E cadherin

Human GC cell lines AGS, SGC7901, HGC-27(Beijing qualityard biotechnology Co., Ltd.) were purchased from the ATCC and routinely checked for mycoplasma contamination using the Vazyme Mycoplasma detection kit. Nude mice were purchased from SLAC Laboratory Animal Center. primers were designed based on the sequences of THUMPD3-AS1, miR-1297, BCAT1, U6 (internal control) and β-actin (internal control) and synthesized by Merck. Fet al bovine serum, Dulbecco’s modified Eagle’s medium (DMEM), streptomycin, β-actin antibody, recombinant basement membrane and Transwell were purchased from Merck. CCK-8 reagent, trypsin, penicillin and streptomycin, and phosphate-buffered saline (PBS) were purchased from MCE and Dual luciferase reporter assay kits and BCA kits were purchased from abcam and Merck. N-cadherin, E-cadherin and glyceraldehyde-3-phosphate dehydrogenase antibodies were purchased from Merck.

Total protein was extracted from cells using RIPA buffer (Thermo Fisher Scientific, Inc.) and quantified using a Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). A total of 50 µg of protein was loaded per lane. Proteins were separated via 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore), which were blocked with 5% skim milk at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4˚C with primary antibodies targeted against: E-Cadherin (cat. no. 14472; 1:1,000; Cell Signaling Technology, Inc.), Vimentin (cat. no. 5741; 1:1,000; Cell Signaling Technology, Inc.) and β-Actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc.). Following primary incubation, the membranes were incubated for 1 h at room temperature with HRP-conjugated anti-mouse IgG (cat. no. 7076; 1:2,000; Cell Signaling Technology, Inc.) and anti-rabbit IgG (cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence reagent (Bio-Rad Laboratories, Inc.) with ChemiDoc MP imaging system (Bio-Rad Laboratories, Inc.). Densitometry was measured using Image Lab Touch Software, version 2.4 (Bio-Rad Laboratories, Inc.). β-Actin was used as the loading control.

The following antibodies were used in this study and were purchased from Santa Cruz Biotechnology unless otherwise noted (including dilutions/amounts used for immunofluorescence, Western blot [WB], and immunoprecipitation [IP]): ERα (HC-20 rabbit; 1:100 immunofluorescence, 1:200 WB; 3 µg IP), ERα (F-10 mouse; 3 µg IP), β1-integrin (LM534 mouse; 1:100 immunofluorescence; EMD Millipore), β1-integrin (M-106 rabbit; 1:300 WB; 3 µg IP), E-cadherin (H-108 rabbit; 1:1,000 WB), β-actin (C4 mouse; 1:10,000 WB), Rab11 (H-87 rabbit; 1:200 WB), Rab7 (sc-376362 mouse; 1:100 immunofluorescence), and caveolin 1 (sc-53564 mouse; 1:600 immunofluorescence; 1:200 WB). LAMP-1 (ab25630 mouse; 1:20 immunofluorescence), clathrin (ab2731 mouse; 1:500 immunofluorescence), and Lamin B1 (ab133741 rabbit; 1:243 immunofluorescence) were purchased from Abcam; and clathrin-HC (clone 23 mouse; 610500; 1:1,000 WB) was purchased from BD. HC-20 peptide was purchased from Santa Cruz Biotechnology. Secondary antibodies used for WB (1:5,000) were goat anti–mouse HRP-conjugated (AP308P) and goat anti–rabbit HRP conjugated (AP132P) purchased from EMD Millipore. Secondary antibodies used for immunofluorescence (1:500) were goat anti–mouse and goat anti–rabbit Alexa Fluor 488–, 555–, and 647–conjugated antibodies, all purchased from Thermo Fisher Scientific.