Journal: Oncogene

Article Title: EGFR-Stat3 signalling in nerve glial cells modifies neurofibroma initiation

doi: 10.1038/onc.2016.386

Figure Lengend Snippet: (a) Western blot of mouse sciatic nerves that are WT, EGFR-overexpressing (EGFR/EGFR), or with diminished levels of Egfr activity (Wa2), showing that Stat3 activity is increased in EGFR/EGFR nerves. (b) Western blot of mouse neurofibroma-derived EGFR+ progenitor-like sphere cells OSI-774. Inhibition of EGFR function inhibits Stat3-Y705 phosphorylation (P-Stat3). Total Stat3 (Stat3) was used as loading control. Statistics B, C, E: unpaired t test, two-tailed. (c–d) Immunofluorescent staining showing that P-Stat3 is detected in Nf1fl/fl;DhhCre mouse neurofibromas (C, CTRL) and decreased in Nf1fl/fl;DhhCre,Wa2 neurofibroma (D, Wa2). Bar = 20 µm. (e) Quantification of P-Stat3+ cells in Nf1fl/fl;DhhCre (white bar) and Nf1fl/fl;DhhCre,Wa2 (black bar). (f) Western blot of neurofibroma lysates from Nf1fl/fl;DhhCre and Nf1fl/fl;DhhCre;Wa2 mice. (g) Western blot of lysates from Nf1fl/fl;DhhCre mouse neurofibromas treated with AZD1480 or vehicle for 5 days. (f and g) Stat3 and anti-β-actin serve as loading controls. Antibodies: P-Stat3, Cell Signaling, #9145; Stat3, Cell Signaling, #4904; EGFR, Santa Cruz, #SC-03; P-EGFR, Santa Cruz, #SC-12351; P-Jak2, Cell Signaling, # 3776; Jak2, Cell Signaling, #3230; β-actin, Cell Signaling, #5125. ***P <0.001.

Article Snippet: Antibodies: P-Stat3, Cell Signaling, #9145; Stat3, Cell Signaling, #4904; EGFR, Santa Cruz, #SC-03; P-EGFR, Santa Cruz, #SC-12351; P-Jak2, Cell Signaling, # 3776; Jak2, Cell Signaling, #3230; β-actin, Cell Signaling, #5125.

Techniques: Western Blot, Activity Assay, Derivative Assay, Inhibition, Two Tailed Test, Staining

Journal: Oncogene

Article Title: EGFR-Stat3 signalling in nerve glial cells modifies neurofibroma initiation

doi: 10.1038/onc.2016.386

Figure Lengend Snippet: (a) A representative western blot of WT mouse sciatic nerve and Nf1fl/fl;DhhCre mouse neurofibroma lysates showing expression of II6 protein (II-6, Abcam, Cambridge, MA, USA; #AB6672). Anti-β-actin served as a loading control. (b) II-6 quantification on OSI-774, FLLL32, and DMSO treated medium conditioned by mouse neurofibroma spheres. Sphere medium without treatment was also used as additional control (n = 3 for each group). Unpaired t test, two-tailed was used. (c) Mouse neurofibroma sphere counts on II-6 antibody (II-6 Ab), OSI-774, II-6 Ab+OSI-774, IgG+DMSO treated mouse neurofibroma spheres. (d) Western blot of P-Jak2, Jak2, P-Stat3 on II-6 antibody (II-6 Ab), OSI-774, II-6 Ab+OSI-774, IgG+DMSO treated mouse neurofibroma spheres. Anti-Stat3 and anti-β-actin serve as controls. (e) Sphere counts show that two shII-6 shRNAs (#1 and #2) each significantly decrease mouse neurofibroma sphere formation, compared to non-target lentivirus yellow fluorescent protein control. (f) Western blot showing knockdown of II-6 in Nf1fl/fl;DhhCre mouse neurofibroma spheres, 4 days after sh II-6 infection using two different shRNA clones (#1, #2). Numbers below indicate the relative band intensity (50% and 40%), normalized to β-actin for each sample. (g) Inhibitory effects of the JAK1/2 inhibitor AZD1480 on Nf1fl/fl;DhhCre, and Nf1fl/fl;DhhCre;EGFR but not Nf1fl/fl;DhhCre;Wa2 mouse neurofibroma spheres. DMSO was used as control. The JAK3 inhibitor CP 690550 did not affect sphere number at concentrations below 1 µM. Three independent experiments were performed, and data are represented as mean ± s.e.m. Statistics: B, one-way ANOVA; E, unpaired t test, two-tailed. *P <0.05, **P <0.01, ***P <0.001.

