Journal: Oncogene

Article Title: An activating mutation in the transmembrane domain of the granulocyte colony-stimulating factor receptor in patients with acute myeloid leukemia.

doi: 10.1038/sj.onc.1205767

Figure Lengend Snippet: Figure 5 Tyrosine phosphorylation of (a) anti-JAK2 and (b) anti-G-CSFR immunoprecipitates from Ba/F3 transfectants sti- mulated with or without 100 ng/ml G-CSF for 3 min or 10 ng/ ml mIL-3 for 10 min. Lysates were immunoprecipitated with anti-JAK2 or anti-G-CSFR antibodies; blots were probed with the phospho-tyrosine specific antibody PY99, stripped and re- probed with antibody against JAK2 or G-CSFR

Article Snippet: Western blots were probed with PY99, a phosphotyrosine specific antibody (Santa Cruz Biotechnology), then stripped and re-probed with antibody against JAK2 or the receptor.

Techniques: Phospho-proteomics, Immunoprecipitation

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: Hypoxia‐induced JAK2/STAT3 activation in a PAH mouse model and HPASMCs. Representative results for coimmunostaining of p‐JAK2 and α‐SMA (A), p‐STAT3 and α‐SMA (B) in lung sections from WT mice exposed to normoxia or hypoxia (n = 3 per group). Confocal immunofluorescence images for coimmunostaining of p‐JAK2 and p‐STAT3 in HPASMCs following normoxic or hypoxic exposure (C). All images were taken at an original magnification of ×400. The data are represented as the mean ± SEM. *** P < .001

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques: Activation Assay, Immunofluorescence

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: Generation of SMC‐specific Jak2 ‐knockout mice. Schematic diagram of transgenic mice used to generate Jak2‐CKO and Jak2‐C mice (A). PCR analysis of tail genomic DNA to determine the presence of the floxed null allele (B). Western blot analysis to confirm Jak2 depletion in pulmonary arteries (C; n = 3 per group) and cardiac muscle (D; n = 3 per group). Immunohistochemistry analysis to confirm Jak2 depletion in PASMCs (E; n = 3 per group). Coimmunostaining results of p‐STAT3 and α‐SMA in lung sections from Jak2‐C and Jak2‐CKO mice (F). All images were taken at an original magnification of ×400. The data are represented as the mean ± SEM. *** P < .001

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques: Knock-Out, Transgenic Assay, Western Blot, Immunohistochemistry

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: SMC‐specific Jak2 deficiency improved the reaction of pulmonary blood vessels to hypoxic condition. RVSP (A), RV/(LV + S) ratio (B), PAT/PT ratio (C), mean systemic arterial BP (D), ejection fraction (E), fractional shortening (F) and body weight change ratio (G) in Jak2‐C and Jak2‐CKO mice after exposure to normoxic (n = 8 per group) or hypoxic (n = 10 per group) conditions for 28 days. The data are represented as the mean ± SEM. ** P < .01. RVSP, right ventricular systolic pressure; RV/ (LV+S), the right ventricle/left ventricle plus septum; PAT/PT, pulmonary acceleration time/pulmonary ejection time

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques:

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: Loss of Jak2 in smooth muscle cells protected against pulmonary vascular remodelling after hypoxia. Representative HE‐stained (top) and EVG‐stained (bottom) sections (A), quantification of pulmonary arteriole wall thickness (B) and α‐SMA immunostaining (C) in the lungs of Jak2‐C and Jak2‐CKO mice after normoxic (n = 8 per group) or hypoxic (n = 10 per group) exposure for 28 days. Ten vessels were analysed per mouse. Coimmunostaining results of α‐SMA and Ki67 in lung sections from Jak2‐C and Jak2‐CKO mice after hypoxic (D; n = 10 per group) exposure for 28 days. All images were taken at an original magnification of ×400. The data are represented as the mean ± SEM. * P < .05; ** P < .01. HE, haematoxylin and eosin; EVG, elastic van gieson; PA, pulmonary artery

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques: Staining, Immunostaining

