Western Blot Analysis and Immunoprecipitation Protein levels were analyzed by Western blot as described previously [41]. Antibodies against calpain, SHP-1, SHP-2 (Santa Cruz Biotechnology), STAT-3, p-STAT-3 (Tyr705 and Ser727) (New England BioLabs), and b-actin (Sigma) were used. For immunoprecipitation, proteins (500 mg) were incubated with specific antibodies and immobilized onto protein A-Sepharose beads. Immunofluorescence and Laser Scanning Confocal Microscopy The immunofluorescence of phosphorylated STAT3 expression in gastric cancer cells and HUVECs was determined by a laser scanning confocal microscope [16]. Images were background- subtracted and merged using the Confocal Assistant MetaMorph software program, and processed with Adobe Photoshop software. RNA Interference and Transfection Assays The delivery of siRNA pools into cells was performed using Lipofectin (Invitrogen). The pooled siRNA duplexes were dis- solved in buffer (20 mM KCl, 6 mM HEPES, pH 7.5, and 0.2 mM MgCl2) to a concentration of 20 mM. Cell transfection was carried out for 24 h at a final siRNA concentration of 100 pM. Control cells were mock-transfected without siRNA, and all experimental results were confirmed using scrambled siRNA. The commercial siRNA products for human SHP-1, calpain II, and scrambled control were used and purchased from Santa Cruz Biotechnology (SHP-1 siRNA, sc-29478; Calpain II siRNA, sc- 41459; Control siRNA, sc-37007). Immunohistochemistry The expressions of SHP-1 and p-STAT-3 proteins in mouse solid tumors were examined by immunohistochemistry. All tumors were fixed in 10% buffered formalin immediately after the animals were sacrificed. After pressure cooker pretreatment in citrate buffer (pH 6.0) for 30 min, 5 mm sections of the tumors were incubated at room temperature for 1 h with a monoclonal antibody for SHP-1, phospho-VEGFR-2 (1:250, Santa Cruz Biotechnology), and CML (1:250, Trans Genic)., (phospho-STAT- 3 (dilution: 1:250, Cell Signaling Technology). After incubation with an appropriate primary antibody enhancer, the slides were incubated with HRP polymer (Laboratory Vision Corporation, Fremont, CA, USA). Reaction products were visualized by immersing the slides in peroxidase-compatible chromogen. Final- ly, the slides were counterstained with hemalaun. Electrophoretic Mobility Shift Assay The electrophoretic mobility shift assay was performed as described previously [16]. The consensus oligonucleotide probes were end-labeled with [c-32P]ATP according to the manufac- References 1. Saif MW, Makrilia N, Zalonis A, Merikas M, Syrigos K (2010) Gastric cancer in the elderly: an overview. 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For the binding reaction, 2 ng of the labeled oligonucleotide (approximately 20,000 cpm) and 2 mg of poly dIdC (Amersham Pharmacia Biotech) carrier were incubated with 2 mg of nuclear protein in a binding buffer (10 mM HEPES, 60 mM KCl, 1 mM DTT, 1 mM EDTA, 7% glycerol, pH 7.6) for 30 min at room temperature. Protein-DNA complexes were separated by electrophoresis on a nondenaturing 6% polyacryl- amide gel and visualized by autoradiography. For competition experiments, a 100-fold excess of the unlabeled oligonucleotides was added 15 min before incubation of nuclear extracts with the end-labeled oligonucleotides. SHP-1 Phosphatase Activity Assay Five hundred mg of supernatant protein were incubated with 2 mg of antiphosphorylated SHP-1 antibody or IgG at 4uC for 2 h, followed by incubation with protein A/G PLUS for 1 h. The precipitates were used to measure SHP-1 phosphatase activity. Phosphatase activities of immunoprecipitated SHP-1 were ana- lyzed using a pNPP protein phosphatase assay kit (AnaSpec) according to the manufacturer’s instructions. The absorbance at 405 nm was determined by a microplate reader. Transmission Electron Microscopy The ER structure in cells was observed by transmission electron microscopy as described previously [44]. Cells were treated with or without drugs for 18 h. Cells were harvested and then fixed with 4% glutaraldehyde and 2.5% paraformaldehyde dissolved in 0.1 M of sodium cacodylate. The sections were prepared with an LKB Ultracut microtome (Leica, Deerfield, IL) and then stained with uranyl acetate and lead citrate in an LKB Ultrostainer. The sections were examined in a JEM 1200 EX transmission electron microscope (JEOL, Peabody, MA) at an accelerating voltage of 80 kV. Analysis of VEGF Generation VEGF concentrations were measured with a human VEGF ELISA kit (R&D Systems). Statistics The values given in this study are presented as mean 6 SEM. Analyses of variance followed by a Fisher’s least significant difference test were performed for all data. P values of less than 0.05 were considered statistically significant. Author Contributions Conceived and designed the experiments: SHL MLS. Performed the experiments: KBW KHL CCS SMW YCP YCC KKL. Analyzed the data: SHL MLS. Contributed reagents/materials/analysis tools: KKL CCS WJL HCP HCC. Wrote the paper: SHL MLS. 7. Fukumura D, Kashiwagi S, Jain RK (2006) The role of nitric oxide in tumour progression. Nat Rev Cancer 6: 521–534. 8. Albini A, Sporn MB (2007) The tumour microenvironment as a target for chemoprevention. Nat Rev Cancer 7: 139–147. 9. Wagner EF, Nebreda AR (2009) Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer 9: 537–549. 10. Yu H, Pardoll D, Jove R (2009) STATs in cancer inflammation and immunity: a leading role for STAT3. Nat Rev Cancer 9: 798–809. 11. 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