Gtx60776

Protein samples (30 μg) from the cells were lysed in radioimmunoprecipitation assay lysis buffer (P0013K, Beyotime Biotechnology, Shanghai, China) and quantified using the bicinchoninic acid assay protein assay kit (P0009, Beyotime). Then, equal amounts of protein samples (30 μg) were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Subsequently, the membranes were blocked with 5% (w/v) skim milk for 2 h, followed by an overnight incubation with antibodies to COL1A2 (1 : 1000, GTX102996, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), TWIST1 (1 : 1000, GTX60776, GeneTex), and EP300 (1 : 1000, ab275378, Abcam, Cambridge, UK) and a 2 h incubation with goat anti-mouse secondary antibody (1 : 15,000, ab205719, Abcam) or goat anti-rabbit IgG (1 : 15,000, ab6721, Abcam) at room temperature. Bands were visualized using a chemiluminescent substrate kit (P0018FS, Beyotime). GAPDH (1 : 1000, ab8245, Abcam) was used as an internal control.

The cells were treated with 4% paraformaldehyde (E672002, Sangon) and 0.5% Triton X-100 (Sangon). The cells were treated with TWIST1 (1 : 500, #GTX60776, GeneTex) and EP300 (1 : 100, ab275378, Abcam) and stained with FITC-conjugated goat anti-rabbit antibody (ab6717, Abcam). The cells were then denatured with 2 M hydrochloric acid at 37°C for 10 min and then stained with Alexa Fluor 647-conjugated goat anti-mouse antibody (1 : 500, ab150083, Abcam). The cells were mounted in medium containing an antifluorescent quenching agent, and the nucleus was stained with 0.25 μg/mL 4′,6-diamidino-2-phenylindole (C1005, Beyotime Biotech). Then, the fluorescent images were captured with a laser scanning confocal microscope (Leica, Frankfurt, Germany) equipped with the appropriate FITC and Texas Red filters.

ChIP was performed using the Magna ChIP™ A/G One-Day ChIP Kit (Catalog #17-10085; Merck) according to the manufacturer's instructions. Cross-linking of chromatin was achieved by using 1% formaldehyde for 10 min at 37°C and neutralizing with glycine for 5 min at room temperature. All GC cells were washed with cold 1 mL PBS + proteinase inhibitor (1 mM phenylmethanesulfonyl fluoride, 1 mg peptidase, and 1 mg gastrase inhibitor A). The cells were centrifuged at 716 × g for 5 min at 4°C and disrupted using sodium dodecyl sulfate (SDS) lysis buffer (1% SDS, 10 mM ethylenediaminetetraacetic acid and 50 mM Tris-Hcl pH = 8.0). Subsequent sonication at 150 Hz was performed with four sets of 10 s pulse shears on ice using a high-intensity ultrasonic processor (Cole-Parmer). Equal amounts of chromatin were immunoprecipitated with antibodies targeting TWIST1 (1 : 500, #GTX60776, GeneTex) or H3K27ac (1 : 500, #GTX50903, GeneTex) overnight at 4°C with anti-mouse IgG (1 : 500, ab18413; Abcam) as an isotype control. The total chromatin was used as input. Immunoprecipitation products were collected after incubation with magnetic beads. Beads were washed using a magnetic separation rack, and bound chromatin was eluted in ChIP elution buffer with a proteinase K mixer according to the manufacturer's instructions.