Smad3

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).

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).

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.

The total protein contents of the cells or tissues were obtained after lysis in RIPA buffer (Solarbio) containing protease inhibitors. The proteins were then boiled in SDS sample buffer and separated on SDS-PAGE before transfer to PVDF membranes (Millipore, USA). After blocking with 5% skim milk, the membranes were treated with primary antibodies at 4℃ overnight. Antibodies against the following proteins were used: SLC7A11 (Proteintech, China), CBS (Proteintech), CTH (Abcam, UK), ATF4 (Abcam), γH2AX (Abcam), p-SMAD3 (Proteintech), SMAD3 (Proteintech), GAPDH (Proteintech), and β-actin (Abcam). The membranes were then incubated with secondary antibodies for 2 h. The bands were visualized using the ECL detection reagent (Advansta, USA).

According to the sequencing data and functional analysis, we analyzed the proteins associated with the transforming growth factor beta (TGF-β) signaling pathway in granulosa cells using the Western blot. Briefly, the whole protein samples were extracted, and their concentrations measured using the BCA Protein Assay Kit (GenStar, Beijing, China). Then, equal amounts of proteins (20 μg per lane) were loaded and separated on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel. Primary antibodies were used after proteins transferred to the 0.45-μm Immobilon-P PVDF Membrane, and GAPDH used as loading control. Signals were obtained in the linear range of detection and quantified with the Bio-Rad ChemiDoc Imaging System. The grey values of target bands were analyzed by ImageJ software and normalized to the reference band. The primary antibodies used were TGF-β2 (19,999–1-AP; 1:2000 diluted; Proteintech), TGF-βR2 (66,636–1-lg; 1:2000 diluted; Proteintech), Smad2 (12,570–1-AP; 1:2000 diluted; Proteintech), Smad3 (25,494–1-AP; 1:2000 diluted; Proteintech).