Journal: HemaSphere

Article Title: AXL Inhibition Extinguishes Primitive JAK2 Mutated Myeloproliferative Neoplasm Progenitor Cells

doi: 10.1097/hs9.0000000000000233

Figure Lengend Snippet: Figure 2. GAS6 levels in MPN; A, Levels of GAS6 were measured in plasma of normal and JAK2 V617F patients by ELISA and the results expressed as ng/ml of plasma (mean±SEM, n=6). RNA expression levels in CD34+ cells were measured using Illumina HiSeq NGS and data shown as the number of normalized RNA reads observed. Results for housekeeping genes (B) and GAS6 (C) are shown (mean±SEM, n=3 normals, n=7 for JAK2 V617F). D, CD34+ cells isolated from MPN patients and healthy controls were incubated with 20mM DCFDA to assess ROS levels. Amalgamated flow cytometry data is displayed as median fluorescent intensity±SEM (n=3 normals, n=7 for JAK2 V617F). The results of a t test are shown, ns indicates not significant.

Article Snippet: Levels of GAS6 were measured using the Human GAS6 Duoset ELISA from R&D Systems (DY885B) as per manufactures instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, RNA Expression, Isolation, Incubation, Cytometry

Journal: HemaSphere

Article Title: AXL Inhibition Represents a Novel Therapeutic Approach in BCR-ABL Negative Myeloproliferative Neoplasms

doi: 10.1097/hs9.0000000000000630

Figure Lengend Snippet: Figure 1. AXL represents a target in MPN patients. Western blot analysis of phosphorylated and total AXL expression was performed in BMMNCs from untreated MPN patients compared with healthy donor samples (n = 6/3). β-Tubulin was used as loading control. Densitometric quantification of (protein expres- sion/β-Tubulin expression) was normalized to healthy samples and represented as percentage (A). GAS6 protein levels were measured by ELISA in BM plasma from MPN patients (n = 11) compared with healthy controls (n = 22) (B) and in PB plasma from MPN patients (n = 30) and healthy controls (n = 14) (C). Subgroup analysis of GAS6 levels in PB plasma from MF patients (n = 13) compared with healthy donors was performed (D). Human peripheral blood mononuclear cells were cultured in semisolid medium with bemcentinib (bem, 1 µM) and number of CFUs was counted after 14 d. Percentage of colony number was normalized to control treatment of the same healthy donor or patient. Quantification of colonies BFU-E, CFU-GEMM, and CFU-GM is shown. The mean number of colo- nies counted in all control-treated samples was 28 ± 5 (mean ± SEM) (E). Representative pictures of BFU-E from patient and healthy control, bar represents 200 µm (F) (*P < 0.05, ***P < 0.001, ns not significant). BFU-E = burst forming unit-erythroid; BM = bone marrow; BMMNC = bone marrow mononuclear cell; CFU = colony-forming unit; CFU-GEMM = colony forming unit - granulocyte, erythrocyte, monocyte, megakaryocyte; CFU-GM = colony forming unit – granulocyte, monocyte; GAS6 = growth arrest-specific gene 6; MF = myelofibrosis; MPN = myeloproliferative neoplasm; ns = not significant; pAXL = phosphorylated AXL; PB = peripheral blood.

Article Snippet: Human GAS6 levels were determined in PB and BM plasma using enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions (R&D Systems).

Techniques: Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Cell Culture