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| Survivin’s Role in Cell Survival | ||
| NFkB Pathway Analysis | ||
| Assessing the Roles of 178 Kinases in the Cell Cycle | ||
RNAi Experimental Steps 1. Order Silencer Kinase siRNA Library (Cat# 81010S)2. Transfect HeLa cells individually with 178 siRNA from the library or Silencer Negative Control #1 siRNA at 100 nM in triplicate 3. Incubate 48 hours 4. Detect of microtubule distribution and chromatin condensation by fluorescence microscopy 5. Determine the percentage of cells undergoing mitosis (mitotic index) |
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Conclusions: Several kinases were identified with apparent roles in cell cycle progression. One kinase siRNA triggered a prometaphase-like arrest state (nearly 16% mitotic index, or ~7-fold higher than normal) wherein spindles were clearly bipolar and chromosomes were well condensed, but with no evidence of successful chromosome alignment (left inset). This likely corresponds to activation of the well-studied spindle assembly checkpoint, which monitors the correct, functional interactions between spindle microtubules and kinetochores in preparation for metaphase and subsequent anaphase onset. A second kinase siRNA that also generated cells in mitotic arrest induced the formation of aberrant spindles displaying either too many or too few apparent poles (e.g. right inset). |
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