The c Kit activation induces cytokines and their receptors, but TrkA does not, suggesting that the part of the signal pathways induced by the two receptors is different. However, TrkA is able to induce common novel downstream tar gets such as KLF2 and SMAD7 which has not been reported in the neuronal system, indicating that NGF induces genes which are involved in stem cell mainte nance similar fairly to c Kit signaling in hematopoietic cells. Furthermore, upregulation of KLF2 may be involved in NGF mediated survival of imatinib treated cells. Methods Cell lines HMC 1 were grown in RPMI1640 medium supplemented with 10 20% fetal calf serum. The presence of V560G mutation and the absence of 816 mutation in c Kit was confirmed by sequencing.
Viability assay HMC 1 cells were grown in medium con taining 10% FCS in the presence Inhibitors,Modulators,Libraries of 5 uM imatinib and or 100 ng ml human recombinant NGF. Cells were counted in a Neubauer chamber using 0. 1% Trypan Blue. TUNEL assay To assess the degree of apoptosis, an in situ cell death detection kit was used for terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining. Growth factor stimulation, and RNA isolation Cells were serum starved for 17 h, then treated with dimethyl sulfoxide or 5 uM imatinib for 4 hours prior to stimulation with 100 ng ml mouse recombinant SCF or NGF, respectively. After 30 or 120 min the stimulation was stopped in ice cold PBS. RNA was isolated from growth factor treated or untreated HMC 1 cells using RNeasy Mini kit according to the manufac turers protocol.
Residual DNA contamination Inhibitors,Modulators,Libraries was removed with DNAseI according to the manufacturers recommenda tions, and the RNA was again purified with RNeasy Mini kit. Microarray analysis The Whole Inhibitors,Modulators,Libraries Human Genome Microarray used in this study con tained 45015 oligonucleotide probes covering the entire human transcriptome. cRNA synthesis was performed with the Low RNA Input Linear Amplification Kit PLUS, Two Color as direc ted by the manufacturer. cRNA fragmentation, hybridiza tion and washing steps were also performed exactly as recommended by the manufacturer Two Color Microar ray Based Gene Expression Analysis Protocol V5. 5 except that 4 ug of each labeled cRNA were used for hybridization. Slides were scanned on Inhibitors,Modulators,Libraries the Agilent Micro Array Scanner G2505 B at two different PMT settings, namely 100% and 5%, to increase the dynamic range of the mea surements.
Data extraction and normalization were performed with the Feature Extraction Software V9. 5. 3. 1 by using the recommended default extraction protocol file, GE2 v5 95 Feb07. xml. Only probes with allocated Inhibitors,Modulators,Libraries gene symbols and arithmetic mean intensity 50 for both chan nels were considered for further analysis. Genes with p value 0. 0001 and fold induction ratio of 2 were con sidered significantly induced. Accession Numbers The complete microarray data have been deposited selleck compound in NCBIs Gene Expression Omnibus and are accessible through GEO series accession number GSE28045.