Th 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 1531364 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 Title Loaded From File 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 PBS.Statistical AnalysisData are expressed as mean 6 SD and were Title Loaded From File analyzed by use of SPSS v11.5 (SPSS Inc., Chicago, IL, USA). The association of clinicopathologic variables and AFP and STAT3 expression was determined by chi-square test, and Yate’s correction was applied in a small number of samples. Chi-square test or two-tailed Student’s t test was used for assessing differences between groups. Analysis of survival involved the log ank test, with Kaplan eier curves. P,0.05 was considered statistically significant.ImmunohistochemistryImmunohistochemistry involved use of biotin-streptavidin-peroxidase with a Vectastain ABC kit (Vector Laboratories, CA, USA). Briefly, tissue sections (4 mm) were prepared from paraffinembedded tissue specimens. The sections were deparaffinized with xylene followed by dehydration in graded alcohol. Sections were heated in a microwave for 2 min at 900 W to retrieve the antigen, and then incubated with 0.3 H2O2 solution in methanol for 30 min to block endogenous peroxidase. After 3 washes with phosphatebuffered saline (PBS), slides were incubated with 10 normal horse serum to block nonspecific background staining, then incubated with primary antibodies rabbit anti-AFP (1:100 dilution) and anti-STAT3 (1:200) in a humid chamber at 4uC overnight. After a washing with PBS, sections were incubated with biotinylated-horse anti-mouse antibodies for 30 min, washedResults Growth Inhibition and Apoptosis Induction in FU97 Cells by As2OFU97 cells were treated with different concentrations of As2O3 (1, 5 and 10 mmol/L) at 24, 48 and 72 h. As2O3 inhibited the proliferation of FU97 cells concentration and time dependently (Fig. 1A). In cells treated with 5 mmol/L and 10 mmol/L As2O3 for 72 h, the growth inhibition was 56.2763.91 and 73.4664.64 , respectively. DNA fragmentati.Th 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 1531364 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 PBS.Statistical AnalysisData are expressed as mean 6 SD and were analyzed by use of SPSS v11.5 (SPSS Inc., Chicago, IL, USA). The association of clinicopathologic variables and AFP and STAT3 expression was determined by chi-square test, and Yate’s correction was applied in a small number of samples. Chi-square test or two-tailed Student’s t test was used for assessing differences between groups. Analysis of survival involved the log ank test, with Kaplan eier curves. P,0.05 was considered statistically significant.ImmunohistochemistryImmunohistochemistry involved use of biotin-streptavidin-peroxidase with a Vectastain ABC kit (Vector Laboratories, CA, USA). Briefly, tissue sections (4 mm) were prepared from paraffinembedded tissue specimens. The sections were deparaffinized with xylene followed by dehydration in graded alcohol. Sections were heated in a microwave for 2 min at 900 W to retrieve the antigen, and then incubated with 0.3 H2O2 solution in methanol for 30 min to block endogenous peroxidase. After 3 washes with phosphatebuffered saline (PBS), slides were incubated with 10 normal horse serum to block nonspecific background staining, then incubated with primary antibodies rabbit anti-AFP (1:100 dilution) and anti-STAT3 (1:200) in a humid chamber at 4uC overnight. After a washing with PBS, sections were incubated with biotinylated-horse anti-mouse antibodies for 30 min, washedResults Growth Inhibition and Apoptosis Induction in FU97 Cells by As2OFU97 cells were treated with different concentrations of As2O3 (1, 5 and 10 mmol/L) at 24, 48 and 72 h. As2O3 inhibited the proliferation of FU97 cells concentration and time dependently (Fig. 1A). In cells treated with 5 mmol/L and 10 mmol/L As2O3 for 72 h, the growth inhibition was 56.2763.91 and 73.4664.64 , respectively. DNA fragmentati.
