Syk (L) isoform regulates cell-cycle progression and cytokinesis.

(A) DLD-1 cells were transduced with Syk shG1 and shL2 shRNAs or shLUC (control shRNA), and the cell cycle distribution of cells one week post-infection was analyzed using flow cytometry. The accumulation of transduced cells in G0-G1, S, G2-M and > 4N phases of each cell line is normalized to its control and is shown as a bar graph. The data are an average of three independent transduction experiments and error bars represent the mean ±SD (*, P<0.05; **, P<0.01). (B) Representative fluorescence microscopy images of DLD-1 cells in mitosis. DLD-1 cells were transduced with shLUC (control shRNA) (panel B1), Syk shG1 shRNA (panels B2-B3) and shL2 shRNA (panels B4-B5). One week post-infection, cells were cultured for 24 hours in Labtek glass chambers and stained with anti-Syk antibody (APC, red), anti-beta Tubulin (Alexa 488, green). Nuclei were stained with Hoechst 33342 dye (blue). Slides were analyzed visually using Zeiss Imager M2 microscope (system magnification: 60x). Two-color fluorescence merge images of representative fields show normal size nuclei and normal bipolar spindles in control cells (panel B1), while cells tranduced with Syk shG1 (panels B2) and shL2 shRNAs (panel B4) showed abnormal metaphases with aberrant accumulation of unaligned chromosomes in midzone (red arrows) and abnormal multipolar spindles, enlarged nuclei and abundant Syk expression (panels B3 and B5). Similar results were observed in three independent experiments. Scale bar: 10 μm.