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: Hypoxia‐induced HPASMC proliferation was suppressed by a JAK2 inhibitor. Western blot analysis of p‐JAK2, JAK2, p‐STAT3 and STAT3 in HPASMCs (A). CCK‐8 analysis of HPASMCs pre‐treated with different concentrations of TG for 1 h following 24 h hypoxic exposure (B). CFSE dilution analysis (C), EdU staining (D) and cell cycle analysis (E) of HPASMCs pre‐treated with DMSO or TG for 1 h following 24 h hypoxic exposure. All images were taken at an original magnification of ×400. The data are represented as the mean ± SEM. * P < .05; ** P < .01; *** P < .001. TG, TG‐101348; CFSE, carboxyfluorescein diacetate succinimidyl ester; EdU, 5‐ethynyl‐uridine; DMSO, dimethyl sulphoxide

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques: Western Blot, CCK-8 Assay, Staining, Cell Cycle Assay

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: JAK2/STAT3 promoted HPASMC proliferation programming by enhancing cyclin A2 expression following hypoxic exposure. Real‐time PCR to determine cyclin A2 (A), cyclin D1 (B), cyclin E1 (C), CDK2 (D) and CDK4 (E) expression in HPASMCs. Western blot analysis of cyclin A2 expression in HPASMCs (F). ChIP‐PCR results for analysis of p‐STAT3 binding activity to the CNNA2 promoter (G). Results for CNNA2 promoter luciferase reporter assays in HPASMCs (H). Coimmunostaining results of α‐SMA and cyclin A2 in lung sections from Jak2‐C and Jak2‐CKO mice after hypoxic (I; n = 10 per group) exposure for 28 days. All images were taken at an original magnification of ×400. The data are represented as the mean ± SEM. * P < .05; ** P < .01; *** P < .001. CDK2, cyclin‐dependent kinase 2; CDK4, cyclin‐dependent kinase 4

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay, Activity Assay, Luciferase

Journal: Cell Proliferation

Article Title: Blockade of JAK2 protects mice against hypoxia‐induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation

doi: 10.1111/cpr.12742

Figure Lengend Snippet: Diagram of the mechanisms underlying JAK2 regulation of PAH. Hypoxia induced PASMC proliferation through promoting the JAK2/STAT3/cyclin A2 pathway, in which STAT3 bound directly to the CCNA2 promoter and transcriptionally activated cyclin A2 under hypoxic condition, ultimately leading to increased pulmonary arterial remodelling in PAH

Article Snippet: Antibodies against JAK2 (Cat. No. #3230s), STAT3 (Cat. No. #79D7), phospho‐STAT3 (Tyr705) (Cat. No. #9145s) and phospho‐STAT3 (Tyr705) (Cat. No. #4113s) were purchased from the Cell Signaling Technology.

Techniques:

Journal: Molecular Cancer

Article Title: GM-CSF mediates immune evasion via upregulation of PD-L1 expression in extranodal natural killer/T cell lymphoma

doi: 10.1186/s12943-021-01374-y

Figure Lengend Snippet: Correlations among p-JAK2, p-STAT5, and PD-L1 expression in ENKTL and PTCL tissues. a The representative immunohistochemical stainings of p-JAK2, p-STAT5, and PD-L1 in ENKTL tissues were shown. b Percent distribution of GM-CSF treatments for mature T-cell lymphoma. c Linear regression analysis of p-JAK2 and p-STAT5 immunohistochemical scores in ENKTL tissue microarray; P = 0.009, r = 0.416. d Linear regression analysis of p-STAT5 and PD-L1 immunohistochemical scores in ENKTL tissue microarray; P = 0.037, r = 0.398. e Immunohistochemical scores of p-JAK2 in patients with short-term progression after GM-CSF treatment or not. f Kaplan-Meier plots for progression-free survival analysis by the optimal cutoff value of p-JAK2 immunohistochemical scores. Samples were grouped as p-STAT5 high (H-score > 4.5), p-STAT5 low (H-score < 4.5). g Kaplan-Meier plots for overall survival analysis by the optimal cutoff value of PD-L1 immunohistochemical scores. Samples were grouped as PD-L1 high (H-score > 5.0), PD-L1 low (H-score < 5.0)

Article Snippet: The antibody against Phospho-Jak2 (Tyr1007/1008), JAK2, Phospho-Stat5 (Tyr694), STAT5, PD-L1 were purchased from Cell Signaling Technology (USA).

