Sc 152

PaC samples and paired adjacent non-tumor tissues with informed consent were collected from 30 patients who underwent pancreatic surgery and were stored at Biobank Center of National Engineering Center for Biochip at Shanghai. Tissue microarray was stained for expression analysis of POSTN (ab14041, Abcam, 1:50 dilution), VEGF (sc-152, Santa Cruz Biotechnology, 1:50 dilution). All immunohistochemically stained sections were dependently scored by two in-house pathologists who were blinded to clinical outcome.

Lung tissues were homogenized in ice-cold buffer containing 50 mM Tris·HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, and a protease inhibitor cocktail (complete minitablets; Roche, Mannheim, Germany). The samples were sonicated and then centrifuged at 500×g for 20 min at 4 °C to remove cellular debris. Proteins (30 μg) were resolved through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 12% SDS-PAGE gel under reducing conditions and electroblotted to a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA, USA). After the membranes were blocked with 5% nonfat dry milk, they were incubated with antibodies against vascular endothelial growth factor (VEGF) (1:750; SC-152, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin (1:1000; Sigma-Aldrich, St. Louis, MO, USA), and they were subsequently incubated with horseradish peroxidase–conjugated goat anti-rabbit or mouse immunoglobulin G (IgG) (Pierce Biotechnology, Rockford, IL, USA). Protein bands were detected using SuperSignal Substrate (Pierce Biotechnology). A densitometric analysis was performed on the AIDA program to measure the intensity of the VEGF and β-actin bands.

After routine deparaffinization and dehydration, the sections were incubated for 10 min. at 37°C with 1% trypsin (Digest All, cat# 003008; Invitrogen, Darmstadt, Germany) for antigen unmasking. After washing with PBS, the tissues were blocked with 5% BSA for 1 hr at RT and incubated with the appropriate primary antibodies diluted in 3% BSA with 0.2% TritonX-100 in PBS overnight at 4°C. Mouse monoclonal Oct-3/4 (C-10), dilution 1:50 (sc5279; Santa Cruz), mouse monoclonal anti-vimentin, 1:500 (C 9080; Sigma-Aldrich), monoclonal anti-α-actin smooth muscle conjugated with Cy3 (α-SMA), 1:500 (C6198; Sigma-Aldrich), anti-PDGFR-α, 1:100 (ab90967; Abcam, Cambridge, UK), rabbit polyclonal PDGFR-β, 1: 100 (sc-432; Santa Cruz), rat antimouse Ly-6A/E/Sca-1, 1:100 (cat # 553333; BD Biosciences, Heidelberg, Germany), rabbit polyclonal C-kit, 1:100 (sc-168; Santa Cruz), rabbit polyclonal VEGF, 1:100 (sc-152; Santa Cruz) were used for the staining. The sections where then incubated with the appropriate secondary antibodies (1:500–1000) for 1 hr at RT, washed with PBS and counterstained with DAPI (1:1000) for 10 min. The slices were mounted with Mowiol and investigated with a Zeiss LSM 710 laser scanning confocal microscope.

Radioimmunoprecipitation lysis buffer (Beyotime, Shanghai, China) freshly mixed with a protease and phosphatase inhibitor cocktail (Thermo Scientific, Rockford, Cambridge, MA) was used to isolate proteins from cells or tissues. Proteins were separated on 8% or 10% SDS-PAGE gels (Bio-Rad). Antibodies for western blotting were as follows: anti-SNIP25 interacting protein antibody (Abcam ab111343, USA), anti-phospho-SRC family (Y416) and anti-phospho-SRC (Y527) were purchased from Cell Signaling Technology (#6943 and #2105, respectively; CST,USA), antibodies against SRC, VEGF and GAPDH were purchased from Santa Cruz Biotechnology (sc-8056, sc-152, and sc-365062, respectively; Santa Cruz, CA, USA).

Paraffin embedded HCC specimens were deparaffinized and blocked with 3% hydrogen peroxide for 10 min. The specimens were then subjected to the antigen retrieval microwave method in 0.01 M citrate buffer for 15 min. The slides were washed with phosphate-buffered saline twice and incubated with anti-SDHB (1:250 dilution; SC-152, Santa Cruz, CA, USA) and anti-CD34 (1:100 dilution; Dakopatts, Copenhagen, Denmark) antibodies for 30 min, followed by horseradish peroxidase conjugated anti-mouse IgG secondary antibody for 30 min, and were then detected using a polymer detection system (catalog no. 87-89431; Zymed, San Francisco, CA, USA). The staining was visualized with 3, 3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich Chemicals, Saint Louis, MO, USA) in 0.1 M Tris, pH 7.2 containing 0.01% hydrogen peroxide. The section slides were counterstained with Gill hematoxylin, dehydrated, and mounted.

ECs (1 × 10⁶) from AVM tissues and normal tissues were incubated in serum-free medium with 10 ng/mL of VEGF (sc-152, Santa Cruz Biotechnology Inc., Santa cruz, CA, USA) for 14 h at 37 °C under normoxic and hypoxic conditions.