JM is a Tier 2 Canada Research Chair in Functional Genomics of Cancer
JM is a Tier 2 Canada Research Chair in Functional Genomics of Cancer. Footnotes Author Contribution P.M.K. we confirmed that synthetic lipopeptide versions of our inhibitors have similarly specific and dosage dependent effects on cancer cell growth. Our screen reveals new drug targets and peptide drug leads and it provides a rich dataset covering phenotypes for inhibition of thousands of interactions. BL21 (DE3) and cultivated to express proteins. Protein expression was induced by 0.5 mM of Isopropyl -D-1-thiogalactopyranoside at mid-log phase. After growing the culture overnight at 16C, cells were harvested by centrifugation at 14,000 g for 10 min. Cells were lysed with a sonicator and proteins were purified using Ni-NTA agarose (Qiagen) according to the product manual. Concentration of the purified proteins was determined by measuring the absorption at 280 nm using extinction coefficients of 16875M?1cm?1 and 1490M?1cm?1 for BIRC5 and INCENP peptide respectively. Isothermal titration calorimetry After the Ni-NTA agarose purification step, protein samples intended for ITC were purified on a Superdex-75 column equilibrated and eluted with the 20mM PF-04457845 Tris, 300 mM NaCl and 5 mM BME buffer. Protein purity was analyzed by SDS-PAGE, concentrated using Amicon Ultra-15 centrifugal units. All protein and peptide samples were dialysed overnight at 4 C against the same buffer, 25 mM Tris, 150 mM NaCl, 5 mM BME, and 5% DMSO at pH 7. Calorimetric titrations were carried out using a MicroCal ITC200 microcalorimeter (Malvern), with an operating cell volume of 300 L. The ITC measurements were performed at 25C and stirred velocity was set at 700 rpm to ensure rapid mixing in Mouse monoclonal to SORL1 the cell. Each titration was initiated by a 0.4 L injection, followed by 20 injections spaced 150s, of 2 L. The titrations were performed using the same protein batch with a concentration of 15 M BIRC5 in the cell and 100 M for INCENP peptide. The same concentrations were used for titrations with the scrambled peptides. The binding parameters were obtained by non-linear regression analysis using a one-independent-type-of-sites binding model implemented in the Origin 7.0. Software. A summary of thermodynamics and curve fitting of INCENP peptide to BIRC5 at 25 C and pH 7.0 is shown in Supplementary Fig. 8. Statistical analyses Data are presented as the means s.d. Significance of functional enrichment of peptides was evaluated using hypergeometric test. To examine the statistical difference between two groups, two-tailed independent Students t-test and Mann-Whitney U test were used. We calculate edge-betweenness of peptide-target network using Python package NetworkX 1.8 (https://networkx.github.io) and compared the properties of our peptide-target network with 1,000 randomly generated peptide-target networks using bootstrap test. P-value 0.05 was considered as statistically significant. All statistical analyses are performed using Python package Numpy 1.7 and Scipy 0.13.2 (http://numpy.scipy.org). Supplementary Material 1Click here to view.(2.0M, pdf) Acknowledgments We thank the members of the Moffat laboratory for valuable technical assistance with lentiviral screening technology, usage of reagents and gear. We thank Dr. Andrew Emili, Dr. Tim Hughes and Dr. Michael Garton for helpful comments around the manuscript. We thank Dr. Andrea Musacchio for providing us the INCENP cDNA clone. PMK acknowledges an Operating Grant from the Canadian Institute of Health Research (CIHR MOP-123526) and an Development Grant from the Canadian PF-04457845 Cancer Society Research Institute (CCSRI# 702884). JM is usually a Tier 2 Canada Research Chair PF-04457845 in Functional Genomics of Cancer. Footnotes Author Contribution P.M.K. designed the project provided study guidance and wrote the bulk of the manuscript. S.N. performed most experiments and contributed to writing of the manuscript. J.J. performed all bioinformatics analysis and interpreted results as well as assisted in manuscript preparation. C.C. and M.S. performed affinity measurements and helped with other biochemical experiments. Y.I. provided oligonucleotide library and provided study guidance. N.T. provided synthetic peptides and provided guidance on their use. J.M. helped design the project and provided guidance on lentiviral screening. Competing financial interests The authors.