Technical Reference #3009

3009.

An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor Melody Tsui; Tiao Xie; James D. Orth; Anne E. Carpenter; Stewart Rudnicki; Suejong Kim; Caroline E. Shamu; Timothy J. Mitchison, Harvard Medical School, PLoS ONE, 4(3009), (2009)
Link To Paper

Abstract:
Kinesin-5 (also known as Eg5 KSP and Kif11) is required for assembly of a bipolar mitotic spindle. Small molecule inhibitors of Kinesin-5 developed as potential anti-cancer drugs arrest cell in mitosis and promote apoptosis of cancer cells. We performed a genome-wide siRNA screen for enhancers and suppressors of a Kinesin-5 inhibitor in human cells to elucidate cellular responses and thus identify factors that might predict drug sensitivity in cancers. Because the drug’s actions play out over several days we developed an intermittent imaging screen. Live HeLa cells expressing GFP-tagged histone H2B were imaged at 0 24 and 48 hours after drug addition and images were analyzed using open-source software that incorporates machine learning. This screen effectively identified siRNAs that caused increased mitotic arrest at low drug concentrations (enhancers) and vice versa (suppressors) and we report siRNAs that caused both effects. We then classified the effect of siRNAs for 15 genes where 3 or 4 out of 4 siRNA oligos tested were suppressors as assessed by time lapse imaging and by testing for suppression of mitotic arrest in taxol and nocodazole. This identified 4 phenotypic classes of drug suppressors which included known and novel genes. Our methodology should be applicable to other screens and the suppressor and enhancer genes we identified may open new lines of research into mitosis and checkpoint biology.

Materials & Methods:
HeLa H2B-GFP cells were reverse transfected with individual siRNA duplexes or pools (both at 15 nM final concentration) in 96 well glass bottom plates (MatTek P96G-0-5-F). 24 hours after transfection K5I was added to the cells so that the final inhibitor concentration was 1 uM. To prevent evaporation during imaging 50 uL of mineral oil was added to each well. Starting immediately after drug and mineral oil addition the cells were imaged at 10 minute intervals for 48 hours on an automated inverted fluorescence microscope (Nikon TE2000E) fitted with an incubation chamber and automated focus. Both GFP and phase contrast images were collected. The chamber was kept at 37uC with 5% CO2 throughout imaging. For each siRNA pool and duplex reagent tested 50–100 cells were scored visually for phenotypic changes during imaging.

Microscopic Technique
Long-term Timelapse Microscopy

Cell Type(s)
HeLa H2B-GFP cells