Techniques: Expressing, Immunohistochemical staining, Microarray

Journal: Molecular Cancer

Article Title: GM-CSF mediates immune evasion via upregulation of PD-L1 expression in extranodal natural killer/T cell lymphoma

doi: 10.1186/s12943-021-01374-y

Figure Lengend Snippet: GM-CSF promotes immunosuppression in vivo. a The representative immunohistochemical staining of CD3, CD8, and granzyme B in EL4 tumor tissues of C57BL/6 mice was shown. b The representative immunohistochemical staining of CD3, CD8, and granzyme B in B16-F10 tumor tissues of C57BL/6 mice were shown. C Immunohistochemistry staining of CD3 in tumor tissues of C57BL/6 mouse-bearing EL4 tumors with different treatments (n = 8). d Immunohistochemistry staining of CD8 in tumor tissues of C57BL/6 mouse-bearing EL4 tumors with different treatments (n = 8). e Immunohistochemistry staining of granzyme B in xenograft tumor tissues of C57BL/6 mouse-bearing EL4 tumors with different treatments (n = 8). f Immunohistochemistry staining of CD3 in tumor tissues of C57BL/6 mice bearing B16-F10 tumors with different treatments (n = 6). g Immunohistochemistry staining of CD8 in tumor tissues of C57BL/6 mice bearing B16-F10 tumors with different treatments (n = 6). h Immunohistochemistry staining of granzyme B in xenograft tumor tissues of C57BL/6 mice bearing B16-F10 tumors with different treatments (n = 8). The staining scores were presented as means ± S. D in the scatter plot. * P < 0.05, ** P < 0.01, *** P < 0.001. i Representative contour plots showing the general gating strategy used to identify the purified CD8 T cells (CD45 + CD3 + CD8+) from EL-4 tumors of C57BL/6 with different treatment (n = 6). j The percentage of Ki-67 + GzmB+ CD8 T cells of EL-4 tumors of C57BL/6 mouse with different treatment (n = 6). k Scatter plots that represent the percentage of Ki-67 + GzmB+ CD8 T cells of EL-4 tumors of C57BL/6 mouse with different treatment (n = 6). l EL-4 and B16-F10 cells were treated with GM-CSF (10 ng/ml, 100 ng/ml, and 500 ng/ml) for 12 h, p-JAK2, JAK2, p-STAT5, STAT5, and PD-L1 protein expression was measured by Western blot. m Relative protein expression levels of PD-L1 were increased by GM-CSF (10 ng/ml, 100 ng/ml, and 500 ng/ml) treatment in EL-4 and B16-F10 cells

Article Snippet: The antibody against Phospho-Jak2 (Tyr1007/1008), JAK2, Phospho-Stat5 (Tyr694), STAT5, PD-L1 were purchased from Cell Signaling Technology (USA).

Techniques: In Vivo, Immunohistochemical staining, Staining, Immunohistochemistry, Purification, Expressing, Western Blot

Journal: Molecular Cancer

Article Title: GM-CSF mediates immune evasion via upregulation of PD-L1 expression in extranodal natural killer/T cell lymphoma

doi: 10.1186/s12943-021-01374-y

Figure Lengend Snippet: JAK/STAT activation directly drives PD-L1 expression in ENKTL. a-c The proliferation of NK-YS, SNK-6, and SNT-8 cells treated by different concentrations of GM-CSF (100 ng/ml and 500 ng/ml). The above assay was determined by CCK8 as described in materials and methods. Each point represents the mean ± standard deviations (SDs) of three independent experiments performed. d Relative mRNA expression levels of PD-L1 were increased by GM-CSF (10 ng/ml, 100 ng/ml, and 500 ng/ml) treatment in NK-YS, SNK-6, and SNT-8 cells. e-g NK-YS, SNK-6, and SNT-8 cells were treated with GM-CSF (100 ng/ml) for 0, 2, 4, 8, 12 h, p-JAK2, JAK2, p-STAT5, STAT5, and PD-L1 protein expression was measured by Western blot. d Relative protein expression levels of PD-L1 were increased by GM-CSF (100 ng/ml) treatment in NK-YS, SNK-6, and SNT-8 cells. i , j The ratio of protein expression of p-JAK2 to JAK and ratio of protein expression of p-STAT5 to STAT5 in ENKTL cell lines (NK-YS, SNK-6, and SNT-8) treated with GM-CSF (100 ng/ml) for 0, 2, 4, 8, 12 h. k ENKTL cell lines NK-YS and SNK-6 were treated by GM-CSF (10 ng/ml, 100 ng/ml, and 500 ng/ml). The protein expressions of p-JAK2, JAK2, p-STAT5, STAT5, and PD-L1 were detected by Western blot. l ENKTL cell lines NK-YS and SNK-6, T-cell lymphoblastic lymphoma/leukemia cell line Jurkat, B-cell lymphoma cell line SU-DHL-6, and melanoma cell line A375 were treated by GM-CSF (100 ng/ml). The protein expressions of p-JAK2, JAK2, p-STAT5, STAT5, PD-L1, and PD-L2 were detected by Western blot. * P < 0.05, ** P < 0.01, *** P < 0.001. Error bars represent SD of three independent experiments

Article Snippet: The antibody against Phospho-Jak2 (Tyr1007/1008), JAK2, Phospho-Stat5 (Tyr694), STAT5, PD-L1 were purchased from Cell Signaling Technology (USA).

