Ab193555

RIPA lysis buffer (Beyotime, Shanghai, China) was applied to extract complete protein based on the specification of the manufacturer. Equal amounts of proteins were subjected to SDS/PAGE (Bio-Rad, Hercules, CA, U.S.A.) and then transferred to a polyvinylidene fluoride (PVDF) (Sigma–Aldrich). Then, PVDF was sealed with skin milk. The membrane was cultured with primary antibodies at 4°C overnight and antibodies were as follows: anti-E-cadherin (ab194982, Abcam, Cambridge, U.S.A.), anti-N-cadherin (ab202030, Abcam), anti-Vimentin (ab193555, Abcam), anti-YY1 (ab109237, Abcam) and anti-GAPDH (ab8245, Abcam). The proteins were visualized via Chemiluminescence detection system (GE Healthcare, Chicago, IL, U.S.A.). Besides, GAPDH was seen as a loading control.

The cells were seeded in a 12-well plate on cover slips at the density of 10⁵ cells/well, and cultured for 24 h. After fixing with 4% paraformaldehyde, the cells were incubated with anti-a-SMA (1:500, ab32575, Abcam), anti-FAP (1:500, ab53066, Abcam), and anti-FSP-1 (1:500, ab32575, Abcam), and anti-Vimentin (1:500, ab193555, Abcam) antibodies, followed by Alexa Fluor® 647-conjugated donkey anti-rabbit IgG H&L (1:500, ab150075, Abcam). The immunostained cells were visualized under a fluorescence microscope.

Total protein of colonic tissues was extracted according to the manufacturer’s protocol (Vazyme, USA). Briefly, protein concentrations were determined through BCA Protein Assay Kit (Vazyme, USA). Samples with equal amounts of protein (25 µg) were fractionated on 10% SDS polyacrylamide gels, transferred to polyvinylidene difluoride (PVDF) membranes, and blocked in 5% skim milk in TBST for 1.5 h at 25°C. The membranes were then incubated at 4°C overnight with 1:1,000 dilutions (v/v) of the primary antibodies. After washing the membranes with TBST, the secondary antibodies with 1:1,000 dilutions were added and incubated for another 2 h at 25°C. Protein expressions were examined using an Enhanced Chemiluminescence Detection System. GAPDH was used as a loading control. Antibodies in western blotting were purchased from Abcam (Cambridge, MA, USA), including Col1a2 (ab96723), Col3a2 (ab196613), MMP-1 (ab52631), MMP-3 (ab52915), TIMP-1 (ab86482), E-cadherin (ab76055), N-cadherin (ab202030), Vimentin (ab193555), a-SMA (ab32575), MMP-9 (ab38898) and GAPDH (ab181602).

Cells were seeded onto sterile cover slips in a Corning 12-well culture plate at density of 10⁴ cells/mL and treated according to the experimental design. At the indicated time point, cells were washed three times with PBS for 10 min each, then fixed with 4% paraformaldehyde at 37 °C for 15 min in a thermostatic water bath, washed with PBS for 10 min each, and then permeabilized using 0.4% Triton X-100 for 30 min at 37 °C. After cells were blocked with goat serum for 30 min, cells were incubated with the primary anti-CK19 (ab52625, Abcam, Cambridge, MA, USA), anti-vimentin (ab193555, Abcam, Cambridge, MA, USA), anti-CD31 (ab134168, Abcam, Cambridge, MA, USA), VEGF (ab32152, Abcam, Cambridge, MA, USA), and anti-vWF (ab6994, Abcam, Cambridge, MA, USA) antibodies overnight, followed by incubation with the corresponding fluorophore-conjugated antibodies for 60 min, then cells were washed with PBST for 10 min each and stained with DAPI for 5 min. The cover slips were carefully removed and then mounted on slides with glycerol. The same protocol was performed in the negative control groups except that the primary antibodies were omitted. The slides were observed by confocal microscopy (DFM-80C, Nikon, Japan), and images were assessed by Nikon auxiliary systems. The results of immunofluorescence were quantified using the Image Pro Plus software.

Western blot analysis was executed based on the methods described previously [25 (link)]. The primary antibodies against Vimentin (ab193555; 1:1000; Abcam, Cambridge, MA. USA), E-Cadherin (E-cad) (ab133597; 1:1000; Abcam), Caspase-3 (ab13847; 1:1000; Abcam), CyclinD1 (ab16663; 1:1000; Abcam), KIF23 (PA5-31773; 1:1000; Invitrogen) and β-actin (ab8227; 1:5000; Abcam) were used in this study, and goat anti-rabbit antobodies (ab205718; 1:5000; Abcam) were acted as the second antibodies. The protein blots were quantified using the Image J software (version 1.46; National Institutes of Health, Bethesda, MA, USA).