Figure 1

Identification of kinases important for mitosis in mouse fibroblasts. (a) Screening of the mouse kinome for mitotic kinases. NIH3T3/H2B-GFP cells were transfected with a small interfering RNA (siRNA) library targeting the mouse kinome and related genes. Each of the 588 genes in the library was targeted by three different Stealth siRNAs. After 24 h, the cells were treated with 0.2 μg/ml of Adriamycin for 2.5 h. Images of the cells were then captured automatically using a fluorescent microscopy and the mitotic index of each transfection was scored. Cutoff was set to candidates with mitotic index above 2 × S.D. of the mean. The mitotic index of these candidate genes are shown. Note that non-protein kinases are also included in the library: Dgkg (diacylglycerol kinase gamma); Mpp2 (MAGUK p55 subfamily member 2 guanylate kinase); Prps1 (ribose-phosphate pyrophosphokinase 1); Sphk2 (sphingosine kinase 2). (b) The candidates obtained from the initial screen (a) were subjected to secondary confirmation assays in the absence of Adriamycin. NIH3T3/H2B-GFP cells were transfected with siRNAs against the indicated candidates. Each gene was targeted by three different Stealth siRNAs. After 26.5 h, the mitotic index was quantified using a fluorescent microscopy. The dotted line indicates the mitotic index of the control. (c) Comparison between mouse and Drosophila kinome required for mitosis. The protein kinases found to be important for mitosis in mouse cells in this study are compared with protein kinases found to contribute to mitosis in Drosophila.² Only four homologous candidates are common in the two screens