Smad2

Propylene glycol alginate sodium sulphate was purchased from Dalian Tianyu Pharmaceuticals Co., Ltd and disposed in saline to obtain different drug doses of 12.5, 25 and 50 mg/kg, and stored at 4°C.¹² CCl₄ was acquired from Sinopharm. Microplate test kits of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute (Jiancheng Biotech). Antibodies against IL‐6, Col‐1, α‐SMA, TGF‐β1, MMP‐2, TIMP‐1, Beclin‐1, p62, Smad2 and Smad3 were purchased from Proteintech, and those against p‐Smad2, p‐Smad3, JAK2, STAT3 and p‐STAT3 were purchased from Cell Signaling Technologies. The polymerase chain reaction (PCR) kit was acquired from Takara Biotechnology.

After LX2, HCCLM3, and HCCLM3 coculture with CM were treated, proteins were extracted in culture and coculture flasks. Then, it was lysed using RIPA buffer (Beyotime, Shanghai, China) and centrifuged at 12,000 g for 10 min keep 4°C, respectively. Bicinchoninic acid (BCA, Beyotime, Shanghai, China) method was used to detect the total protein content. 50 mg protein from each group was transferred onto nitrocellulose (NC) membranes after through 10%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, Shanghai, China). Membranes were blocked with 5% fat-free milk for 60 min and incubated continuously with the following primary antibodies overnight at 4°C: CD133, PCNA, TGF-β, Collagen1, Smad2, Smad3, Snail, E-cadherin, N-cadherin, and β-actin, purchased from Proteintech Group (Wuhan, China). Finally, they were incubated with secondary antibodies (LI-COR Biosciences, Nebraska, USA) and imaged with an Odyssey Fc imaging system (LI-COR Biosciences, Nebraska, USA).

The experimental procedures for the extraction and detection of protein expression were performed following the protocols mentioned in the previous study [10 (link)]. The detailed information of antibodies were listed as follows: Nanog (Cat No. 14295-1-AP, 1:1000, Proteintech, Wuhan, China), ALG10 (Cat # ab124711, 1:3000, Abcam), Oct4 (Cat No. 11263-1-AP, 1:1000, Proteintech), Sox2 (Cat No. 11064-1-AP1:1000, Proteintech), TGFBR2 (Cat No. 66636-1-Ig, 1:1000, Proteintech), TGFBR1 (Cat # ab31013, 1:1000, Abcam), p-Smad2 (Cat # ab28088, 1:1000, Abcam), Smad2 (Cat No. 12570-1-AP, 1:1000, Proteintech), GAPDH (Cat No. 60004-1-Ig, 1:1000, Proteintech), Histone H3 (Cat No. 17168-1-AP, 1:1000, Proteintech) were used in this study. The full length uncropped original western blots used in their manuscript were shown in Supplementary Figure 1.

Antibodies against PIM1, p-Smad2 (S467), p-Smad3 (S423 and S425) and p-c-Myc (S62) were purchased from Abcam (Cambridge, MA, USA). Antibodies against E-cadherin, N-cadherin, Vimentin, ZEB1, ZEB2, Snail1, Snail2 (Slug), Twist, MMP2, MMP9, Smad2, Smad3, c-Myc, PCNA, Ki67 and GAPDH were purchased from Proteintech Group (Chicago, USA). Wright-Giemsa was purchased from Solarbio (Beijing, China). The inhibitors 10058-F4 and SGI-1776 were purchased from MedChem Express (New Jersey, USA). TGF-β was purchased from PeproTech (New Jersey, USA). The Alexa Fluor 488- and 594-conjugated secondary antibodies were purchased from Invitrogen (CA, USA).

Protein lysate was obtained from the cultured cells using the RIPA lysis buffer (Solarbio Corporation, Beijing, China). The protein concentration was measured with a bicinchoninic acid assay. Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membrane was first incubated with rabbit anti-rat primary antibodies, such as E-cadherin (1:2,000; no. 20874-1-AP), fibronectin (FN; 1:3,000; no. 15613-1-AP), vimentin (1:2,000; no. 10366-1-AP), Smad2 (1:2,000; no. 12570-1-AP), Smad4 (1:2,000; no. 51144-1-AP), zinc-finger-enhancer-binding protein 1 (ZEB; 1:2,000; no. 21544-1-AP) (all from Proteintech Group Inc., Chicago, IL, USA), Smad3 (1:2,000, no. AF6362; Affinity BIO, Scoresby, Australia), Smad7 (1:2,000; no. AF5147; Affinity BIO), and β-actin (1:5,000; no. Ab8226; Abcam, Cambridge, UK), and then incubated with goat anti-rabbit horseradish peroxidase-conjugated-labeled secondary antibodies (1:5,000, # A0208, Beyotime Institute of Biotechnology, Haimen, China) for 1 h at room temperature. The blots were detected using enhanced chemiluminescence.

Cells were lysed, and proteins in the supernatant extracts were quantified using a BCA Protein Assay Kit (Beyotime). Total cell lysates containing 50 µg of protein were separated using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto nitrocellulose (NC) membranes (PALL). After blocking with 5% non‐fat dry milk in Tris‐buffered saline‐Tween‐20 (TBST) for 2 hours, the membranes were incubated with primary antibodies [the glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibody diluted at 1:3000; collagen I, collagen III, lysyl oxidase (LOX), osteopontin (OPN), vimentin, α‐SMA, smad2, p‐smad2, smad3, p‐smad3, smad4, smad7 and p‐smad7 antibodies diluted at 1:2000 or 1:1000] at 4°C overnight. The collagen I, collagen III, p‐smad2, p‐smad3 and p‐smad7 antibodies were purchased from Abcam, and the GAPDH, β‐tubulin, smad2, smad3, smad4, smad7, LOX, OPN, vimentin and α‐SMA antibodies were purchased from Proteintech. After three washes with TBST, the membranes were incubated with the corresponding horseradish peroxidise (HRP)‐conjugated secondary antibody (1:5000, GE, HyClone) at 37°C for 2 hours. The protein bands were visualized with enhanced chemiluminescence (ECL; Advansta) and detected using a ChemiDoc^TM MP imaging system (BIO‐RAD). The protein bands were then scanned using Image Lab^TM Software Version 4.1.