Vimentin

Cardiac fibroblasts in the confocal dish were washed twice with PBS and fixed with 4% paraformaldehyde for 20 min. Cells were washed with PBS for 5 min and perforated by 0.5% TritonX-100 for 15 min. Then, cells were washed twice with PBS for 5 min each time and blocked with 20% goat serum at 37 °C for 30 min. After that, cells were incubated with diluted primary antibodies of vimentin (Bioss) and cTnT (Abcam) overnight at 4 °C. The next day, cells were washed twice and incubated with Alexa Fluor 488 and Alexa Fluor 555-conjugated secondary antibodies at room temperature for 1 h away from light and washed twice. Finally, cells were stained with 4, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 5 min and washed again. Cells were observed using an inverted fluorescence microscope (Olympus IX71, Japan) after mounting under glycerol.

Cells were lysed in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc., Gyeonggi-do, Korea) to prepare protein lysates according to the manufacturer’s instructions. Cell lysates were centrifuged at 12,000 rpm for 10 min at 4 °C. Protein concentration was measured using Bradford Easy Protein Quantitative Kit (TransGene). Equal amounts of protein were separated in 10% polyacrylamide gels (Sigma-Aldrich) and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 10% non-fat milk and incubated with anti-α-SMA (Abcam, Cambridge, UK), anti-collagen I (Abcam), anti-E-cadherin (Abcam), anti-N-cadherin (Bioss, Beijing, China), albumin (Bioss), matrix metalloproteinase 9 (MMP9) (Bioss), vimentin (Bioss), pan-cytokeratin (a wide spectrum cytokeratin antibody, Bioss), fibronectin (Bioss), β-catenin (Sigma) and anti-GAPDH (TransGene) antibodies at 4 °C overnight. After washing the membranes with Tris-buffered saline containing Tween 20 three times for 5 min each time, the membranes were incubated with secondary antibodies. Finally, immunoreactive proteins were detected using an enhanced chemiluminescence detection kit (Beyotime, Shanghai, China).

For immunohistochemical examination, paraffin-embedded colonic sections were deparaffinized in xylene and hydrated in gradient alcohol. Then, antigen retrieval was performed by heating in pre-boiling buffer in a microwave for 10 min. Next, slides were incubated in 3% hydrogen peroxide solution for 15 min to quench endogenous peroxidase activity and then blocked by 10% goat serum in PBS (pH 7.4) for 15 min at room temperature. Subsequently, slides were incubated with primary antibodies in a humidified chamber at 4°C overnight: cyclooxygenase-2 (COX-2; 1:300), inducible nitric oxide synthase (iNOS; 1:300), β-catenin (1:200), E-cadherin (1:200), N-cadherin (1:200; BOSTER, Wuhan, China), proliferating cell nuclear antigen (PCNA; 1:100), Vimentin (1:300), Snail (1:100; Bioss, Beijing, China), or p53 (1:50), p-p53 (1:50; Santa Cruz, Dallas, TX, USA). After incubation with biotinylated goat anti-rabbit secondary antibody (1:200; Beyotime, Jiangsu, China) and avidin-biotin-horseradish peroxidase (HRP; Beyotime, Jiangsu, China), slides were visualized using diaminobenzidine (DAB), counterstained with haematoxylin and observed under an optical microscope.

C4-2, HEK293, Phoenix and 22Rv1 cell lines were purchased from ATCC. AURKA, Actin and YBX1 antibodies were bought from Santa Cruz Biotech (Santa Cruz, CA, USA). N-cadherin, CD44, Slug and Snail antibodies were bought from One World Lab (San Diego, CA, USA). E-cadherin, MMP-2 and Vimentin antibodies were obtained from Bioss (Woburn, MA, USA). All validated antibodies were used at 1-1000 dilution. Details of antibodies are provided in Supplementary Table S1.

Proteins from SCC15 cells and xenograft tumor tissues were extracted using immunoprecipitation assay buffer. The concentration of total protein was determined using the Lowry method. Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% gels and transferred to nitrocellulose membranes. The membranes were then blocked and incubated with primary antibodies against Prx1 (1:1000; Abcam, Cambridge, MA, United States), Snail (1:500; Abcam), E-cadherin (1:1000; Cell Signaling Technology, Beverly, MA, United States), vimentin (Bioss, Beijing, China), and GAPDH (1:2000; Sigma–Aldrich, United States). The protein bands were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using an enhanced chemiluminescence detection system.

Human breast cancer cell lines HS578T and BT20 were purchased from American Type Culture Collection. Cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM, HyClone/Thermo) supplemented with 10% fetal bovine serum (Biowest), 50 IU/ml of penicillin, and 50 µg/ml of streptomycin. Cells were kept at 37 °C in a humidified incubator with 5% CO₂. Phosphate-buffered saline (PBS) with 0.05% trypsin (Invitrogen) was used for cell harvesting and passage. Antibodies for MAGP2 (MFAP5), Smad2/Smad3 and p-Smad2/Smad3 were purchased from Abcam (Cambridge, UK). Antibodies for E cadherin, N-cadherin, vimentin and Type I Collagen were purchased from Bioss (Shanghai, China). TGF-β inhibitor SB431542 and the Notch inhibitor ly-411575 were purchased from Selleck (USA).

For formalin‐fixed paraffin‐embedded tissues, slides were de‐waxed by washing with xylenes, rehydrated and blocked. For cultured cells, cells were seeded in chamber slides (Cat. #: C6807; Sigma Chemical Co.), fixed with 4% formaldehyde, permeabilized in 0.1% Triton‐X100, and blocked. Slides were then incubated with primary antibodies for α‐SMA (Cat. #: bs‐0189R; Bioss, Beijing, China), Vimentin (Cat. #: 5741; Cell Signaling Technology, Danvers, MA, USA), E‐cadherin (E‐cad, Cat. #: 14472; Cell Signaling Technology), or SP‐C (Cat. #: sc‐7706; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), for overnight at 4°C. Slides were then washed and stained with fluorophore‐conjugated secondary antibodies for 1 hr at room temperature. Cover slips were mounted using mounting medium (Cat. #: C9368; Sigma Chemical Co.), allowed to harden overnight at 4°C and then sealed. Images were acquired on Olympus IX70 (Japan) fluorescence microscope.