Techniques: Activation Assay, Expressing, Western Blot

Journal: Molecular Cancer

Article Title: GM-CSF mediates immune evasion via upregulation of PD-L1 expression in extranodal natural killer/T cell lymphoma

doi: 10.1186/s12943-021-01374-y

Figure Lengend Snippet: Downregulation of JAK/STAT pathway decreases PD-L1 expression in ENKTL. NK-YS, SNK-6, and SNT-8 cells were treated with JAK2 inhibitor Fedratinib (TG-101348, 3 nM) alone, or GM-CSF (100 ng/ml) alone, or Fedratinib combined with GM-CSF for 12 h. a-c Protein expression of p-JAK2, JAK2, p-STAT5, STAT5, and PD-L1 of ENKTL cell lines NK-YS, SNK6, and SNT-8 were measured by Western blot. d-f And relative mRNA expression of PD-L1 was measured by quantitative polymerase chain reaction (qPCR). g-j SNK-6 and SNT-8 cells expressing shSTAT5 or control were evaluated for STAT5 and PD-L1 protein expression by Western blot, and mRNA expression by qPCR. k The − 620 to − 500 nucleotide sequence of the 5′-flanking region of PD-L1 is shown. Underlined sequences are putative STAT5A and STAT5B transcription factor binding sites, as predicted by JASPAR database and PROMO. And PD-L1 promoter fragments cloned into pGL3-Basic vector. l Analysis of PD-L1 promoter fragment A constructs in 293 T cells transiently transfected with STAT5A or STAT5B for 48 h. Relative luciferase activity was determined as described. Error bars represent the SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. Error bars represent the SD of three independent experiments

Article Snippet: The antibody against Phospho-Jak2 (Tyr1007/1008), JAK2, Phospho-Stat5 (Tyr694), STAT5, PD-L1 were purchased from Cell Signaling Technology (USA).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Control, Sequencing, Binding Assay, Clone Assay, Plasmid Preparation, Construct, Transfection, Luciferase, Activity Assay

Journal: Molecular Cancer

Article Title: GM-CSF mediates immune evasion via upregulation of PD-L1 expression in extranodal natural killer/T cell lymphoma

doi: 10.1186/s12943-021-01374-y

Figure Lengend Snippet: STAT5A mutation increases the PD-L1 overexpression in the presence of GM-CSF. A A proposed working model to illustrate how GM-CSF induced disease progression in ENKTL. STAT5 mutations in ENKTL increased the auto-phosphorylation of STAT5. JAK2 hyperphosphorylation combined with STAT5 mutations led to aggressive up-regulation of PD-L1 expression after GM-CSF treatment, which could induce disease progression in ENKTL

Article Snippet: The antibody against Phospho-Jak2 (Tyr1007/1008), JAK2, Phospho-Stat5 (Tyr694), STAT5, PD-L1 were purchased from Cell Signaling Technology (USA).

Techniques: Mutagenesis, Over Expression, Biomarker Discovery, Phospho-proteomics, Expressing

Journal: Molecular Medicine Reports

Article Title: Effect of modified Xiaochaihu decoction-containing serum on HepG2.2.15 cells via the JAK2/STAT3 signaling pathway

doi: 10.3892/mmr.2017.7561

Figure Lengend Snippet: Primer sequences.

Article Snippet: The membranes were then exposed to the primary antibodies against JAK2 (1:2,000; cat. no. 17670-1-AP; ProteinTech Group, Inc.) and STAT3 (1:1,000; cat. no. 60199-1-lg; ProteinTech Group, Inc) overnight at 4°C. β-actin (1:1,000; cat. no. 60008-1-lg; ProteinTech Group, Inc) was used as an internal control for protein loading.

