980
J. Lee et al. / Bioorg. Med. Chem. 17 (2009) 977–980
4.6. RNA interference (RNAi) experiments
Supplementary data
Jurkat cells (2 · 106 cells/dish) were plated into 10 cm
dishes and polybrene (Sigma, 4 lg/mL) was added.
The cells were infected with lentiviruses producing shR-
NAs targeting c-parvin or adenosine kinase, incubated
for 84 h, and harvested for a transwell cell migration as-
say. Transcript levels of c-parvin and adenosine kinase
were measured by TaqMan quantitative RT-PCR.
Supplementary data associated with this article can be
References and notes
1. Shimizu, Y.; Rose, D. M.; Ginsberg, M. H. Adv. Immunol.
1992, 72, 325.
Sequences of shRNAs targeting c-parvin
2. von Andrian, U. H.; Mackay, C. R. N. Engl. J. Med. 2000,
343, 1020.
3. Holzmann, B.; Gosslar, U.; Bittner, M. Curr. Top.
Microbiol. Immunol. 1998, 231, 125.
4. Jackson, D. Y. Curr. Pharm. Des. 2002, 8, 1229.
5. Podolsky, D. K.; Lobb, R. R.; King, N.; Benjamin, C. D.;
Pepinsky, B.; Sehgal, P.; deBeaumont, M. J. Clin. Invest.
1993, 92, 372.
6. Hesterberg, P. E.; Winsor-Hines, D.; Briskin, M. J.; Soler-
Ferran, D.; Merrill, C.; Mackay, C. R.; Newman, W.;
Ringler, D. J. Gastroenterology 1996, 111, 1373.
7. Fotouhi, N.; Joshi, P.; Fry, D.; Cook, C.; Tilley, J. W.;
Kaplan, G.; Hanglow, A.; Rowan, K.; Schwinge, V.;
Wolitzky, B. Bioorg. Med. Chem. Lett. 2000, 10, 1171.
8. Jackson, D. Y.; Quan, C.; Artis, D. R.; Rawson, T.;
Blackburn, B.; Struble, M.; Fitzgerald, G.; Chan, K.;
Mullins, S.; Burnier, J. P.; Fairbrother, W. J.; Clark, K.;
Bersini, M.; Chui, H.; Renz, M.; Jones, S.; Fong, S.
J. Med. Chem. 1997, 40, 3359.
9. Souers, A. J.; Virgilio, A. A.; Schurer, S. S.; Ellman, J. A.;
¨
Kogan, T. P.; West, H. E.; Ankener, W.; Vanderslice, P.
Bioorg. Med. Chem. Lett. 1998, 8, 2297.
10. Ambroise, Y.; Yaspan, B.; Ginsberg, M. H.; Boger, D. L.
Chem. Biol. 2002, 9, 1219.
shRNA#1: GGACGTCTTTGATGAATTA (sense
sequence)
shRNA#2: GGGCCTGTCTGTGCAGAAT (sense
sequence)
shRNA#3: GCTTCTTCCTGCACTTAAA (sense
sequence)
shRNA#4: GAAGGCTTCTTCCTGCACT (sense
sequence)
Sequences of shRNAs targeting ADK
shRNA#1: GCCACACAAAGCAGCAACA (sense
sequence)
shRNA#2: GGCTTTGAGACTAAAGACA (sense
sequence)
shRNA#3: GGTCCCTCATAGCTAATCT (sense
sequence)
4.7. TaqMan quantitative RT-PCR
Jurkat cells were harvested and total RNA was extracted
using Trizol (Invitrogen) and an RNeasy purification kit
(Qiagen). cDNAs of the total mRNAs were synthesized
using the cDNA Archive kit (Applied Biosystems), and
transcript levels of c-parvin were measured using Taq-
Man Universal PCR Master Mix (Applied Biosystems)
and TaqMan Gene Expression Assays (primers & probe
sets, Applied Biosystems) following the manufacturer’s
protocol. GAPDH was used as an internal control.
11. Collins, C. S.; Hong, J.; Sapinoso, L.; Zhou, Y.; Liu, Z.;
Micklash, K.; Schultz, P. G.; Hampton, G. M. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 3775.
12. Yarrow, J. C.; Perlman, Z. E.; Westwood, N. J.; Mitch-
ison, T. J. BMC Biotechnol. 2004, 4, 21.
13. Liu, S.; Thomas, S. M.; Woodside, D. G.; Rose, D. M.;
Kiosses, W. B.; Pfaff, M.; Ginsberg, M. H. Nature 1999,
402, 676.
14. Ding, S.; Gray, N. S.; Wu, X.; Ding, Q.; Schultz, P. G.
J. Am. Chem. Soc. 2002, 124, 1594.
15. Trexler, P.; Bold, G.; Lang, M.; Frei, J. PCT Int. Appl.
WO 9807726.
16. Sandy, P.; Ventura, A.; Jacks, T. Biotechniques 2005, 39,
215.
Acknowledgments
This work was supported by the Novartis Research
Foundation and The Skaggs Institute of Chemical Biol-
ogy (P.G.S.), by the National Institutes of Health
(Grant AR 27214 to M.H.G) and partially by the Post-
doctoral Fellowship of Korea Research Foundation
(J.L., 04-12-07-1). We thank Ms. Myleen Medina
(Genomics Institute of the Novartis Research Founda-
tion) for providing us with both c-parvin and ADK
shRNA clones.
17. Yoshimi, R.; Yamaji, S.; Suzuki, A.; Mishima, W.;
Okamura, M.; Obana, T.; Matsuda, C.; Miwa, Y.; Ohno,
S.; Ishigatsubo, Y. J. Immunol. 2006, 176, 3611.
´
18. Maqueda, A.; Moyano, J. V.; Gutierrez-Lopez, M. D.;
Ovalle, S.; Rodrıeguez-Frade, J. M.; Caban˜as, C.; Garcia-
´
´
Pardo, A. J. Cell. Physiol. 2006, 207, 746.
19. Chu, H.; Thieessen, I.; Sixt, M.; La¨mmermann, T.;
Waisman, A.; Braun, A.; Noegel, A. A.; Fa¨ssler, R.
Mol. Cell. Biol. 2006, 26, 1817.