Figure 3
Effects of anti-FRβ immunotoxin on cell death induction and NO production of FRβ-expressing cells. (A)The schema shows the construction of VH-PE38 and dsFv anti-FRβ-PE38. The purity of VH-PE38 and dsFv anti-FRβ-PE38 were analyzed using sodium dodecyl sulphate- 6% to 15% gradient gel electrophoresis in non-reducing (-SH) and reducing conditions (+SH). Molecular weight markers are shown at left. (B) Rat FRβ-transfected B300-19 cells were cultured at the indicated concentrations of VH-PE38 (open triangle) for 72 hours or at the indicated concentrations of dsFv anti-FRβ-PE38 (filled circle) for 36, 48, or 72 hours. Non-transfected B300-19 cells were also cultured at the indicated concentrations of dsFv anti-FRβ-PE38 (open circle) for 36, 48, or 72 hours. The number of dead cells was measured using propidium iodide staining and flow cytometric analysis, and the percentage of dead cells was calculated as the number of dead cells divided by total cell number. Values are the mean ± SEM of induced cell death from three separate experiments. The right panels are representative of propidium iodide staining patterns at the indicated concentrations of VH-PE38 or dsFv anti-FRβ-PE38. Values in panels show the percentages of dead cells. (C) Thioglycollate-elicited macrophages were cultured at the indicated concentrations of VH-PE38 or dsFv anti-FRβ-PE38 for 48, 72, or 96 hours. Induced cell death (%) was determined by subtracting the percentage of dead cells induced by VH-PE38 from that induced by dsFv anti-FRβ-PE38 in each sample. Values are the mean ± SEM of induced cell death of three separate experiments. (D) Thioglycollate-elicited macrophages were cultured at the indicated concentrations of VH-PE38 (open circle) or dsFv anti-FRβ-PE38 (filled circle) for 24 hours and stimulated using IFN-γ and LPS for an additional 48 hours. NO in the supernatants was measured using the Griess reaction. Values are the mean ± SEM of three separate experiments. * P < 0.05 compared to the VH-PE38 group. FRβ, folate receptor β; IFN-γ, interferon-γ; LPS, Lipopolysaccharide; SEM, standard error of the mean.