Techniques: Sequencing, Amplification

Journal: Molecular Medicine Reports

Article Title: Effect of modified Xiaochaihu decoction-containing serum on HepG2.2.15 cells via the JAK2/STAT3 signaling pathway

doi: 10.3892/mmr.2017.7561

Figure Lengend Snippet: Effect of mXCHD serum on the mRNA expression levels of JAK2 and STAT3 in HepG2.2.15 cells. Following treatment for 48 h, the cells were collected, and the mRNA levels of JAK2 and STAT3 were determined using reverse transcription-quantitative polymerase chain reaction analysis. β-actin was used as the internal control. The data are presented as the mean ± standard deviation of three independent experiments. ▲ P<0.05 and # P<0.01 vs. healthy group; ■ P<0.01, vs. entecavir group; °P<0.01, vs. 10% mXCHD group. mXCHD, modified Xiaochaihu decoction; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3.

Article Snippet: The membranes were then exposed to the primary antibodies against JAK2 (1:2,000; cat. no. 17670-1-AP; ProteinTech Group, Inc.) and STAT3 (1:1,000; cat. no. 60199-1-lg; ProteinTech Group, Inc) overnight at 4°C. β-actin (1:1,000; cat. no. 60008-1-lg; ProteinTech Group, Inc) was used as an internal control for protein loading.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Standard Deviation, Modification

Journal: Molecular Medicine Reports

Article Title: Effect of modified Xiaochaihu decoction-containing serum on HepG2.2.15 cells via the JAK2/STAT3 signaling pathway

doi: 10.3892/mmr.2017.7561

Figure Lengend Snippet: Effect of mXCHD serum on the protein expression levels of JAK2 and STAT3 in HepG2.2.15 cells. Following treatment with mXCHD serum, entecavir serum and healthy control serum for 48 h, the cells were collected. The protein levels of JAK2 and STAT3 were determined using western blot analysis. β-actin was used as the internal control. (A) Representative images of the results ofwestern blot analysis. (B) Data are presented as the mean ± standard deviation of three independent experiments. Δ P<0.05 vs. healthy group. mXCHD, modifiedXiaochaihu decoction; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3.

Article Snippet: The membranes were then exposed to the primary antibodies against JAK2 (1:2,000; cat. no. 17670-1-AP; ProteinTech Group, Inc.) and STAT3 (1:1,000; cat. no. 60199-1-lg; ProteinTech Group, Inc) overnight at 4°C. β-actin (1:1,000; cat. no. 60008-1-lg; ProteinTech Group, Inc) was used as an internal control for protein loading.

Techniques: Expressing, Control, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Leptin Resistance in the Ovary of Obese Mice Is Associated with Profound Changes in the Transcriptome of Cumulus Cells

doi: 10.1101/729657

Figure Lengend Snippet: Abundance of mRNA (grey box) and protein of leptin signalling pathway components in ovarian extracts collected from animals injected with saline (C) or 100 μg of leptin (L) for 9 or 16 days (d) and sacrificed in oestrus stage. Expression of (A) leptin receptor (ObR), phosphorylation of (B) tyrosine 985 of leptin receptor, (C) tyrosine 1077 of leptin receptor, (D) tyrosine 1138 of leptin receptor, (E) Janus kinase 2 (JAK2), (F) signal transducer and activator of transcription 3 (STAT3), (G) STAT5, expression of (H) protein tyrosine phosphatase 1B (PTP1B) and (I) suppressor of cytokine signaling 3 (SOCS3) determined by real-time PCR and Western blot. (J) SOCS3 ovarian quantification in animals in oestrus stage determined by ELISA. mRNA expression of Rpl37 and protein expression of β-actin was used to normalize the expression data. Each bar represents the mean ± SD. Differences between groups were analysed by Mann-Whitney test. N=4-8 for immunoblots and N=8 for RT PCR analysis and ELISA. * p<0.05; ** p<0.01; ***p<0.001; + p=0.09.

Article Snippet: The expression of other leptin signalling pathway components was assessed using the following antibodies: RP against JAK2 (1:200, cat# sc-294, Santa Cruz Biotechnology), RP against phosphorylated Tyr 1007/1008 JAK2 (pJAK2; 1:200, cat# sc-16566-R, Santa Cruz Biotechnology), RP against STAT3 (1:200, cat# sc-482, Santa Cruz Biotechnology), MM against phosphorylated Tyr 705 pSTAT3 (1:200, cat# sc-8059, Santa Cruz Biotechnology), RP against STAT5 (1:200, cat# sc-835, Santa Cruz Biotechnology), MM against phosphorylated Tyr 694/699 pSTAT5 (1:200, cat# sc-81524, Santa Cruz Biotechnology), GP against PTP1B (1:200, cat# sc-1718, Santa Cruz Biotechnology), MM against SOCS3 (1:500, cat# sc-51699, Santa Cruz Biotechnology) in cell lysates.

