Journal: Annals of hematology

Article Title: A novel germline hyperactivating JAK2 mutation L604F.

doi: 10.1007/s00277-023-05423-y

Figure Lengend Snippet: Fig. 1 The pedigree of the fam- ily with the JAK2 L604F muta- tion. The patient and her sister have the L604F mutation (blue) in a homozygous state, and her parents have heterozygous L604F. The germline L604F probably arises from a common ancestor pair (encircled in blue). The presence of the mutation in the nontested family members is presumed (light blue) as a highly likely way to yield the results obtained in the tested members

Article Snippet: Plasmids with JAK2 wild-type (WT) or JAK2 V617F were constructed by PCR-based techniques of molecular cloning by incorporating JAK2 WT or JAK2-V617F sequences from pDONR223 plasmids containing respective genes [Addgene plasmids # 23,915 and # 81,756 [23, 24]] into plasmid pEGFP-N2 (originally Clontech, Mountain View, CA, USA) designed for exogenous expression of proteins with a green fluorescent protein (eGFP) tag.

Techniques: Mutagenesis

Journal: Annals of hematology

Article Title: A novel germline hyperactivating JAK2 mutation L604F.

doi: 10.1007/s00277-023-05423-y

Figure Lengend Snippet: Fig. 3 Effect of endogenous JAK2 mutations in HeLa cells. Western blot analysis of HeLa cells with V617F or L604F JAK2 mutations introduced by CRISPR. The expected gene modification was confirmed by sequencing. Eight independent harvests were performed for each modified subline and the wild type (WT) parental line. The cell lysates were analyzed in groups – each western-blot membrane contained 4 WT samples and 4 samples from one mutated subline. The measured band intensities were normal- ized to ACTIN and related to the mean value from WT samples included in the given membrane (100%). Top: repre- sentative examples of JAK2 and pJAK2 Tyr1007/1008 signals. ACTIN was used as the loading control. Bottom: means ± SD of the relative band intensities from 8 independent samples for each JAK2 variant. The differences between modified sublines and the WT control were evaluated by unpaired Student´s t test (** p < 0.01, *** p < 0.001)

Article Snippet: Plasmids with JAK2 wild-type (WT) or JAK2 V617F were constructed by PCR-based techniques of molecular cloning by incorporating JAK2 WT or JAK2-V617F sequences from pDONR223 plasmids containing respective genes [Addgene plasmids # 23,915 and # 81,756 [23, 24]] into plasmid pEGFP-N2 (originally Clontech, Mountain View, CA, USA) designed for exogenous expression of proteins with a green fluorescent protein (eGFP) tag.

Techniques: Western Blot, CRISPR, Modification, Sequencing, Membrane, Control, Variant Assay

Journal: Scientific Reports

Article Title: Distinct effects of V617F and exon12-mutated JAK2 expressions on erythropoiesis in a human induced pluripotent stem cell (iPSC)-based model

doi: 10.1038/s41598-021-83895-6

Figure Lengend Snippet: Verification of JAK2 gene mutations and expression in the modified induced pluripotent stem cells (iPSCs). ( A ) Conventional polymerase chain reaction (PCR) using transgene-specific primers showed exogenous JAK2 genes in the two modified iPSC lines. The normal iPSC line was used as a negative control. The full gel is presented in the Supplementary Fig. A. ( B ) DNA sequencing confirmed the point mutation p.V617F in exon 14 and the p.N542_E543del in exon 12 of JAK2 gene in the respective iPSC lines. ( C ) Exogenous JAK2 gene expression levels in iPSCs after transfection comparing normal, JAK2V617F and JAK2 exon 12 mutation with and without doxycycline (DOX) induction for 24 h and analyzed by real-time quantitative RT-PCR. Data are presented as means ± standard deviations (SD) from three independent experiments. The asterisks (*) and (**) denoted p < 0.05 and p < 0.01.

Article Snippet: The details on iPSC characterization of pluripotency were described in the Supplementary Fig. . Firstly, the wild-type JAK2 -containing plasmid (Addgene, Cambridge, MA, USA) was altered using the Site-directed mutagenesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Modification, Polymerase Chain Reaction, Negative Control, DNA Sequencing, Mutagenesis, Gene Expression, Transfection, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Distinct effects of V617F and exon12-mutated JAK2 expressions on erythropoiesis in a human induced pluripotent stem cell (iPSC)-based model

doi: 10.1038/s41598-021-83895-6

Figure Lengend Snippet: Erythroid cell differentiation from modified induced pluripotent stem cells (iPSCs) via ES-Sacs. ( A ) ES-derived sacs containing hematopoietic progenitor cells were generated from modified iPSCs on day 14 at ×100 and ×400 magnifications. ( B ) The percentages of CD34 + cells derived from JAK2V617F-iPSCs and JAK2exon12-iPSCs were similar with or without doxycycline. ( C ) Erythroid cells in a culture plate and red blood cell pellets after centrifugation. ( D ) Expression mRNA levels of exogenous JAK2 in hematopoietic progenitor cells determined by real-time quantitative RT-PCR. ( E ) Expression mRNA levels of exogenous JAK2 in erythroid cells. ( F ) The capillary Western immunoassay showed total JAK2 proteins from JAK2V617F-iPSCs and JAK2exon12-iPSCs at the induced pluripotent stem cell, hematopoietic progenitor cell (HPC) and erythroid cell (RBC) stages in the conditions with vs. without doxycycline. Each band was electrophoresed in a separate capillary tube. The full blot is presented in the Supplementary Fig. A.

