Effect of Skp2 silencing alone or in combination with p27^KIP1 in A549 carcinoma cell line. (A) Western blot analysis of Skp2, p27^KIP1, cyclin E and low-molecular-weight isoforms, Ser15 phosphorylated p53 levels in A549 mock, siSkp2-, and siSkp2/p27^KIP1-treated cells. (B) Impact of deregulated cyclin E expression on centrosome status after Skp2 and Skp2/p27^KIP1 silencing in A549 cells. Immunofluorescence analysis (Texas Red = cyclin E, Oregon Green = γ-tubulin) and counterstaining with DAPI. A549 mock cells demonstrate moderate nuclear staining of cyclin E accompanied by centrosome amplification (top panel, magnification ×600). A549 siSkp2 cells display normal centrosomal profile (arrow) and suppression of cyclin E expression (middle panel). A549 siSkp2/p27^KIP1 cells showed increased, cyclin E levels (accumulation of both nuclear and cytoplasmic isoforms) and centrosome aggregates (arrows; bottom panel). (C) Abnormal mitoses, micronuclei, nuclear lobulation, and nucleoplasmic bridges in A549 siSkp2/p27^KIP1 cells. Cells were counterstained with DAPI. Histograms depict percentages of abnormal mitoses (P = .001, ANOVA) and micronuclei (P < .001, ANOVA) in A549 mock and A549 siSkp2/p27^KIP1-treated cells. Images in panels B and C were viewed through a Zeiss Axiolab microscope with 63 times 0.80, Zeiss Achroplan lens (both Carl Zeiss, AntiSel). Cell spreads were mounted in Fluoromount G. Texas Red was used to detect cyclin E, Oregon Green to detect γ-tubulin, and DAPI as a counterstain (Invitrogen, AntiSel). Images were photographed with a SenSys camera (Photometrics, Tucson, AZ) and processed with SmartCapture VP software version 1.4 (Digital Scientific, Cambridge, United Kingdom).