Fig 1: DPYD 3′-UTR activity in wild-type MCF7 and tamoxifen-resistant MCF7 cells.Wild-type (wt)-MCF7 and TAM-resistant MCF7 (MCF7/T) cells were transfected with the pGL3-DPYD3′ UTR, and luciferase activity was measured as described in the Materials and Methods. The data shown were normalized with the internal control. The error bars represent the standard deviations of the values obtained in the experiments performed in triplicate. The experiments were repeated independently at least three times, and one representative result is provided in the figures. NS, not significant; **p < 0.01 by one-way ANOVA with Tukey’s multiple comparisons.
Fig 2: The expressions of thymidine synthase, thymidine phosphorylase, and dihydropyrimidine dehydrogenase in ER-positive breast cancer cell lines and their tamoxifen-resistant sublines.The mRNA expression of thymidine synthase (A), thymidine phosphorylase (B), and DPYD (C) were quantified by real-time RT-PCR in ER-positive wild-type cell lines (MCF7, T47D, and BT474) and their TAM-resistant sublines (MCF7/T, T47D/T, and BT474/T). ß-actin was used as an internal control. The error bars in each graph represent the standard deviations of the values obtained in the experiments performed in triplicate. The experiments were repeated independently at least three times, and one representative result is provided in the figures. NS, not significant; *p < 0.05, by unpaired Student’s t-tests.
Fig 3: Effects of a demethylating agent on DPYD mRNA expression and sensitivity to tamoxifen in wild-type MCF7 and tamoxifen-resistant MCF7 cells.Alteration of DPYD mRNA expression and sensitivity to 5-fluorouracil exerted by a demethylating agent, 5-azacytidine, was tested in wild-type and TAM-resistant MCF7 (MCF7/T) cells. (A) DPYD mRNA expression in wt-MCF7 and MCF7/T cells treated with 5 µM 5-azacytidine for 96 h was analyzed by real-time RT-PCR. Relative expression levels were calculated as ratios of the expression in the treated cells to those in untreated cells. The error bars represent the standard deviations of the values obtained in the experiments performed in triplicate. The experiments were repeated independently at least three times, and one representative result is provided in the figures. NS not significant, **p < 0.01 by one-way ANOVA with Tukey’s multiple comparisons. (B) Effects of 5-azacytidine treatment on sensitivity to 5-fluorouracil was tested in MCF7/T cells using WST assay. The black line with closed circles (?) indicates control, and the dotted line with closed triangles (?) indicates cells treated with 5 µM of 5-azacytidine. Error bars represent standard deviations of the values obtained from triplicate experiments. Each experiment was independently performed and repeated at least three times, and one representative result is provided in the figures.
Fig 4: Anti-tumor effects of capecitabine in mouse xenograft models.The anti-tumor effect of capecitabine was tested in the wt-MCF7 and TAM-resistant MCF7 (MCF7/T) tumor xenograft model. Distilled water (control), 1/2 maximum tolerated dose (MTD) of capecitabine, or 2/3 MTD of capecitabine were orally administered with an orogastric probe once a day for five days, and then they were given a two-day washout as one course. Four courses of treatment were performed. (A) Representative photographs of immunohistochemistry (×200) for ERα and DPD in tumors obtained from control groups on day 29. Scale bars = 100 μm. (B) Representative photographs of mice bearing wt-MCF7-tumor (left) and MCF7T-tumor (right) in each treatment group on days 1 and 29. Each scale bar represents 1 cm. (C) The mean tumor volumes were plotted from day 1 to day 29, with measurements taken every two or three days (left; wt-MCF7 tumors, right; MCF7/T tumors). Closed squares (■) indicate control, closed triangles (▲) indicate 1/2 MTD of capecitabine, and closed circles (●) indicate 2/3 MTD groups. *p < 0.01 (control group vs. 2/3 MTD group), # p < 0.01 (control group vs. 1/2 MTD group) using unpaired Student’s t-tests. (D) The mean body weights were plotted from day 1 to day 29. Closed squares (■) indicate control, closed triangles (▲) indicate 1/2 MTD of capecitabine, and closed circles (●) indicate 2/3 MTD groups.
Fig 5: DPYD promoter activity in wild-type MCF7 and tamoxifen-resistant MCF7 cells.Exogenous promoter activity of DPYD was measured by transient transfection assay of the 5' region of DPYD with the luciferase reporter gene. Relative luciferase activity normalized to pGL3-Basic in each cell line is expressed. The experiments were repeated independently at least three times, and one representative result is provided in the figures. NS: not significant, **p < 0.01, using unpaired Student’s t-tests.
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