Fig 7.

Knockdown of SMC1 blocks MVC infection-induced intra-S-phase arrest. (A) Western blot analysis of SMC1 expression. WRD cells were infected with MVC. At the indicated times p.i., the cells were collected and analyzed for expression of SMC1 and SMC1 phosphorylated at serine 957, p-SMC1(Ser957). Mock-infected cells were used as a control. (B to D) Knockdown of SMC1 reduces cell population in S phase and viral DNA replication. WRD cells were transfected with siRNA control (siScrambled) or SMC1 siRNA (siSMC1). At 2 days posttransfection, the cells were mock or MVC infected. At the indicated times p.i., cells were analyzed as follows. (B) One-third of the cells were collected and analyzed for SMC1 by Western blotting. (C) One-third of the cells were incubated with BrdU for 1 h, denatured by HCl, and costained with an anti-BrdU antibody and DAPI for flow cytometry analysis. Numbers show percentages of the cell population in S phase and G₂/M phase, respectively. The statistical analysis of the percentage of cells in S phase from three independent experiments is shown. Data are shown as means ± standard deviations. P values were determined using Student's t test. (D) One-third of the cells were collected for Hirt DNA extraction and Southern blot analysis.