Techniques: Injection, Saline, Expressing, Phospho-proteomics, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Reverse Transcription Polymerase Chain Reaction

Journal: ACS Omega

Article Title: Discovery of JAK2/3 Inhibitors from Quinoxalinone-Containing Compounds

doi: 10.1021/acsomega.2c04769

Figure Lengend Snippet: (A) Docking results of quinoxalinone derivatives toward JAK2/3 derived from FlexX docking. (B) Summary of screened compounds toward JAK2/3.

Article Snippet: Primary antibodies against JAK2 (ab108596), p-JAK2 (ab32101; Y1007/1008) were purchased from Abcam.

Techniques: Derivative Assay

Journal: ACS Omega

Article Title: Discovery of JAK2/3 Inhibitors from Quinoxalinone-Containing Compounds

doi: 10.1021/acsomega.2c04769

Figure Lengend Snippet: In vitro study of focused compounds toward JAK2/3 by kinase assay and cell-based assay. (A) In vitro IC 50 values of the potent quinoxalinone derivatives (MN341P, MN390, ST3i, and ST4j) and drugs (tofacitinib and ruxolitinib) toward JAK2/3 (ND = not detected), the IC 50 value of ST4j compound and drugs toward (B) TF1 and HEL, (C) Vero and HepG2 cells, and (D) Western blot analysis in TF1 cells after treatment with ST4j and drugs at various concentrations. * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001 vs tofacitinib, # p ≤ 0.05 and ### p ≤ 0.001 vs ruxolitinib.

Article Snippet: Primary antibodies against JAK2 (ab108596), p-JAK2 (ab32101; Y1007/1008) were purchased from Abcam.

Techniques: In Vitro, Kinase Assay, Cell Based Assay, Western Blot

Journal: ACS Omega

Article Title: Discovery of JAK2/3 Inhibitors from Quinoxalinone-Containing Compounds

doi: 10.1021/acsomega.2c04769

Figure Lengend Snippet: (A) Per-residue decomposition free energy (Δ G bind residue ), the van der Waals (vdW) and electrostatic energy contributions of the domain of JAK2 for the binding of tofacitinib and ST4j. (B) the binding orientation of ruxolitinib and ST4j within the binding pocket drawn from the last MD snapshot. The lowest and highest energies are colored from purple to dark red, respectively.

Article Snippet: Primary antibodies against JAK2 (ab108596), p-JAK2 (ab32101; Y1007/1008) were purchased from Abcam.

Techniques: Residue, Binding Assay

Journal: ACS Omega

Article Title: Discovery of JAK2/3 Inhibitors from Quinoxalinone-Containing Compounds

doi: 10.1021/acsomega.2c04769

Figure Lengend Snippet: (A) Percentage of hydrogen bond occupation of ruxolitinib and ST4j within the JAK2 binding pocket. Note that the hydrogen bond with value >50% was selected to represent in 3D and (B) B factor of JAK2, the flexible and rigid regions are ranged from blue to green and red, respectively. The data were derived from the last 100 ns of the one simulation of JAK2 with ruxolitinib (run 1) and ST4j (run 2).

Article Snippet: Primary antibodies against JAK2 (ab108596), p-JAK2 (ab32101; Y1007/1008) were purchased from Abcam.

Techniques: Binding Assay, Derivative Assay

Journal: Cancers

Article Title: Inhibition of USP9X Downregulates JAK2-V617F and Induces Apoptosis Synergistically with BH3 Mimetics Preferentially in Ruxolitinib-Persistent JAK2-V617F-Positive Leukemic Cells