Article Snippet: Antibodies: P-Stat3, Cell Signaling, #9145; Stat3, Cell Signaling, #4904; EGFR, Santa Cruz, #SC-03; P-EGFR, Santa Cruz, #SC-12351; P-Jak2, Cell Signaling, # 3776; Jak2, Cell Signaling, #3230; β-actin, Cell Signaling, #5125.

Techniques: Western Blot, Expressing, Two Tailed Test, Infection, shRNA, Clone Assay

Journal: Molecular Oncology

Article Title: A newly identified 45‐kDa JAK2 variant with an altered kinase domain structure represents a novel mode of JAK2 kinase inhibitor resistance

doi: 10.1002/1878-0261.13566

Figure Lengend Snippet: Ruxolitinib‐resistant Ba/F3 cell clones harbor a truncated JAK2 variant. (A) Immunoblot analysis of STAT5, AKT, and ERK signaling in 4 μ m ruxolitinib‐resistant Ba/F3 cell clones R1–R5 ( n = 5 different resistant clones). Par‐parental Ba/F3 cells. (B) Immunoblot analysis of resistant clones using a JAK2 antibody recognizing amino acids (aa) 841–845 shows presence of a 45 kDA form ( n = 5 different resistant clones). (C) Immunoblot analysis of resistant clones using a FLAG antibody detecting the n‐terminal sequence next to JAK2‐V617F cDNA ( n = 5 different resistant clones). (D) An antibody raised against aa 750–757 fails to identify the 45 kDa JAK2 variant in drug‐resistant clones in an immunoblot ( n = 5 different resistant clones). A representative image of n = 2 two independent experiments is shown (A, B, C and D). (E) PCR analysis of the JAK2 cDNA isolated from 4 μ m ruxolitinib‐resistant clones ( n = 10 different resistant clones). (F) Schematic representation of sequencing results that revealed an in‐frame deletion of aa 77 to aa 814.

Article Snippet: JAK2 c‐terminal antibody (D2E12 XP R ), pSTAT5, pAKT, pERK, ERK, AKT were purchased from Cell Signaling Technologies (Leiden, The Netherlands).

Techniques: Clone Assay, Variant Assay, Western Blot, Sequencing, Isolation

Journal: Molecular Oncology

Article Title: A newly identified 45‐kDa JAK2 variant with an altered kinase domain structure represents a novel mode of JAK2 kinase inhibitor resistance

doi: 10.1002/1878-0261.13566

Figure Lengend Snippet: FERM‐JAK2 transforms Ba/F3 cells and activates STAT5 via a non‐canonical pathway. (A) Left panel: Proliferation of parental Ba/F3 cells and Ba/F3 cells expressing FERM‐JAK2 or JAK2‐V617F in the absence of IL‐3 was quantified by the relative optical density (OD) after 96 h using an MTS (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide)‐based assay. Right panel: Absolute cell numbers over time were measured in the absence of IL‐3 by trypan blue exclusion ( n = 3). *** P < 0.001 compared to parental cells by Student's t test. Data are shown as mean ± standard deviation (SD). (B) Immunoblot analysis of serum‐starved Ba/F3 cells expressing FERM‐JAK2 or JAK2‐V617F. A representative image of n = 2 two independent experiments is shown. (C) IL‐3Rβ immunoprecipitation (IP) analysis of Ba/F3 cells expressing FERM‐JAK2 or JAK2‐V617F. pY, phosphotyrosine; WCL, whole cell lysate. A representative image of n = 3 three independent experiments is shown. (D) Immunoblot analysis of Gamma2A cells stably expressing mock vector, FERM‐JAK2 or JAK2‐V617F in combination with or without IL‐3Rβ chain. A representative image of n = 3 three independent experiments is shown.

Article Snippet: JAK2 c‐terminal antibody (D2E12 XP R ), pSTAT5, pAKT, pERK, ERK, AKT were purchased from Cell Signaling Technologies (Leiden, The Netherlands).

