The correlation between the cytotoxic activity of enzastaurin and the corresponding gene, RTKs phosphorylation and miRNA expression patterns has been examined to clarify the Enzastaurin was kindly provided by Ely Lilly. Growth inhibition was assessed by MTS assay to examine the effect of enzastaurin on lung cancer cell lines. Cell suspensions were seeded Imatinib into 96 well plates and increasing concentrations of enzastaurin were added. After incubation for 72 h at 37 1C, MTS was added to each well and incubated for 2 h at 37 1C, after which absorbance was measured using a microplate reader with a test wavelength of 450 nm. The IC50 value was defined as the concentration needed for 50% reduction of the growth by treatment with enzastaurin. JAK inhibitor was purchased from Calbiochem .
A549 and RERF LC KJ cells were seeded into 96 well plates. After 24 h, the cells were incubated for 72 h in the various concentrations of enzastaurin , with or without low dose JAK inhibitor. RNA isolation, cDNA array, RTKs phosphorylation antibody array and miRNA array Total RNA was isolated from lung cancer cell Benazepril clinical trial lines with the use of TRIzol reagent , according to the manufacturer’s instructions. High density oligonucleotide array analysis was carried out using Affymetrix HG U133A expression array, as previously described . Scanning was performed with the GeneChip Scanner 3000 , and GeneChip analysis was based on the Affymetrix GeneChip Manual with GeneChip Operating Software version 1.0 , and Microarray Database software. We also performed human RTKs phosphorylation antibody array, including 71 antibodies .
MicroRNA expression profiles were analysed by TaqMan MicroRNA Array set version 2.0 containing 667 miRNAs and validated by TaqMan MicroRNA assay Benazepril structure . Western blot analysis Cells were lysed in buffer containing 50mM Tris HCl, pH 7.6, 150mM NaCl, 0.1% sodium dodecyl sulphate, 1% Nonidet P 40 and 0.5% sodium deoxycholate. The lysates were kept on ice for 30 min, and then centrifuged at 13 000 g for 30 min. The supernatant was collected and 10 mg of protein were separated by gel electrophoresis on 10% gels, transferred to nitrocellulose membranes and detected by immunoblotting using a chemiluminescence system . The antibodies detecting JAK1, STAT3, phospho STAT3 and b actin were purchased from Cell Signaling Technology .
Lentiviral mediated JAK1 overexpressing cells Expression plasmid vector pEZ Lv151 was Benazepril solubility used for lentiviral vector production . The coding sequence of human JAK1 or enhanced green fluorescent protein was inserted under the transcriptional health insurance control of the CMV promoter in pEZ Lv151. The human JAK1 lentiviral expression plasmid or EGFP plasmid was cotransfected into 293Ta cells with the Lenti Pac HIV Packaging Mix . Lentivirus containing supernatants were harvested 48 h after transfection. The lentivirus particles were purified and stored at 80 1C in aliquots until use. To establish stable JAK1 overexpressing cell lines, A549 cells were transduced with serial dilutions of lentiviral supernatant in the presence of 5 mgml 1 polybrene and selected by 0.8 ng ml 1 geniticine. After antibiotic selection for 3 weeks, stable overexpressing JAK1 cells were obtained. Statistical analyses .