Akt inhibitor IV, Akt inhibitor VIII, PI3Ka inhibitor VIII, PI3Kb inhibitor VI, PI3Kg inhibitor VII and Raf1 kinase inhibitor I were purchased from Merck. OSU 03012 was obtained from Tebu bio. All solutions have been stored at 20 C. Thymidine uptake, cell cycle analysis and detection of apoptotic cells Assays of thymidine incorporation have been executed as follows, 1. 25 ? 104 cells were seeded in triplicate in 96 properly flat bottom microtiter plates. Inhibi tors were added as 2x concentrated answer inside a 100 ul volume. For the final 3 h with the incubation period, 1 uCi thymidine was added to each and every nicely. Apoptotic cells were detected and quantified with all the annexin V PI approach utilizing the TACS Annexin V FITC kit in accordance with the makers directions.
Binding of fluorescein isothiocyanate labeled annexin V and PI staining in the cells was deter mined by flow cytometry on the FACSCalibur. For cell cycle selleck chemical evaluation, cells had been fixed with 70% ethanol, washed with phosphate buffered saline, and stained with PI. DNA content material of the cells was deter mined by flow cytometry. Sequencing in the BCR ABL1 kinase domain, of CBL exons 7 9 and of PIK3CA exons ten and 21 Exclusively to amplify the kinase domain of BCR ABL1, hemi nested PCR was performed based on Hochhaus et al. For cell lines carrying b2 a2 and b3 a2 BCR ABL1 fusion, the following primers were used in 1st round PCR, BCR exon 13 forward, For cell lines with e1 a2 and e6 a2 BCR ABL1 trans location, exactly the same ABL1 exon 7 reverse primer was combined with the BCR exon 1 forward primer, Very first round PCRs had been performed at 60 C, respectively 59 C for 35 cycles.
The PCR solutions had been diluted read full article and applied within a second round PCR at 59 C for 25 cycles applying reverse primer A7 plus the ABL1 exon 4 forward primer, Purified PCR products had been sequenced applying the second round primers. The following primers have been used to amplify and to sequence CBL exons 7 9 from cDNA. CBL exon 6 for ward, RT PCR was performed for Quantitative real time PCR evaluation Quantitative PCR was performed on a 7500 Applied Biosystems real time PCR method working with the manufacturers protocol. RNA was prepared applying the RNeasy Mini kit. For mRNA quantification, reverse transcription was per formed making use of the SuperScript II reverse transcriptase kit. Expression of BCR ABL1 e1 a2 and b2 a2 fusion mRNAs, of CBL and of p85b had been assessed employing the SYBR GREEN PCR Master Mix with GAPDH as internal manage.
BCR forward, Relative expression levels have been calcu lated making use of the Ct technique. Expression analysis of Ikaros splice variant 6 For detection of Ikaros splice variant 6, we per formed PCR utilizing the following primers, Ikaros exon two forward, The PCR was performed with an annealing temperature of 62 C. Splice variants had been detected by electrophoresis on a 1. 2% agarose gel and verified by sequencing with the PCR items.