Kinome siRNA Screen Identifies Regulators of Ciliogenesis and Hedgehog Signal Transduction

Marie Evangelista,^1* Tze Yang Lim,¹ James Lee,¹ Leon Parker,² Amir Ashique,¹ Andrew S. Peterson,¹ Weilan Ye,² David P. Davis,¹ Frederic J. de Sauvage^1*

¹Department of Molecular Biology, Genentech, South San Francisco, CA 94080, USA.

²Department of Tumor Biology and Angiogenesis (TBA), Genentech, South San Francisco, CA 94080, USA.

Supplementary Materials (PDF File)

• Materials and Methods
• Fig. S1. Analysis of the Hedgehog kinome siRNA screen.
• Fig. S2. Candidate hits are specific to Hh signaling.
• Fig. S3. Pooled siRNAs do not affect the SV40 luciferase reporter stably transfected in CH310T1/2 cells.
• Fig. S4. Cotransfection of siRNAs to candidate genes and Gli3 indicate that all nine candidates act upstream of Gli3 in S12 cells since none repressed luciferase activity induction by Gli3 in the absence of Hh.
• Fig. S5. Effect of siRNA knockdown of candidates on ciliogenesis.
• Fig. S6. Nek1 and Prkra affect ciliogenesis.
• Fig. S7. Candidate hits are not transcriptionally regulated by the Hh pathway in S12 cells.
• Fig. S8. qRT-PCR of Ptch1^-/+-driven medulloblastoma tumors treated in vivo with a Hh antagonist indicate that, unlike Ptch1 and Gli1, none of the hits are down-regulated by treatment with the Hh antagonist.
• Fig. S9. qRT-PCR of Smo, Plk1, and Cdc2l1 knockdown in S12 cells in cell cycle experiments in Fig. 4F.
• Fig. S10. Failure of Flag-tagged Cdc2, another Cdk-related kinase, to associate with Myc-tagged Sufu in coimmunoprecipitation experiments.
• Fig. S11. Cdc2l1 activates the Hh pathway in a dose-dependent manner.
• Table S1. Summary of primary hits from the Hh siRNA screen.
• Table S2. Summary of nine validated hits from the Hh siRNA screen.
• Table S3. List of qRT-PCR probe sets.
• Table S4. List of PCR primers for DNA construct design.
• Table S5. List of PCR primers for in situ hybridization probes.
• References

Format: Adobe Acrobat PDF

Size: 1,317 KB

[Download PDF]

Supplementary Materials (Excel File)

• Table S6. Raw and normalized values for Hh SiRNA screen.

Format: Microsoft Excel

Size: 324 KB (compressed); 900 KB (decompressed)

Excel file is packaged as a compressed archive, in *.zip format; users should download the compressed file to their machine and decompress the file on their local hard drive, using the instructions below.

Instructions for downloading and decompressing files:

1. Create a temporary folder on your machine's hard drive.
2. Save the compressed archive to the temporary folder you created, using the links above.
3. Decompress the compressed file in the temporary folder using decompression software such as WinZip (Windows; www.winzip.com) or StuffIt Expander (Windows and Mac; www.stuffit.com).

Excel files can be opened and viewed using Microsoft Excel, the spreadsheet module of the freely downloadable Open Office suite, or the freely downloadable Excel Viewer available from Microsoft.

[Download Excel File]

---

*To whom correspondence should be addressed. E-mail: evangelm{at}gene.com (M.E.) and sauvage{at}gene.com (F.J.d.S.)

Citation: M. Evangelista, T. Y. Lim, J. Lee, L. Parker, A. Ashique, A. S. Peterson, W. Ye, D. P. Davis, F. J. de Sauvage, Kinome siRNA screen identifies regulators of ciliogenesis and hedgehog signal transduction. Sci. Signal. 1, ra7 (2008).

© 2008 American Association for the Advancement of Science