Techniques: Expressing, Standard Deviation, Western Blot, Immunoprecipitation, Stable Transfection, Plasmid Preparation

Journal: Molecular Oncology

Article Title: A newly identified 45‐kDa JAK2 variant with an altered kinase domain structure represents a novel mode of JAK2 kinase inhibitor resistance

doi: 10.1002/1878-0261.13566

Figure Lengend Snippet: FERM‐JAK2 directly binds STAT5 via constitutive dimerization. (A) FLAG immunoprecipitation (IP) analysis of Ba/F3 cells expressing FLAG‐FERM‐JAK2 or FLAG‐JAK2‐V617F. A representative image of n = 3 three independent experiments is shown. (B) Immunoblot analysis of in vitro translated FERM‐JAK2 or JAK2‐V617F. A representative image of n = 3 three independent experiments is shown. (C) Immunoblot analysis of in vitro translated FERM‐JAK2 or JAK2‐V617F incubated with purified STAT5 after washing. A representative image of n = 3 three independent experiments is shown. (D) Myc IP analysis of HEK‐293T cells co‐expressing FLAG‐tagged and Myc‐tagged FERM‐JAK2 or JAK2‐V617F. WCL, whole cell lysate. A representative image of n = 3 three independent experiments is shown.

Article Snippet: JAK2 c‐terminal antibody (D2E12 XP R ), pSTAT5, pAKT, pERK, ERK, AKT were purchased from Cell Signaling Technologies (Leiden, The Netherlands).

Techniques: Immunoprecipitation, Expressing, Western Blot, In Vitro, Incubation, Purification

Journal: Molecular Oncology

Article Title: A newly identified 45‐kDa JAK2 variant with an altered kinase domain structure represents a novel mode of JAK2 kinase inhibitor resistance

doi: 10.1002/1878-0261.13566

Figure Lengend Snippet: FERM‐JAK2 confers resistance to JAK2‐ATP competitive inhibitors. (A) MTS (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide)‐based cell proliferation analysis of Ba/F3 cells expressing JAK2 mutants cultured with indicated concentration of ruxolitinib for 48 h. OD, optical density. Data are shown as mean ± standard deviation (SD) ( n = 3). (B) Immunoblot analysis of Ba/F3 cells expressing JAK2 mutants cultured with indicated concentration of ruxolitinib for 48 h. A representative image of n = 2 two independent experiments is shown. (C) MTS‐based cell proliferation analysis of Ba/F3 cells expressing JAK2 mutants cultured with indicated concentration of TG101348 (TG) for 48 h. Data are shown as mean ± standard deviation (SD) ( n = 3). OD, optical density. (D) Immunoblot analysis of Ba/F3 cells expressing JAK2 mutants cultured with indicated concentration of TG101348 (TG) for 48 h. A representative image of n = 2 two independent experiments is shown.

Article Snippet: JAK2 c‐terminal antibody (D2E12 XP R ), pSTAT5, pAKT, pERK, ERK, AKT were purchased from Cell Signaling Technologies (Leiden, The Netherlands).

Techniques: Expressing, Cell Culture, Concentration Assay, Standard Deviation, Western Blot

Journal: Molecular Oncology

Article Title: A newly identified 45‐kDa JAK2 variant with an altered kinase domain structure represents a novel mode of JAK2 kinase inhibitor resistance

doi: 10.1002/1878-0261.13566

Figure Lengend Snippet: Activation loop phosphorylation is dispensable for FERM‐JAK2 activation. (A) MTS (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide)‐based cell proliferation analysis of Ba/F3 cells expressing FERM‐JAK2 or JAK2‐V617F plus activation loop mutations Y1007F and/or Y1008F. A representative result ( n = 2) from three independent experiments is shown. Data are shown as mean ± standard deviation (SD). OD, optical density. (B) Immunoblot analysis of serum starved Ba/F3 cells expressing FERM‐JAK2 or JAK2‐V617F phospho‐deficient mutants Y1007F and/or Y1008F. A representative image of n = 2 two independent experiments is shown.

Article Snippet: JAK2 c‐terminal antibody (D2E12 XP R ), pSTAT5, pAKT, pERK, ERK, AKT were purchased from Cell Signaling Technologies (Leiden, The Netherlands).

