Following ncubatoand successve actions of washng, the arrays had been dred and maged usng a Fujfm LAS 3000 magng Procedure.Duplcate spot ntenstes have been quantfed from every array mage usng the mage quantfcatosoftware.Blood samples were collected from patents newly dagnosed wth CML orhCL as part of ansttutonally accredited cellular sample collectoprotocol.nformed consent was obtaned accordng to nsttutonal gudelnes.Mononuclear cells had been solated from blood samples by densty centrfugaton, washed wth PBS, 5% SVF, and 2 mM EDTA, and theresuspended cell culture medum and ncubated overnght at 37 C a 5% CO2 ncubator.CML cells had been labeled wth CD34 mcrobeads solated by magnetc postve selecton.Purty was estmated to be at least 90% by FACS analyss.Experments had been carried out usng aMDM for CML andhCL cells.SkE was additional to the K562 CML cell lnes growng sem offered methylcellulose medum.MethoCulth4100 was used for cell lnes.Colones have been detected after ten days of culture by addng one mg ml of three 2,five dphenyltetrazolumbromde reagent and had been scored by mage quantfcatosoftware.
Tumors from management or SkE taken care of mce were eliminated, frozeand reduce preparatofor mmunostanng.Sldes contanng a representatve sectoof just about every tumor had been fxed, permeabzed and ncubated wth ant phospho ERK 1 2 or ant ERK antbodes duted PBS and 1% BSA at RT for 1h.Then, cells have been washed and ncubated wth a secondary ant Rabbt antbody.Fnally, DAP was employed to label the nucle, and the sldes have been mounted and theanalyzed find more information under a fluorescence mcroscope.Female athymc NMR Mce had been randomzed nto 3 expermental groups, each contanng seven anmals.Two sets of anmals receved a 200 ul njectoof five.106 K562 Luc leukema cells both flanks.Whetumors reached one hundred mm3, the anmals have been njected ntrapertoneally wth vehcle, matnb or SkE at dose levels of 60 mg kg and one mg kg entire body weght, respectvely.The development of your leukemc cells comprsng the tumor was vsualzed the anmal at dfferent days following ntrapertoneal njectoof 30 mg kg lucferby CX4945 bolumnescence magng wth a Photomager, as descrbed elsewhere.
The vvo study was carried out accordng to French legslatoolaboratory anmal use and care.Followng U0126, PLX 4720 or SkE therapy,hCL cells were staned wth propdum odde, along with the staned cells were analyzed

wth a cytometer.SkE was extracted and purfed from Quassa amara as prevously descrbed.K562 cells had been ncubated at 37 C wth 250 nM SkE to the ndcated tmes.Ras actvty was determned immediately after GST pull down.Ras GTlevels have been determned usng GST c Raf RBD to pull dowactve GTbound Ras from cell extracts by glutathone beads.The beads were washed four tmes and subjected to SDS Web page.Ras and Phospho ERK1 two protens have been detected by Westerblot analyss as descrbed prevously.All information are presented because the mea SD of no less than three ndependent determnatons.