doi: 10.3390/cancers12020406

Figure Lengend Snippet: The USP9X inhibitor WP1130 or G9 induces apoptosis more prominently in JAK2-V617F-dependent cells than in cells dependent on BCR/ABL or cytokine-activated JAK2. ( A ) HEL, PVTL-2, and K562 cells were treated with indicated concentrations of WP1130 for 24 h, and viable cell numbers were measured by the CCK-8 colorimetric assay. Each data point represents the mean of triplicate cultures, with error bars indicating standard errors, and is expressed as percentage of the cell numbers cultured without WP1130. The asterisks indicate significant differences between K562 and HEL or PVTL-2 determined by one-way ANOVA followed by Dunnett’s post-hoc test (* p < 0.05). ( B ) 32DE or UT7 cells transduced with either JAK2-V617F or empty vector (Control) and cultured with Epo were left untreated as control or treated for 24 h with 3 μM WP1130 (WP) or 2 µM G9, as indicated, in triplicate, and viable cell numbers were measured. The asterisks indicate significant differences between JAK2-V617F and control cells determined by Student’s t-test (* p < 0.05). ( C ) HEL, PVTL-2, and K562 cells were treated for 24 h with indicated concentrations of WP1130. Cells were analyzed for cellular DNA content by flow cytometry. Percentages of apoptotic cells with the sub-G1 DNA content are indicated. ( D ) 32DE/JAK2-V617F cells (JAK2-V617F) or vector-control cells (Control) cultured with Epo were treated for 5 h with indicated concentrations of WP1130 or G9 and analyzed.

Article Snippet: For immunoblot analysis, monoclonal antibodies against phospho-JAK2 (Tyr1007/1008) (#3776), JAK2 (#3230), phospho-STAT5 (Tyr694) (#9359), phospho-MEK1/2 (Ser217/221) (#9121), phospho-p90RSK (Ser380) (#9335), phospho-p70 S6 Kinase (Thr389) (#9234), phospho-4EBP1 (Thr37/46) (#2855), MEK1/2 (#9122), HA-Tag (#3724), phospho-SAPK/JNK (Thr183/Tyr185) (#9251), phospho-p38 MAP Kinase (Thr180/Tyr182) (#9211), phospho-Histone H2A.X (Ser139) (#9718), phospho-ATM (Ser1981) (#4526), phospho-Chk2 (Thr68) (#2661), JAK1 (#3344), JAK3 (#8827), Bcl-xL (#2461), Mcl-1 (#5453), and LC3B (#2775) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: CCK-8 Assay, Colorimetric Assay, Cell Culture, Transduction, Plasmid Preparation, Control, Flow Cytometry

Journal: Cancers

Article Title: Inhibition of USP9X Downregulates JAK2-V617F and Induces Apoptosis Synergistically with BH3 Mimetics Preferentially in Ruxolitinib-Persistent JAK2-V617F-Positive Leukemic Cells

doi: 10.3390/cancers12020406

Figure Lengend Snippet: WP1130 enhances K63-linked polyubiquitination and preferentially downregulates the phosphorylated form of JAK2-V617F to inhibit downstream signaling. ( A , B , C ) HEL ( A , C ) or PVTL-2 ( B ) were treated with 5 µM WP1130 (WP) for indicated times. Cells were lysed and subjected to immunoblot analysis with antibodies against indicated proteins. HSP90 was used for a loading control. ( D ) 293T cells transfected with plasmids coding for either JAK2-V617F (V617F) or wild-type JAK2 (WT) and HA-tagged ubiquitin were treated for 3 h with or without 5 µM WP1130, as indicated, and analyzed. β-actin was used for a loading control. Short and long exposure results are shown where indicated. Relative expression levels of JAK2 as compared with those in untreated cells analyzed by densitometry and normalized by that of ß-actin (J2/β -a) are indicated. The vertical line indicates the smeary pattern characteristic of polyubiquitination. ( E ) 32DE/JAK2-V617F cells or vector control cells cultured with Epo were treated with or without 5 µM WP1130 for 2 h, as indicated, and analyzed. ( F ) 293T cells transduced with HA-tagged ubiquitin along with either JAK2-V617F or wild-type JAK2 were treated for indicated times with 5 µM WP1130 and lysed. Immunoprecipitates (IP) obtained with anti-JAK2 were analyzed. The arrow indicates the position corresponding to JAK2. ( G ) 293T cells transduced with JAK2-V617F along with HA-tagged ubiquitin (WT) or its K48R or K63R mutant were treated with 5 µM WP1130 for indicated times and analyzed. ( H ) HEL cells were left untreated as control or treated for 30 min with 5 µM WP1130 or 3 µM G9 as indicated. K63-polyubiquitinated proteins were isolated by immunoprecipitation with anti-Flag after incubation of cell lysates with FLAG-K63-TUBE (K63-TUBE). Immunoprecipitates, as well as total cell lysates (TCL), were analyzed.

