Figure 4.
Figure 4. Role of Ku80 in B19 infection in vitro. (A) Ku80 expression on cell surface. The indicated cell lines were reacted with 5 μg/mL mouse monoclonal anti-Ku80 antibody (line) or 5 μg/mL isotype-matched mouse monoclonal antibody 1F5 (shadow), followed by FITC-labeled anti–mouse IgG antibodies. Cells were washed with PBS, and cell-surface expression of Ku80 was analyzed by flow cytometry. (B) Blocking of B19 adsorption by anti-Ku80 antibody or antigloboside antibody. KU812Ep6 cells (2 × 106) were infected with B19 (2 × 1011 copies of B19 DNA) on ice for 30 minutes in the presence of the indicated antibodies (5 μg/mL) and extensively washed with PBS 3 times. To activate α5β1 integrin, anti-integrin antibodies were used in the presence of divalent ions (1 mM Mn2+, 1 mM Mg2+). B19 DNA in each group was quantified by quantitative PCR. The blocking ability of B19 binding by each antibody was expressed as percent decrease of B19-DNA in each group compared to that in antibody-untreated cells. **P < .01, *P < .05 by Student t test. (C) Blocking of B19 replication by anti-Ku80 antibody or antigloboside antibody. KU812Ep6 cells were infected with B19 and washed as described. Cells were further incubated for 48 hours at 37°C and washed with PBS 3 times before the quantitative study of B19 DNA. To activate α5β1 integrin, anti-integrin antibodies were used in the presence of divalent ions (1 mM Mn2+, 1 mM Mg2+). The blocking ability of B19 replication by each antibody was expressed as described. **P < .01, *P < .05 by Student t test. (D) RNA interference of Ku80 in KU812Ep6 cells. Cell-surface expression of Ku80 was examined by flow cytometry in scramble RNA or siRNA of Ku80-transfected KU812Ep6 cells (left panel). KU812Ep6 cells treated with indicated RNA were reacted with 5 μg/mL mouse monoclonal anti-Ku80 antibody or 5 μg/mL isotype-matched mouse monoclonal antibody 1F5 (shadow), followed by FITC-labeled anti–mouse IgG antibodies. B19 association of siRNA-transfected KU812Ep6 cells was evaluated by quantitative PCR (right panel). Sample DNA was prepared from extensively washed scramble RNA or siRNA of Ku80-transfected KU812Ep6 cells after 2 hours of incubation with B19. *P < .01 by Student t test.

Role of Ku80 in B19 infection in vitro. (A) Ku80 expression on cell surface. The indicated cell lines were reacted with 5 μg/mL mouse monoclonal anti-Ku80 antibody (line) or 5 μg/mL isotype-matched mouse monoclonal antibody 1F5 (shadow), followed by FITC-labeled anti–mouse IgG antibodies. Cells were washed with PBS, and cell-surface expression of Ku80 was analyzed by flow cytometry. (B) Blocking of B19 adsorption by anti-Ku80 antibody or antigloboside antibody. KU812Ep6 cells (2 × 106) were infected with B19 (2 × 1011 copies of B19 DNA) on ice for 30 minutes in the presence of the indicated antibodies (5 μg/mL) and extensively washed with PBS 3 times. To activate α5β1 integrin, anti-integrin antibodies were used in the presence of divalent ions (1 mM Mn2+, 1 mM Mg2+). B19 DNA in each group was quantified by quantitative PCR. The blocking ability of B19 binding by each antibody was expressed as percent decrease of B19-DNA in each group compared to that in antibody-untreated cells. **P < .01, *P < .05 by Student t test. (C) Blocking of B19 replication by anti-Ku80 antibody or antigloboside antibody. KU812Ep6 cells were infected with B19 and washed as described. Cells were further incubated for 48 hours at 37°C and washed with PBS 3 times before the quantitative study of B19 DNA. To activate α5β1 integrin, anti-integrin antibodies were used in the presence of divalent ions (1 mM Mn2+, 1 mM Mg2+). The blocking ability of B19 replication by each antibody was expressed as described. **P < .01, *P < .05 by Student t test. (D) RNA interference of Ku80 in KU812Ep6 cells. Cell-surface expression of Ku80 was examined by flow cytometry in scramble RNA or siRNA of Ku80-transfected KU812Ep6 cells (left panel). KU812Ep6 cells treated with indicated RNA were reacted with 5 μg/mL mouse monoclonal anti-Ku80 antibody or 5 μg/mL isotype-matched mouse monoclonal antibody 1F5 (shadow), followed by FITC-labeled anti–mouse IgG antibodies. B19 association of siRNA-transfected KU812Ep6 cells was evaluated by quantitative PCR (right panel). Sample DNA was prepared from extensively washed scramble RNA or siRNA of Ku80-transfected KU812Ep6 cells after 2 hours of incubation with B19. *P < .01 by Student t test.

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