Se 3 (1:500), Bcl-2 (1:500), BAX(1:500) and GAPDH (1:1000; all Cell signaling technology), then horseradish peroxidase-conjugated anti-mouse/ rabbit IgG antibody (Santa Cruz Biotechnology) after a final wash. Reactions were developed with use of 4-chloro-1-naphthol (Sigma) and H2O2. Signals were detected with use of an enhanced chemiluminescence kit (Amersham Pharmacia, Buckinghamshire, UK). GAPDH level was an internal standard.MTT Cytotoxicity AssayThe effect of As2O3 on inhibiting in vitro growth of FU97 cells was determined by measuring MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) dye absorbance of living cells. FU97 cells were seeded in 96-well plates at 1.66 
103 cells per well in 100 mL DMEM containing 10 FBS overnight. After exposure to various concentrations of As2O3 for 24, 48 and 72 h, 20 mL (5 g/L) MTT (Sigma, St. Louis, MO) solution was added to each well and plates were incubated for an additional 4 h at 37uC. Formazine was dissolved in 150 mL/well dimethyl sulfoxide (DMSO) and the absorbance was detected at 490 nm. Inhibitory rate ( ) = (12A value in experimental group/A value in control group) 6 100 . The 0 mmol/L group was used as blank control.DNA Fragmentation Analysis by ElectrophoresisA total of 106 cells was gently scraped from dishes, washed twice in cold PBS, and centrifuged at 15000 rpm for 10 min, then lysed in 200 mL lysis buffer (1 mL of 1 M Tris Cl buffer, pH 7.4, 0.2 mL of 0.5 M ethylenediaminetetraacetic acid [EDTA],Immunoassay of AFP Concentration in SupernatantThe supernatant of FU97 cells were collected after treatment with As2O3 or negative control for 24, 48 and 72 h.AFPNovel Therapy for AFP-Producing Gastric CancersFigure 1. Arsenic trioxide (As2O3)-induced growth inhibition and apoptosis of gastric cancer FU97 cells. (A) Cellular growth inhibition measured by MTT assay. Data are mean 6 SD of 3 independent experiments. (B) Agarose gel analysis of DNA fragmentation in FU97 cells treated with As2O3 for 72 h.(C) Apoptotic nuclei stained with Hoechst 33258 show intense fluorescence corresponding to AN-3199 supplier chromatin condensation and fragmentation.(D) Western blot analysis of caspase3 protein in total cell extracts of FU97 cells treated with the indicated concentration of As2O3 for 72 h. GAPDH expression served as loading control. doi:10.1371/journal.pone.0054774.gconcentration in supernatant was determined by two-site immunoenzymometric assay in an TOSOH AIA system (Japan). The cut-off value for AFP was 10 ng/ml.PatientsWe examined data from surgical and pathological records for 24 patients with AFPGC and 24 randomly selected patients with normal levels of serum AFP and matched to AFPGC patients by gastric cancer stage. Patients had undergone surgical resection at the Clinical Hospital of Shandong University, China, from January 1996 to December 2011. AFPGC patients showed elevated serum AFP level but no concomitant liver diseases. Histopathological presence of AFP positivity was confirmed by immunohistochemistry. We contacted each patient to confirm survival or date of death.times with PBS, and incubated with streptavidin-conjugated peroxidase for 30 min. Sections were visualized by incubation with 3, LED-209 chemical information 39-diaminobenzidine solution (0.3 H2O2 and 0.05 3, 39-diaminobenzidine) and counterstained with hematoxylin. Omission of the primary antibody was a negative control. Every run included a positive control and a negative control. For the negative control, the primary antibody was replaced with.Se 3 (1:500), Bcl-2 (1:500), BAX(1:500) and GAPDH (1:1000; all Cell signaling technology), then horseradish peroxidase-conjugated anti-mouse/ rabbit IgG antibody (Santa Cruz Biotechnology) after a final wash. Reactions were developed with use of 4-chloro-1-naphthol (Sigma) and H2O2. Signals were detected with use of an enhanced chemiluminescence kit (Amersham Pharmacia, Buckinghamshire, UK). GAPDH level was an internal standard.MTT Cytotoxicity AssayThe effect of As2O3 on inhibiting in vitro growth of FU97 cells was determined by measuring MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) dye absorbance of living cells. FU97 cells were seeded in 96-well plates at 1.66 103 cells per well in 100 mL DMEM containing 10 FBS overnight. After exposure to various concentrations of As2O3 for 24, 48 and 72 h, 20 mL (5 g/L) MTT (Sigma, St. Louis, MO) solution was added to each well and plates were incubated for an additional 4 h at 37uC. Formazine was dissolved in 150 mL/well dimethyl sulfoxide (DMSO) and the absorbance was detected at 490 nm. Inhibitory rate ( ) = (12A value in experimental group/A value in control group) 6 100 . The 0 mmol/L group was used as blank control.DNA Fragmentation Analysis by ElectrophoresisA total of 106 cells was gently scraped from dishes, washed twice in cold PBS, and centrifuged at 15000 rpm for 10 min, then lysed in 200 mL lysis buffer (1 mL of 1 M Tris Cl buffer, pH 7.4, 0.2 mL of 0.5 M ethylenediaminetetraacetic acid [EDTA],Immunoassay of AFP Concentration in SupernatantThe supernatant of FU97 cells were collected after treatment with As2O3 or negative control for 24, 48 and 72 h.AFPNovel Therapy for AFP-Producing Gastric CancersFigure 1. Arsenic trioxide (As2O3)-induced growth inhibition and apoptosis of gastric cancer FU97 cells. (A) Cellular growth inhibition measured by MTT assay. Data are mean 6 SD of 3 independent experiments. (B) Agarose gel analysis of DNA fragmentation in FU97 cells treated with As2O3 for 72 h.(C) Apoptotic nuclei stained with Hoechst 33258 show intense fluorescence corresponding to chromatin condensation and fragmentation.(D) Western blot analysis of caspase3 protein in total cell extracts of FU97 cells treated with the indicated concentration of As2O3 for 72 h. GAPDH expression served as loading control. doi:10.1371/journal.pone.0054774.gconcentration in supernatant was determined by two-site immunoenzymometric assay in an TOSOH AIA system (Japan). The cut-off value for AFP was 10 ng/ml.PatientsWe examined data from surgical and pathological records for 24 patients with AFPGC and 24 randomly selected patients with normal levels of serum AFP and matched to AFPGC patients by gastric cancer stage. Patients had undergone surgical resection at the Clinical Hospital of Shandong University, China, from January 1996 to December 2011. AFPGC patients showed elevated serum AFP level but no concomitant liver diseases. Histopathological presence of AFP positivity was confirmed by immunohistochemistry. We contacted each patient to confirm survival or date of death.times with PBS, and incubated with streptavidin-conjugated peroxidase for 30 min. Sections were visualized by incubation with 3, 39-diaminobenzidine solution (0.3 H2O2 and 0.05 3, 39-diaminobenzidine) and counterstained with hematoxylin. Omission 
of the primary antibody was a negative control. Every run included a positive control and a negative control. For the negative control, the primary antibody was replaced with.
