H 300

Tissue specimens from clinical GC samples and xenograft tumors derived from GC cells were fixed with 10% neutralbuffered formalin, and 4-μm paraffin sections were prepared. After rehydration, sections were stained with hematoxylin and eosin for histologic assessment, or were immunostained after antigen retrieval using a Bond-max automated immunostainer (Leica Microsystems, Newcastle upon Tyne, UK). The primary antibodies used were against FOXO1 (1:40, C29H4, Cell Signaling Technology), phospho-FOXO1^Ser256 (pFOXO1; 1:60, Cell Signaling Technology), CD31 (1:100, M20, Santa Cruz Biotechnology), HIF-1α (1:50, provided by Dr. Jong-Wan Park at Seoul National University), VEGF (1:200, C1, Santa Cruz Biotechnology), and SIRT1 (1:100, H300, Santa Cruz Biotechnology). Antibody binding was detected with the Bond Polymer Refine Detection Kit (Leica Microsystems). All immunostained sections were lightly counterstained with Mayer’s hematoxylin. Throughout the above analysis, negative controls were prepared by omitting the primary antibody. The results of immunostaining were evaluated by two pathologists (Y.K. and J.-S.P.), who were blinded to the origin of the samples. For statistical analysis, the results of immunostaining for proteins were considered positive if immunoreactivity was seen in ≥ 10% (cytoplasmic pFOXO1 and nuclear SIRT1) or ≥ 5% (nuclear HIF-1α) of the neoplastic cells.

Western blot analysis was performed as we described previously [10 (link)]. Cell lysates in sodium dodecyl sulfate (SDS) lysis buffer (125 mM Tris-HCl [pH 6.8], 4% SDS, 0.004% bromophenol blue, and 20% glycerol) were separated on 10% SDS–polyacrylamide gel and electrophoretically transferred to PVDF membranes (Millipore Co., Billerica, MA) blocked with 5% non-fat dry milk in phosphate buffered saline– Tween-20 (0.1%, vol/vol) for 1 hour. The membranes were then incubated with a primary antibody against FOXO1 (1:1,000, C29H4, Cell Signaling Technology, Beverly, MA), hypoxia inducible factor-1α (HIF-1α; 1:500, H1α67, BD Biosciences, San Jose, CA), vascular endothelial growth factor (VEGF; 1:1,000, C1, Santa Cruz Biotechnology, Santa Cruz, CA), SIRT1 (1:1,000, H300, Santa Cruz Biotechnology), or β-actin (1:1,000, C4, Santa Cruz Biotechnology). Horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000, Santa Cruz Biotechnology) or anti-mouse IgG (1:2,000, Santa Cruz Biotechnology) was used as a secondary antibody. Enhanced chemiluminescence (Amersham, Arlington Heights, IL) was used to detect the immunoreactive proteins. Equal protein loading was confirmed by β-actin.