Techniques: Activation Assay, Phospho-proteomics, Expressing, Standard Deviation, Western Blot

Journal: Molecular Oncology

Article Title: A newly identified 45‐kDa JAK2 variant with an altered kinase domain structure represents a novel mode of JAK2 kinase inhibitor resistance

doi: 10.1002/1878-0261.13566

Figure Lengend Snippet: FERM‐JAK2 + mice succumb to an accelerated MPN with myelofibrosis. Two independent experiments were analyzed, including a total of n = 17 mice receiving bone marrow (BM) transduced with FERM‐JAK2, n = 18 JAK2‐V617F + bone marrow (BM) and n = 10 empty vector control bone marrow (BM) cells. (A) Kaplan–Meier survival plot of recipient mice, FERM‐JAK2 mice display accelerated disease ( n = 5 for all groups). ** P < 0.001 by Logrank test. Tx, transplantation. (B) FERM‐JAK2 mice show a significant decrease of total body weight 60 days after transplantation compared to MiG (MSCV‐ires‐GFP) empty vector control mice ( n = 5). *** P < 0.001, n.s., not significant, both compared to MiG (MSCV‐ires‐GFP) by Student's t test. Data are shown as mean ± standard deviation (SD). (C, D) Flow cytometric analysis of (C) splenocytes and (D) BM cells taken 60 days after transplantation. Values represent Mean ± SEM of the transplanted animals. *** P < 0.001 compared to MiG by Student's t test. (E) Histopathologic analysis (hematoxylin and eosin staining, ×400) revealed hyperplastic, left‐shifted myelopoiesis granulopoiesis, erythropoiesis, and moderate increased megakaryopoiesis. Scale bar represents 15 μ m . Notably, infiltrates are very dense in FERM‐JAK2 mice compared to JAK2‐V617F mice. (F) Hematoxylin/eosin and reticulin staining of representative tissue samples 60 days after transplantation (400×). BM obtained from FERM‐JAK2 mice shows left‐shifted increase of myeloid cells and a marked presence of collagen fibers, similar to the increase of reticulin fibers in human myeloproliferative disorders. Slides were viewed with a Zeiss Axioplan 2 microscope (Göttingen, Germany) (40×/0.75 NA Plan‐Neofluar air objective). Scale bar represents 15 μ m . Images were acquired using a Zeiss Axiocam MRc 5 camera and were processed with axiovision rel 4.6 scanning software (CarlZeissMicroscopy GmBH, Jena, Germany).

Article Snippet: JAK2 c‐terminal antibody (D2E12 XP R ), pSTAT5, pAKT, pERK, ERK, AKT were purchased from Cell Signaling Technologies (Leiden, The Netherlands).

Techniques: Transduction, Plasmid Preparation, Control, Transplantation Assay, Standard Deviation, Staining, Microscopy, Software

Journal: Experimental hematology

Article Title: Phosphorylated c-MPL tyrosine 591 regulates thrombopoietin-induced signaling

doi: 10.1016/j.exphem.2014.02.007

Figure Lengend Snippet: (A) Top: Western blot analysis of phosphorylation levels of JAK2, STAT5, Akt and ERK1/2 in Ba/F3-MPL wild-type and YSF cells stimulated with 10ng/mL rhTPO for 0, 15, 60 or 120 minutes. Bottom: densitometry of phospho-ERK1/2 blot (*P < 0.05; ***P < 0.001). Error bars represent ±SEM. (n=3). (B) Top: Western blot analysis of phosphorylation levels of JAK2, STAT5, Akt and ERK1/2 in Ba/F3-MPL wild-type and Y591F cells stimulated for 5 minutes with 0, 0.1, 1 or 10ng/mL rhTPO. Bottom: densitometry of phospho-ERK1/2 blot. Error bars represent ±SEM. (n=3). (C) Top: Detection of active, GTP-bound Ras in Ba/F3-MPL wild-type and Y591F cells. Bottom: Densitometry of GTP-bound Ras blot (*P < 0.05). Error bars represent ±SEM. (n=3). Densitometry was performed using ImageJ

Article Snippet: Phosphospecific and total JAK2 (phospho-JAK2 clone C80C3 and total JAK2 clone D2E12), STAT5 (phospho-STAT5 clone D47E7), Akt (phospho-AKT clone D9E), ERK1/2 (phosphor-ERK1/2 clone D13.14.43 and total ERK clone 137F5), SYK (phospho-SYK Y323, phospho-SYK Y352 and phospho-SYK Y525/526 clone C87C1 and total SYK clone D1I5Q) and SHP-1 (phospho-SHP-1 clone D11G5 and SHP-1 clone C14H6) in addition to total BTK antibodies were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Western Blot