Article Snippet: The details on iPSC characterization of pluripotency were described in the Supplementary Fig. . Firstly, the wild-type JAK2 -containing plasmid (Addgene, Cambridge, MA, USA) was altered using the Site-directed mutagenesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Cell Differentiation, Modification, Derivative Assay, Generated, Centrifugation, Expressing, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Distinct effects of V617F and exon12-mutated JAK2 expressions on erythropoiesis in a human induced pluripotent stem cell (iPSC)-based model

doi: 10.1038/s41598-021-83895-6

Figure Lengend Snippet: Characteristics of modified induced pluripotent stem cells (iPSC)-derived erythroid cells. ( A ) Flow cytometry of erythroid-specific surface molecules on iPSC-derived erythroid cells without vs. with doxycycline. Cells were stained with PE-conjugated anti-human CD71 and FITC-conjugated anti-human Glycophorin A (GPA) antibodies. ( B ) Total CD71 + GPA + erythroid cell numbers from erythroid differentiation via ES-Sacs with vs. without doxycycline. ( C ) Five stages of erythroid series were classified by Wright-Giemsa stain consisting of proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts and erythrocytes. The images were captured by Leica DM 1000 microscopy using LAS49 software and the scale bars represented 10 µm for all panels. ( D ) The percentage of erythroid cell differentiation stages. ( E ) Chromatograms from the ion exchange high performance liquid chromatography (HPLC) Bio-Rad Variant II showed mainly embryonic hemoglobin in modified iPSC-derived erythroid cells with and without doxycycline. ( F ) The relative expression of beta-similar globin genes which were epsilon, gamma and beta in iPSC-derived erythroid cells comparing overexpression of JAK2V617F vs. JAK2 exon 12 mutants and analyzed by real-time quantitative RT-PCR. Data were presented as mean ± SD from three independent experiments. The asterisks (**) denoted p < 0.01.

Article Snippet: The details on iPSC characterization of pluripotency were described in the Supplementary Fig. . Firstly, the wild-type JAK2 -containing plasmid (Addgene, Cambridge, MA, USA) was altered using the Site-directed mutagenesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Modification, Derivative Assay, Flow Cytometry, Staining, Giemsa Stain, Microscopy, Software, Cell Differentiation, High Performance Liquid Chromatography, Variant Assay, Expressing, Over Expression, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Distinct effects of V617F and exon12-mutated JAK2 expressions on erythropoiesis in a human induced pluripotent stem cell (iPSC)-based model

doi: 10.1038/s41598-021-83895-6

Figure Lengend Snippet: Signal transduction and drug treatment of modified induced pluripotent stem cells (iPSCs). ( A ) The capillary Western immunoassay of phosphorylated and total signaling proteins which were JAK2, STAT1, STAT3, STAT5, ERK1/2 and AKT in the absent and presence of doxycycline in JAK2V617F-iPSCs and JAK2exon12-iPSCs. Each band was electrophoresed in a separate capillary tube. The full blot is presented in the Supplementary Fig. B. ( B ) The relative changes of phosphorylated signaling proteins after doxycycline induction in JAK2V617F-iPSCs and JAK2exon12-iPSCs compared with those without doxycycline. The levels of phosphoproteins were corrected by the amounts of respective total proteins. ( C ) Erythroid cell numbers after incubations without (untreated) vs. with arsenic trioxide, interferon alpha and the combination of both drugs. ( D ) The relative increases in numbers of apoptotic cells in modified iPSCs in the presence of arsenic trioxide, interferon alpha and the combination of both drugs. Data were presented as mean ± SD from three independent experiments. The asterisks (*), (**) and (***) denoted p < 0.05, p < 0.01 and p < 0.001, respectively.

Article Snippet: The details on iPSC characterization of pluripotency were described in the Supplementary Fig. . Firstly, the wild-type JAK2 -containing plasmid (Addgene, Cambridge, MA, USA) was altered using the Site-directed mutagenesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Transduction, Modification, Western Blot

Journal: Scientific Reports

Article Title: Distinct effects of V617F and exon12-mutated JAK2 expressions on erythropoiesis in a human induced pluripotent stem cell (iPSC)-based model

doi: 10.1038/s41598-021-83895-6

Figure Lengend Snippet: The primer sets for exogenous JAK2 , pluripotency testing of stem cells and hemoglobin expression.

