Fibronectin

Cellular proteins were lysed using standard methods. Cellular proteins were separated by 10% SDS‐PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with TBS containing 0.1% Triton X‐100 and 5% nonfat milk for 2 hours at RT and then were incubated with Trop2 (R＆D SYSTEMS), E‐cadherin (Invitrogen), fibronectin (Affinity), vimentin (Affinity), β‐catenin (Santa Cruz Biotechnology), and β‐actin antibody (Abcam) at 4°C overnight. After being washed, the membranes were incubated with HRP‐conjugated anti‐IgG at RT for 1 hour. Signal detection was carried out with an ECL system (Tanon, shanghai, China). BGC823 and MGC803 were transfected with different plasmids and then lysed in cellular lysis buffer (Sangon Biotech, Shanghai, China) for 15 minutes on ice. Extracts were clarified by centrifugation at 13 000 g for 25 minutes at 4°C for co‐immunoprecipitation. After centrifugation, the supernatant was collected, and then incubated with protein G PLUS‐agarose immunoprecipitation beads (Santa Cruz Biotechnology) at 4°C for 1 hour. Then, they were incubated with special antibodies against Trop2, β‐catenin at 4°C on a rocker platform. The antibody‐coated beads were then incubated with the lysates at 4°C overnight. Immunoprecipitates were collected, washed, lysed, and boiled. The boiled samples were analyzed by Western blot analysis as describe above.

Immunohistochemical analyses were performed on 5μm paraffin sections with an indirect immunoperoxidase method by antibodies against JAM-A (Life Technologies, 1:100 dilution), E-Cadherin, Vimentin, Fibronectin (Affinity biosciences, Piscataway, NJ, USA, 1:200 dilution) and NODAL (Abcam, 1:200 dilution). Immunofluorescent assays were performed on acetone-fixed cells with rabbit anti-human-E-Cadherin, Vimentin and Fibronectin as primary antibodies, and Alexa Fluor-conjugated donkey anti-mouse-IgG and anti-rabbit-IgG antibodies (Invitrogen, Carlsbad, CA, USA) as secondary antibodies. Nuclei were stained by DAPI.
The protein expression levels were scored based on staining intensity (SI) and distribution using the immunoreactive score (IRS). Briefly, IRS = SI × PA (positive area). The intensity of immunohistochemical and immunofluorescence staining was measured by the Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Inc., Silver Spring, USA)^{29 (link)}.

Deg-AZM (≥98.0%) was manufactured by chemical laboratory, College of Pharmacy, Nankai University. Recombinant Human TGF-β1 was purchased from PEPROTECH (USA). Antibodies to α-SMA, Collagen I, Fibronectin, P-Smad3/Smad3, P-Akt/Akt, P-ERK/ERK, P-P38/P38 were from Affinity Biosciences (Cincinnati, OH, USA). GAPDH, E-cadherin, Vimentin, N-cadherin antibodies were from Cell Signaling Technology (Danvers, MA, USA). Goat pAb to Rb IgG (HRP) and Rb pAb to Ms IgG were from ImmunoWay (Shanghai, China). TRIzol reagent was acquired from Ambion Life Technology (Shanghai, China). DEPC Treated H₂O and Rnase Away H₂O were purchased from Life Technologies. UNICON^® Qpcr SYBR Green Master Mix was from YEVSEN (Shanghai, China). BLM was from Nippon Kayaku (Tokyo, Japan). Hematoxylin-eosin solution and Masson’s Trichrome Stain Kit were from Solarbio.