Altered localization of cohesin and its regulators in Vpr-expressing cells. (A) Expression of the cohesin proteins Smc1, Smc3, and hRad21 in DOX-treated MIT-23 versus ΔVpr cells at 48 h. Ch, isolated chromatin; Wh, whole-cell lysate. Loading controls: histone H3 (HH3) and β-tubulin (βTub). Data are representative of at least three independent experiments. (B) Anti-hRad21 staining in DOX-treated MIT-23 cells versus ΔVpr cells. Arrows indicate hRad21 signals at the interface of sister chromatids within the centromere, and arrowheads indicate undetectable hRad21 signals at loosely aligned sister chromatids. Blue, DNA; red, hRad21. Similar results were obtained in three independent experiments. (C) Anti-hSgo1 immunostaining. (D and E, top) Immunostaining of AurB (D) or INCENP (E) in early mitosis. (insets) Magnifications of the centromere. (bottom) Intensities of AurB (D) or INCENP (E) at the inner centromere region. *, P < 0.0001 (AurB) and P = 0.0002 (INCENP) versus Vpr-expressing cells in prophase. Values represent the mean ± SD. (C–E) Results are representative of three independent experiments. Bars: (B and C [right]) 5 µm; (C [middle], D, and E) 10 µm; (D and E, insets) 0.5 µm.