N. for JAK3L875H. This different cytokine receptor requirement correlated with different practical properties and with unique level of sensitivity to JAK inhibitors. Transduction of murine hematopoietic cells with JAK3V674A led homogenously to lymphoblastic leukemias in BALB/c mice. In contrast, transduction with JAK3L857P induced various types of lymphoid and myeloid leukemias. Moreover, ruxolitinib, which preferentially blocks JAK1 and JAK2, abolished the proliferation of cells transformed from the receptor-dependent JAK3V674A, yet proved much less potent on cells expressing JAK3L857P. These Acarbose particular cells were, in contrast, more sensitive to JAK3-specific inhibitors. Completely, our results showed that different JAK3 mutations induce constitutive activation through unique mechanisms, pointing to specific restorative perspectives. at space temperature). This last step was repeated the day after to increase the transfection level. Expression of the markers was analyzed by FACS (BD FACSCaliburTM circulation cytometer), using a phycoerythrin-coupled antibody against hCD4 (catalog no. 555347; BD Pharmingen) diluted 1/30. For murine bone marrow cell transduction, mutated JAK3 pMSCV-GFP viral vectors were produced in HEK293T cells using an EcoPack packaging plasmid and TurboFect transfection reagent (Fermentas). Disease was harvested after 48 h. Dual Luciferase Assay STAT5 transcriptional activity Acarbose was assessed by measurements of luciferase manifestation in HEK293 cells upon transient transfection of appropriate cDNA constructs and pLHRE-luc vector harboring tandem copies of the STAT5-inducible lactogenic hormone response element (LHRE) of the rat -casein promoter, put upstream from a luciferase gene (14). Another reporter plasmid, luciferase (pRLTk; Promega), was co-transfected as an internal transfection control. Transient Acarbose transfection of HEK293 cells by Lipofectamine (Invitrogen) was previously explained (15). 24 h after transfection, luciferase assays were performed using the dual luciferase reporter assay system (Promega). Western Blots For Western blot analysis of JAK3 manifestation in HEK293 cells, 4 105 cells were transfected with 1 g of different JAK3 constructs with Lipofectamine (Invitrogen) Itga2b as previously explained (15). Cell lysate preparation, gel electrophoresis, and transfer to nitrocellulose membranes were performed as previously explained (16). Phosphorylation of signaling proteins was investigated with following phospho-specific antibodies from Cell Signaling Technology: anti-pY1034/1035 JAK1 (catalog no. 3331), anti-pY980/981 JAK3 (catalog no. 5031), anti-pY705 STAT3 (catalog no. 9131), anti-pY694 STAT5 (catalog no. 9351), and anti-pT202/Y204 ERK1/2 (catalog no. 9101). Blots were reprobed with anti-JAK1 (catalog no. 3332), anti-JAK3 (catalog no. 3775), anti-STAT3 (catalog no. 9132) (Cell Signaling Technology, Beverly, MA), anti-STAT5 (catalog no. SC-835; Santa Cruz Biotechnology), or anti–actin (catalog no. A5441; Sigma) antibodies, like a control. Murine Bone Marrow Transplantation Balb/c mice were purchased from Charles River Laboratories. Harvest of bone marrow cells from male donor mice, lineage bad cells enrichment, transduction with viral supernatant, and injection into irradiated female recipient mice was performed as previously explained (13). After sacrifice of the mice, single-cell suspensions were prepared from peripheral blood, bone marrow, spleen, thymus, and lymph nodes. Cells were analyzed on a FACSCanto circulation cytometer (BD Biosciences). Proliferation Assays For knockdown experiments, 1.5 106 IL-3-dependent control Ba/F3 cells or growth factor-independent Ba/F3 cells acquired after transduction with ALL-associated JAK3 mutants (V674A and L857P) and increase mutant (L857P/Y100A) were resuspended in 400 l of medium comprising 200 nm siRNA duplexes (Silencer Select Il2rg mouse, Ambion catalog no. s68269; Stealth siRNA Jak1 mouse, Invitrogen catalog no. mss205625; Silencer Select Bad Control No. 1 siRNA, Ambion catalog no. 4390843; and Stealth RNAiTM siRNA Bad Control, Invitrogen catalog no. 12935C400) and then transferred to 4-mm cuvettes (Bio-Rad). Cells were electroporated (260 V, 90 , 1500 microfarad) and seeded in 96-well plates at 10,000 cells/well. For JAK inhibitors treatment, Ba/F3 cells stably transduced.
