Anti aeg 1

THP-1 cells in suspension were collected, centrifuged and resuspended in 100 μl fresh medium. These cells and tissue samples were embedded in Tissue-Tek OCT compound (Sakura Seiki) and cryosectioned. THP-1 cells and THP-1-derived macrophages were fixed in 4% paraformaldehyde. Tissue specimens and macrophages were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes. They were blocked at room temperature for 1 hour and then incubated with primary antibodies overnight at 4°C. Co-immunofluorescence staining was done with anti-AEG-1 (Abcam) and anti-CD68 (ZSJB-BIO) antibodies as previously described [42 (link)]. Sections were rinsed in PBS and incubated with either Alexa Fluor 546-conjugated goat anti-rabbit (Invitrogen) or DyLight-488 conjugated goat anti-mouse secondary antibodies (ZSJB-BIO) for 1 hour at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The images were then captured by confocal microscope (TCS SPE; Leica, Germany).

Total proteins of cells were extracted according to the protocol of protein extraction kit (KeyGen, China), and the protein concentration of each sample was determined using the bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL). Equivalent quantities of protein were separated by 12% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 10% defatted milk (10% BSA for p‐protein) and then incubated with the appropriate primary antibody overnight at 4°C. They were next washed and incubated with the corresponding horseradish peroxidase (HRP)‐conjugated secondary antibody for 1 h. Bound secondary antibody was visualized using an enhanced chemiluminescence (ECL) system (Pierce Biotechnology). The primary antibodies used were anti‐GAPDH (1:1000, Cell Signal Technology, Danvers, MA), anti‐AEG‐1 (1:10000, Abcam, USA), anti‐E‐cadherin(1:200 Boster bio., Shanghai, China), anti‐β‐catenin (1:1000, Cell Signal Technology), anti‐MMP2 (1:1000, Abcam, USA), and anti‐MMP9 (1:1000, Cell Signal Technology). The results were normalized to GAPDH to correct for loading.

Sections (4 μm) of formalin-fixed, paraffin-embedded tissues were made using a rotary microtome (Leica, Wetzlar, Germany) and labeled with anti-AEG-1 (Abcam, Cambridge, UK), anti-E-cadherin (Abcam, Cambridge, UK) and anti-Vimentin (Abcam, Cambridge, UK) primary antibodies. We used the known positive slice in the SP kit (Maxim-Bio, Fuzhou, China) as a positive control. The number of immunopositive cells was semiquantitatively estimated. The staining index was calculated using Aperio ImageScope software (Aperio Technologies).

For the Western blot analyses, cells were lysed in RIPA buffer (Sigma, St Louis, MO, USA). Protein concentrations were determined with a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Equal amounts of total proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difuoride membranes (Millipore, Bedford, MA, USA). The membranes were subsequently immunoblotted with the appropriate primary antibody. Anti-AEG-1 was purchased from Abcam Ltd. (Abcam, Cambridge, MA, USA), anti-CXCR4 was purchased from eBioscience Ltd. (eBioscience, San Diego, CA, USA), and antibodies against Akt, phosphorylated Akt (S473), caspase-3, cleaved caspase-3, Bcl-2, Bad, phosphorylated Bad (S99) and GAPDH were obtained from Epitomics company (Epitomics, CA, USA). A secondary anti-rabbit antibody (Promoter Biological Company, Wuhan, Hubei, China) was used for detection with an ECL kit (Pierce) according to the manufacturer's instructions. Bands were scanned and analyzed. Each experiment was repeated three times.