Article Snippet: The details on iPSC characterization of pluripotency were described in the Supplementary Fig. . Firstly, the wild-type JAK2 -containing plasmid (Addgene, Cambridge, MA, USA) was altered using the Site-directed mutagenesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing

Journal: Experimental Hematology & Oncology

Article Title: The SMAC mimetic LCL-161 selectively targets JAK2 V617F mutant cells

doi: 10.1186/s40164-019-0157-6

Figure Lengend Snippet: JAK2 V617F mutant cells lines are more sensitive to killing by LCL-161 under certain circumstances. a – c . L929 cells expressing JAK2 WT , JAK2 V617F or empty vector were incubated with increasing concentrations of a LCL-161 alone, b LCL-161 with the addition of 0.25 ng/ml mTNFα, c LCL-161 with the addition of 400 ng/ml mTNFa neutralizing antibody for 48 h and then analyzed with a resazurin based viability assay. **** P < 0.0001, 2way ANOVA. d Western blot of L929 cell lines harvested 24 h after exposure to combinations of LCL-161 and mTNFα. e – g HEL and K562 cells were incubated with e LCL-161 alone, f LCL-161 + 1 ng/ml hTNFα, or g LCL-161 + 10 ng/ml hTNFα for 48 h. Apoptosis was measured with Annexin V and PI staining. ** P < 0.01, **** P < 0.0001 unpaired t test

Article Snippet: Here, we found that mouse cell lines ectopically expressing JAK2 V617F , the JAK2 V617F mutated human cell line HEL, JAK2 V617F knock-in mice, and primary MPN samples were more sensitive to killing by the SMAC mimetic LCL-161 compared to their JAK2 WT counterparts.

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Incubation, Viability Assay, Western Blot, Staining

Journal: Experimental Hematology & Oncology

Article Title: The SMAC mimetic LCL-161 selectively targets JAK2 V617F mutant cells

doi: 10.1186/s40164-019-0157-6

Figure Lengend Snippet: JAK inhibitors rescue hypersensitivity of JAK2 V617F mutant cells to LCL-161. L929 cells expressing JAK2 WT , JAK2 V617F or empty vector were incubated with increasing concentrations of a LCL-161 alone, b LCL-161 with the addition of 1 µM ruxolitinib, or c LCL-161 with the addition of 1 µM pacritinib. After 48 h in culture a resazurin-based cell viability assay was performed. **** P < 0.0001, 2way ANOVA

Article Snippet: Here, we found that mouse cell lines ectopically expressing JAK2 V617F , the JAK2 V617F mutated human cell line HEL, JAK2 V617F knock-in mice, and primary MPN samples were more sensitive to killing by the SMAC mimetic LCL-161 compared to their JAK2 WT counterparts.

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Incubation, Viability Assay

Journal: Experimental Hematology & Oncology

Article Title: The SMAC mimetic LCL-161 selectively targets JAK2 V617F mutant cells

doi: 10.1186/s40164-019-0157-6

Figure Lengend Snippet: LCL-161 preferentially decreases MPN colony formation. Methylcellulose colony formation of a , b whole bone marrow from JAK2 V617F or wild-type mice and c , d peripheral blood mononuclear cells from JAK2 V617F mutated MPN patients or normal controls with increasing concentrations of LCL-161 alone ( a , c ) or LCL-161 + 0.25 ng/ml TNFα ( b , d ). * P < 0.05, *** P < 0.001, unpaired t test

Article Snippet: Here, we found that mouse cell lines ectopically expressing JAK2 V617F , the JAK2 V617F mutated human cell line HEL, JAK2 V617F knock-in mice, and primary MPN samples were more sensitive to killing by the SMAC mimetic LCL-161 compared to their JAK2 WT counterparts.

Techniques:

Journal: Experimental Hematology & Oncology

Article Title: The SMAC mimetic LCL-161 selectively targets JAK2 V617F mutant cells

doi: 10.1186/s40164-019-0157-6

Figure Lengend Snippet: Impact of LCL-161 treatment in a transduction–transplantation model of JAK2V617F mutated MPN. a Percentage of GFP+ (empty vector or JAK2 V617F ) cells in peripheral blood. b White blood cell (WBC), c hematocrit (HCT), d platelet (PLT) and e hemoglobin (HGB) counts in wild-type (empty vector) or JAK2 V617F mice treated with LCL-161. f Spleen weights in JAK2 V617F mice, P < 0.05, unpaired t test. g Percentage of GFP+ (JAK2 V617F ) cells in the spleen and bone marrow. h Snook’s reticulin staining of paraffin-embedded bone marrows in wildtype (empty vector) and JAK2 V617F mice treated with LCL-161. n = 3–4 mice per group

Article Snippet: Here, we found that mouse cell lines ectopically expressing JAK2 V617F , the JAK2 V617F mutated human cell line HEL, JAK2 V617F knock-in mice, and primary MPN samples were more sensitive to killing by the SMAC mimetic LCL-161 compared to their JAK2 WT counterparts.

Techniques: Transduction, Transplantation Assay, Plasmid Preparation, Staining