Ab8978

Cells were cultured on the glass slide, which were then rinsed with PBS for three times and fixed with the 4% paraformaldehyde. Next, cells were treated with 0.5% Triton X-100 (Beyotime, China) to increase the permeability of cells. After that, cells were blocked with BSA (Beyotime, China) for 2 hours and incubated with the primary antibodies at 4°C overnight. Vimentin (Abcam, ab8978), α-SMA (Abcam, ab5831) and β-catenin (Abcam, ab223075) were applied. Followingly, cells were incubated with the secondary antibodies including Goat polyclonal Secondary Antibody to Rabbit IgG (Abcam, ab150077) and Goat Anti-Mouse IgG (Abcam, ab205719) in dark room for 2 hours. Finally, cell nucleus of was stained with the DAPI (Invitrogen, USA) and the fluorescence was observed using the laser scanning confocal microscope (Olympus, Japan).

JIMT-1 cells were plated on poly-L-lysine-coated glass slides and treated with 50 nM compound or 0.0005 % DMSO as control for 72 h. After fixation in 3.7 % paraformaldehyde (in PBS) for 15 min and subsequent washing in PBS, the cells were permeabilized with PBS containing 1 % Tween 20 and 1 % bovine serum albumin in a single step. The cells were incubated with primary antibody against vimentin (1:100), β-catenin (1:500) or E-cadherin (1:100) for 1 h at room temperature. Antibodies against vimentin (ab8978) and E-cadherin (ab1416) were purchased from Abcam. Antibody against β-catenin (610154) was purchased from BD Biosciences. After washing, the cells were incubated for 1 h with the Alexa Fluor 488 goat anti mouse (1:300) antibody (Invitrogen). Slides were counter-stained with bisbenzimide (Hoechst 33258) (1 μg/ml in PBS) for 2 min and finally washed with PBS before mounting. The cells were viewed in an Olympus/Nikon epifluorescence microscope (Olympus Optical Co. Ltd.) and photos were taken with a digital camera (Nikon Imaging Japan Inc.). Each slide was photographed at randomly chosen areas (at least 8 areas).

NFs, CAFs and GC cells (AGS and SGC-7901) were harvested and lysed by the RIPA (containing 1% PMSF) lysis solution (Beyotime, Shanghai, China), followed by the total protein extraction with centrifugation (14000 rpm for 30 min). The BCA protein concentration kit (Beyotime, Shanghai, China) was applied for protein concentration quantification. Then, the proteins were isolated via SDS-PAGE and transferred to PVDF membranes at a constant current of 200 mA for 90 min. After being sealed with 5% skim milk solution for 2 hours, the PVDF membranes were incubated with primary antibodies of anti-E-cadherin (Abcam, ab1416, 1:1000) and anti-Vimentin (Abcam, ab8978, 1:1000) overnight at 4°C. Subsequently, the membranes were flushed with TBST three times (15 min each time) and maintained with the secondary antibodies at room temperature (RT) for 2 hours. After cleaning with TBST again, the protein bands were exposed using an ECL solution (Beijing Xinjingke Biotechnologies Co., Ltd, China). The densitometry analysis was performed using Image J 3.0 (IBM, USA), and GAPDH served as a control.

Scoparone (E‐0358) was purchased from Tauto (Shanghai, China). Ang II (A9525) was purchased from Sigma‐Aldrich (St. Louis, MO, USA), Alzet osmotic minipump (model 2004) was purchased from DURECT (California, USA), tail‐cuff instrument was purchased from IITC Inc (Woodland Hills, California, USA), and the ultrasound imaging system(Vevo2100) was purchased from VisualSonics (Toronto, Canada). Antibodies against 4HNE (ab46545), RAC1‐GTP (ab203884), α‐actinin (ab9465), vimentin (ab8978), collagen I (ab34710), collagen III (ab7778) and connective tissue growth factor (CTGF, ab6992) were purchased from Abcam (Cambridge, MA, USA). Antibody against α‐smooth muscle actin (α‐SMA, A2547, for Western blot use) was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Antibodies against total‐RAC1 (66122‐1‐lg), NOX2 (19013‐1‐AP), NOX4 (14347‐1‐AP), α‐SMA (14395‐1‐AP, for immunofluorescence stain use), cardiac troponin I (CTNI, 66376‐1‐Ig) and β‐actin (60008‐1‐Ig) were purchased from Proteintech (Hubei, China). ³H‐leucine (NET135H) was purchased from Perkin Elmer (Waltham, MA, USA), ROS assay (S0033) was purchased from Beyotime (Shanghai, China), and the Cytotoxicity Detection Kit (11644793001) was purchased from Roche (Hoffmann, Germany).