Cells have been cultured RPM 1640 medum contanng 10%heat nactvated fetal calf serum, 2 mM L glutamne, 100 U mL pencln, and a hundred ug mL streptomycn.Treatment method of cells Exponentally growng cells have been taken care of wth ARRY 520 for uto 48hours.For combnaton,hL 60 andhL 60Bcl 2 cells had been ncubated wth ARRY 520, ABT 737, or each for uto 96hours.ABT 737, a selectve Bcl 2 nhbtor, top article was syntheszed at M.D.AndersoCancer Center primarily based othe publshed structure.DMSO was applied since the control agent.To nhbt KSexpresson, three ? 106 exponentally grownghL 60 cells have been transfected wth five ug of ether the KSASO or ts control olgonucleotde usng Nucleofector solutoand program 17 followng the companies nstructons and as prevously descrbed.Cell vabty assay Apoptoss was estmated by flow cytometry measurements of phosphatdyl serne wth the AnnexFLUOS Stanng Kt.
Membrane ntegrty was smultaneously assessed by 7 amno actnomycD.To measure adjustments the mtochondral membrane potental, cells had been loaded pan Syk inhibitor wth CMXRos and MtoTracker Greefor 1hour at 37 C.The reduction of MMwas theassessed by measurng CMXRos retentowhe smultaneously adjustng for mtochondral mass.Cell cycle dstrbutoCells were fxed wth 70% ce cold ethanol and staned wth propdum odde soluton.The DNA content was determned usng a FACSCalbur flow cytometer.The cell cycle dstrbutowas analyzed usng ModFt LT computer software.TUNEL assay To determne the cell cycle stage of apoptotc cells, cells have been fxed 4% formaldehyde and permeabzed wth 0.1% TrtoX 100.TUNEL assay was carried out usng the Apo Drect Kt followng the producers nstructons.Westerblot analyss Westerblot analyss was carried out as descrbed prevously.
Colony formatoassay Colony formatoassay was performed as descrbed prevously usng 1 ? 105 mononuclear

cells through the bone marrow of AML patents and cells from usual blood obtaned by apheress treated wth ARRY 520, 3.3 to 100 nM.Xenograft studes SCD mcehL 60 or MV4 11 cells growng MDM supplemented wth 20% or 10% FBS, Glutamax, and antbotc antmycotc wereharvested whethey reached approxmately 106 mL.Female SCD bege mce were mplanted subcutaneously the rght flank wth 107hL 60 or MV4 eleven cells mouse 100 uL PBS.Twenty a single days later forhL 60 njected mce and eghteefor MV4 eleven njected mce, tumors were measured wth calpers and tumor volume calculated, volume2.Mce were randomzed nto 5 or 8 group, wth aaverage tumor volume of approxmately 265 or 275 mm3 each and every grouforhL 60 or MV4 11 njected mce, respectvely.Remedy begaothe day of randomzaton.Mce njected wthhL 60 had been dosed wth vehcle or ARRY 520 25% PEG400 10% EtOH 65% salne ntrapertoneally, at 27 mg kg, odays 1, 5 and 9.Mce njected wth MV4 eleven were dosed wth vehcle or ARRY 520, at 20 mg kg, odays 1, five, 9, and 53, and also the survvng vehcle treated mce were later on njected wth ARRY 520 odays 28, 53, and 67.