Anti zeb2

ISH was performed on paraffin-embedded tissues sections containing 20 pairs of GC tissues and corresponding non-cancerous stomach tissue. The miR-338-3p probes were double digoxigenin (DIG)-labeled mercury locked nucleic acid probes [miRCURY LNA^TM detection probes (Exiqon, Vedbaek, Denmark)]. The sequences were as follows: 5′-CAA CAA AAT CAC TGA TGC TGG A-3′. IHC procedures were conducted in 20 GC tissues samples as previously described [15 (link),25 ]. The following primary antibodies were used: anti-ZEB2, anti-N-cadherin, and anti-vimentin (Abcam); anti-MACC1 (ImmunoWay, Newark, DE, USA). Staining patterns were evaluated by two independent reviewers, and the half-quantitative scoring system was as previously described [15 (link), 25 ].

U‐251MG and U‐87MG cells were treated with MCCK1 (2.5 μmol/L) or TMZ (100 μmol/L) for 48 hours. Cells were collected and tumors tissues were homogenized. Protein was extracted with RIPA lysis buffer adding cocktail (Sigma‐Aldrich) and phenylmethanesulfonyl fluoride (PMSF; Sigma‐Aldrich). Equal amounts of protein (40 µg) were resolved on (6%‐12%) SDS‐PAGE, transferred onto PVDF membranes (Millipore, Billerica, MA), probed with primary antibodies. Antibodies were as follows: antiVimentin (Abcam, Cambridge, MA), antiGAPDH (Abcam), antiKi67 (Abcam), antiCaspase‐3 (Abcam), antiVEGF (Abcam), antiMMP‐14 (Abcam), antiEcadherin (Abcam), antiTwist1 (Abcam), antizeb1 (Abcam), antizeb2 (Abcam), antiNcadherin (Abcam), antiSnail1 (Cell Signaling Technology, Beverly, MA). After incubation with the proper horseradish peroxidase (HRP)‐conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA), proteins were detected using a ChemiDoc xRS imaging system and Quantity One analysis software (Bio‐Rad Laboratories, San Francisco, CA). Antibodies were visualized the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, UK).

Western blot analysis of total cell lysates (30 μg) was performed as previously described [30 (link)], using the following antibodies: anti-KLF7 (sc-101,034, Santa Cruz, Santa Cruz, CA, USA, 1:1000); anti-SNAIL (#3879, Cell Signaling Technology Inc., Danvers, MA, 1:1000); anti-ZEB2 (ab25837, Abcam, Cambridge, UK, 1:1000); anti-VIM monoclonal antibody (sc-73,259, Santa Cruz, 1:1000); anti-CD44 (M7082, Dako Agilent Technologies, Santa Clara, CA, USA, 1:500); anti-GAPDH (ab8245, Abcam, 1:5000). Blots were visualized by enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK) using a Chemidoc imaging system (Bio-Rad). Experiments were performed at least three times. Proteins were densitometrically quantified using Imager ChemiDoc™ XRS+ Software (Bio-Rad, Version 6.0.1.34). All proteins were normalized to GAPDH loading control.

Total protein was extracted from tissues and transfected cells using RIPA buffer including protease inhibitor cocktail (Cell Signaling Technology), and western blotting was carried out as described previously [42 (link)]. The primary antibodies used were as follows: anti-DHFR (1:400, Cell Signaling Technology), anti-ZEB2 (1:1000, Abcam), anti-E-cadherin (1:250, Abcam), anti-VIM (1:1500, Cell Signaling Technology), anti-ITGAV (1:1000, Cell Signaling Technology), anti-ITGB1 (1:2000, Cell Signaling Technology), anti-ITGB3 (1:2000, Cell Signaling Technology), anti-CD47 (1:2000, Abcam), and anti-β-actin (1:5000, Sigma-Aldrich).

Western blotting was performed as previously described [37 (link)]. In addition, cytoplasmic and nuclear extracts were separated and prepared using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions and a previous study [36 (link)]. Primary antibodies (anti-E-cadherin [Cat# ab15148, 1:500], anti-N-cadherin [Cat# ab18203, 1:1000], anti-ZEB1 [Cat# ab124512, 1:1000], anti-ZEB2 [Cat# ab138222, 1:1000], anti-peroxiredoxin 1/PAG [Cat# ab109498, 1:10,000] and anti-GAPDH [Cat# ab181602, 1:1000] [Abcam, USA]; anti-Vimentin [Cat# D21H3, 1:1000, CST, USA]; anti-H3 [Cat# AH433, 1:1000, Beyotime, China]; and anti-HA Tag [Cat# 26183, 1:10000, Thermo Fisher Scientific]) were used, as well as anti-mouse and anti-rabbit secondary antibodies (IR Dye-labeled secondary antibodies [1:10000, Sigma, USA] and HRP-labeled secondary antibodies [1:10000, CST]). The signals were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences, USA) or with ECLUltra (New Cell and Molecular Biotech, Suzhou, China).

The protein for subsequent detection was extracted from the previously transfected cells under the action of Radio Immunoprecipitation Assays lysis buffer (RIPA, Boster, China). The amounts of protein were assessed by bicinchoninic acid (BCA, Boster, China). Owing to the different molecular weights of the proteins being measured, they can be isolated and formed into bands in the 10% SDS-PAGE. The isolated proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Boster, China). The PVDF membranes was incubated with 5% skim milk. The cut membranes were then placed in the primary antibodies, respectively. The primary antibodies purchased from Abcam (MA, USA) were as following: anti-CDK6, anti-cyclin D1, anti-CDK4, anti-GAPDH, anti-ZEB2, anti-Snail, anti-Twist, anti-TGFBR2, anti-AKT, anti-p-AKT, anti-ERK, anti-p-ERK, anti-smad2, anti-p-smad2. All antibodies were diluted in skim milk at the concentration of 1:1000. The membranes were immersed completely in secondary antibody (abcam, USA) at room temperature. The blots acquired in our study were dripped with ECL chemiluminescent reagent (Servicebio, Wuhan, China) to produce a light-emitting reaction that can be captured and visualized by the ChemiDoc XRS+ (Bio-Rad), allowing us to analyze each blot with Image Lab Software.