF‐β1 (Cell Signaling Technology). Cells were then incubated with FITC‐conjugated or APC‐conjugated secondary antibodies (Santa Cruz Biotechnology) at the room temperature for 1 hour and mounted with DAPI (Invitrogen). Images were taken using Olympus FV1200 microscope.

HSC-T6 was purchased from Beijing Friendship Hospital (Beijing, China). TGF-β1, LY-364947 [4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline HTS 466284 transforming growth factor β Type I receptor kinase inhibitor, molecular formula: C17H12N4, weight: 272.30, and purity: ≥95% (HPLC)], DMSO, and MTT were purchased from Sigma (St. Louis, USA). AST [molecular weight: 248.36386; purity: ≥98%] was purchased from Pure-One Bio Technology (Shanghai, China). Malotilate was purchased from Yabang Epson Pharmaceutical (Jiangsu, China). Kits of ALT, AST, TBil, Alb, MDA, GSH, SOD, COL-I, COL-III, α-SMA, MMP-2, TIMP-2, hydroxyproline, H&E staining, Masson's trichrome staining, Sirius red staining, and Annexin V-FITC/PI double staining were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Antibodies against α-SMA, TGF-β1, TβR-I, Smad 2, p-Smad 2 (S465/467), Smad 3, p-Smad 3 (S423/425), Smad 7, and β-actin were purchased from Cell Signaling Technology (MA, USA). HRP-labeled secondary antibodies were purchased from ZSGB-BIO (Beijing, China). RevertAid™ First Strand cDNA Synthesis Kit and SYBR Green Real-Time PCR Kit were purchased from Thermo Fisher (MA, USA).

Total proteins were extracted from cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich) and quantified by Bicin Choninic Acid (BCA) protein assay kit (Thermo). The Western blot was performed according to standard procedures. The following antibodies were used: EphA8 (Abcam, USA), TGFβ1, TGFβR1, smad2, smad3 and smad4 (Cell Signaling Technology, Beverly, MA, USA); β-actin (Proteintech, USA) was used as loading control. The blots were incubated with goat anti-rabbit or anti-mouse secondary antibodies (Bioss, Beijing, China). The proteins were visualized using a chemiluminescence method (ECL Plus Western Blotting Detection System; Amersham Biosciences, Foster City, CA, USA). The bands were quantified by ImageJ software.

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.

Antibodies against CXCR7 (ABcam; ab38089), GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA; sc-25778), E-cadherin (Cell Signaling Technology, Danvers, MA, USA; 3195), Ep-CAM (Santa Cruz Biotechnology; sc-25308), N-cadherin (Cell Signaling Technology; 13116), α-smooth muscle actin (Sigma Aldrich, St. Louis, MO, USA; A5228), Slug (Cell Signaling Technology; 9585), Twist (Santa Cruz Biotechnology; sc-81417), Vimentin (Cell Signaling Technology; 3932), phosphorylated-AKT at Ser⁴⁷³ (Cell Signaling Technology; 9271), AKT (Cell Signaling Technology; 9272), phosphorylated-ERK1/2 (Cell Signaling Technology; 9101), ERK1/2 (Cell Signaling Technology; 9102), phosphorylated-JNK (Cell Signaling Technology; 4668), JNK (Cell Signaling Technology; 9251), phosphorylated-p38 (Cell Signaling Technology; 9211), p38 (Cell Signaling Technology; 9212), TGF-β1 (Cell Signaling Technology; 3711), phosphorylated-Smad2/3 (Cell Signaling Technology; 8828), Smad2/3 (Cell Signaling Technology; 8685), MMP2 (ABcam; ab37150), and MMP9 (ABcam; ab38898) were used in Western blotting and immunofluorescence. Small interfering (si) RNAs for controls, CXCR7, and Smad2/3 were purchased from Santa Cruz Biotechnology and Thermo Fisher Scientific (St. Louis, MO, USA). For inhibition of protein kinases, LY294002 and wortmannin were purchased from Sigma Aldrich.