Among the NRASQ61L mutant cutaneous group we chose the resistant M244 and the

Pui Kai Lis laboratory, STAT3 SH2 inhibitors Stattic and S3I 201, JAK2 inhibitor AG490 was purchased from Calbiochem, Curcumin was purchased from Sigma Aldrich Che mical Co, Western blot analysis FLLL32 and curcumin were dissolved in DMSO. Cancer cells were treated with the listed concentrations of these agents or DMSO JAK 阻害剤 for 24 hours, then lysed in cold RIPA lysis buffer containing protease inhibitors and subjected to SDS PAGE. The primary antibodies were purchased from Cell Signaling Technologies, including phospho specific STAT3, phos pho specific STAT3, phospho specific JAK2, phospho specific STAT1, phospho specific ERK1 2, phospho specific mTOR, cleaved Poly polymerase, cleaved caspase 3, cyclin D, Bcl 2, survivin, TWIST1 and GAPDH.<br><br> DNMT1 primary antibodies were purchased from abcam Inc, Membranes were ana lyzed with enhanced chemiluminescence Plus reagents and scanned with a Storm PhosphorI mager, The possible effects of FLLL32 on ten purified human protein kinases were purchase LDE225 performed at Reaction Biology Corp. using Kinase profiler assay. The IC50 inhibitory values of FLLL32 on the kinase activity were determined using 10 different concentrations of FLLL32 with 100 uM as the highest concentration. IL 6 induction of STAT3 phosphorylation MDA MB 453 breast cancer cells were seeded and serum starved overnight. The cells were then left untreated or were treated with FLLL32, curcu min or DMSO for indicated hours. After stimu lation with IL 6 or IFN g for 30 min, the cells were harvested and ana lyzed by western blot.<br><br> STAT3 DNA binding assays After treatment with FLLL32, curcumin, or DMSO for 24 hours, the nuclear extract kit was used to prepare cell LY2109761 臨床試験 nuclear extracts following the manufacturers protocol. Nuclear extracts were analyzed for STAT3 DNA binding activity using the TransFactor Universal STAT3 specific kits with an ELISA based method. MTT cell viability assay Cells were seeded in 96 well plates in triplicate, and treated with FLLL32, cur cumin, WP1066, Stat tic, S3I 201, or AG490 for 72 hours. Twenty five ul of 3 2,5 diphenyltetrazolium bromide was added to each sample and incubated for 3. 5 hours. After this, 100 ul of N, N dimethylforma mide solubilization solution was added to each well. The absorbance at 595 nm was read the following day. Half Maximal inhibitory concentrations were determined using Sigma Plot 9.<br><br> 0 software, Mouse xenografts All animal studies were conducted in accordance with the standard procedures approved by IACUC at the Research Institute at nationwide childrens hospital. MDA MB 231 breast cancer cells were implanted subcutaneously into the flank region of 4 6 week old female NOD SCID mice. After tumors developed, the mice were randomized into two groups and treated with 50 mg kg FLLL32 or DMSO intraperitoneally daily for 18 days. Tumor growth was determined by measuring the major and minor diameter with a caliper. The tumor volume was calculated according to the formula, Tumor volume 0. 5236 × L × W2. The mammalian Pim family of serine threonine specific kinases consists of three highly homologous proteins, Pim 1, Pim 2 and Pim 3, whose functions and expres sion patterns are partially overlapping, Crystalliza tion studies have revealed that Pim kinases constitutively reside in an active conformation, so that their activities are mainly regulated at the level of expression.