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Thus, we decided to expand our studies to not only include silen cing of ERK1 andor ERK2, but to compare and contrast the biological responses and bypass mechanisms trig gered by exposing A375 melanoma cells to PLX4032, as well as a MEK inhibitor. The results clearly demonstrate that not only is a combination of ERK1 and ERK2 superior in triggering a caspase depen dent mode of killing JAK 阻害剤 FDA approved A375 melanoma cells compared to PLX4032 or PD0325901, but drug resistant clones infre quently appear by directly targeting ERK. The ability of using ERK shRNAs to not only kill melanoma cells, but to block emergence of treatment resistant clones likely involves not only reductions in levels of phospho ERKs, but also in upstream reductions in BRAF, CRAF and phospho MEK thereby interrupting a feedback loop cri tical to melanoma survival.<br><br> ERK shRNAs were also shown to increase the sensitivity of melanoma cells to killing by PLX4032 paving the way for combination therapeutic approaches in melanoma. LDE225 溶解度 These results demonstrate that targeting ERK in melanoma can overcome the apoptotic resistance of this highly aggres sive and difficult to cure tumor. Methods Cell culture and chemicals The human melanoma cell line A375 was purchased from American Type Culture Collection and maintained in DMEM plus 10% FCS in a humidified incubator. Annexin FITC was purchased from Biovision Research Products, and tetra methyl rhodamine ethyl ester was purchased from Invitrogen Molecular Probes. Pan caspase inhibitor ZVAD was purchased from BD Bios ciences. Propidium iodide was purchased from Sigma Chemical Co.<br><br> PD0325901 and PLX4032 were purchased from Biovision Research Products and Selleck Chemicals, respectively. Abs against ERK1, ERK2, pERK1, pERK2, MEK, pMEK, p Bad, Bak, オーダー LY2157299 Bim, PUMA were purchased from Cell Sig naling Technology. whereas Bcl XL, Mcl 1, Bad, PARP, caspase 3, Raf 1, Raf B and GAPDH were purchased from Santa Cruz. Ab against Bcl 2 was from DAKO, ab against actin from Chemicon Int. Ab against Bax from Calbiochem, and against XIAP and activated Bax from BD Transduc tion Lab. Primary Abs incu bated overnight at 4 C, and secondary Abs were incubated at room temperature for 1 hr. Production of lentiviral supernatants Mission TCR shRNAs targeting human ERK1 and ERK2 were purchased from Sigma Chemical Co. pLKO. 1 Scramble control shRNA plasmid, psPAX2 packaging plasmid and pMD2.<br><br> G envelope plasmid were provided by Addgene. To make lentiviral particles, HEK 293 T cells were plated into 10 cm plates, 2 106 cellplate, with 8 ml of DMEM plus 10% FBS and no antibiotics. On the next day, for each plate 3 ug of pLKO. 1 shRNA plasmid together with 2. 25 ug of psPAX2 and 0. 75 ug of pMD2. G plasmid were transfected with FuGen 6 reagents according to the manufactures instruction. The transfection reagent was removed by replacing the medium with fresh DMEM containing FBS and penicillinstreptomycin on the following day. The cells were incubated at 37 C, 5% CO2 for 24 hr for another 2 days. Supernatants from 24 hr and 48 hr incubations were harvested and combined followed by centrifugation to remove cell debris and stored at 80 C.