Article Snippet: For immunoblot analysis, monoclonal antibodies against phospho-JAK2 (Tyr1007/1008) (#3776), JAK2 (#3230), phospho-STAT5 (Tyr694) (#9359), phospho-MEK1/2 (Ser217/221) (#9121), phospho-p90RSK (Ser380) (#9335), phospho-p70 S6 Kinase (Thr389) (#9234), phospho-4EBP1 (Thr37/46) (#2855), MEK1/2 (#9122), HA-Tag (#3724), phospho-SAPK/JNK (Thr183/Tyr185) (#9251), phospho-p38 MAP Kinase (Thr180/Tyr182) (#9211), phospho-Histone H2A.X (Ser139) (#9718), phospho-ATM (Ser1981) (#4526), phospho-Chk2 (Thr68) (#2661), JAK1 (#3344), JAK3 (#8827), Bcl-xL (#2461), Mcl-1 (#5453), and LC3B (#2775) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Control, Transfection, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Cell Culture, Transduction, Mutagenesis, Isolation, Immunoprecipitation, Incubation

Journal: Cancers

Article Title: Inhibition of USP9X Downregulates JAK2-V617F and Induces Apoptosis Synergistically with BH3 Mimetics Preferentially in Ruxolitinib-Persistent JAK2-V617F-Positive Leukemic Cells

doi: 10.3390/cancers12020406

Figure Lengend Snippet: WP1130 induces aggresomal translocation of JAK2 preferentially for the V617F mutant most likely through inhibition of USP9X. ( A , B ) HEL ( A ) or primary post-myeloproliferative neoplasms (MPN) secondary acute myeloid leukemic (sAML) cells expressing JAK2-V617F ( B ) were treated with 5 μM WP1130 (WP) for indicated times. Cells were lysed and detergent-soluble and –insoluble proteins were extracted and analyzed. GAPDH and β-actin were used for loading controls and confirmation of appropriate fractionation. ( C ) Primary post-MPN sAML cells expressing JAK2-V617F were treated for 2 h with indicated concentrations of WP1130 and analyzed. HSP90 was used for a loading control. ( D ) 293T cells were transfected with plasmids coding for JAK2-V617F, -WT, or empty vector (Control). Cells were left untreated as control or treated with 5 μM WP1130 for 3 h, followed by processing for confocal microscopy as described in the Materials and Methods. Images represent a 60× optical zoom with a 3× digital zoom. DAPI staining shows the position of the nucleus. Representative images of cells are shown. Positions of aggresomes are indicated by arrows. ( E ) UT7/JAK2-V617F or vector-control cells (Control) were treated with 5 μM WP1130 for indicated times and analyzed. Relative levels of JAK2 as compared with that in cells not treated with WP1130 were determined by densitometric analyses. ( F ) HEL cells were left untreated as control or treated for 3 h with 5 μM WP1130, 3 μM G9, 1 μM b-AP15, or 25 μM PR-619, as indicated. Cells were lysed and detergent-soluble and -insoluble protein were extracted and analyzed. ( G) HEL cells were treated for 4 h with or without 2 μM ruxolitinib (Rux), as indicated. Immunoprecipitates (IP) with anti-USP9X antibody or normal rabbit IgG (Cont.) and total cell lysate (TCL) were analyzed.

Article Snippet: For immunoblot analysis, monoclonal antibodies against phospho-JAK2 (Tyr1007/1008) (#3776), JAK2 (#3230), phospho-STAT5 (Tyr694) (#9359), phospho-MEK1/2 (Ser217/221) (#9121), phospho-p90RSK (Ser380) (#9335), phospho-p70 S6 Kinase (Thr389) (#9234), phospho-4EBP1 (Thr37/46) (#2855), MEK1/2 (#9122), HA-Tag (#3724), phospho-SAPK/JNK (Thr183/Tyr185) (#9251), phospho-p38 MAP Kinase (Thr180/Tyr182) (#9211), phospho-Histone H2A.X (Ser139) (#9718), phospho-ATM (Ser1981) (#4526), phospho-Chk2 (Thr68) (#2661), JAK1 (#3344), JAK3 (#8827), Bcl-xL (#2461), Mcl-1 (#5453), and LC3B (#2775) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Translocation Assay, Mutagenesis, Inhibition, Expressing, Fractionation, Control, Transfection, Plasmid Preparation, Confocal Microscopy, Staining

Journal: Cancers

Article Title: Inhibition of USP9X Downregulates JAK2-V617F and Induces Apoptosis Synergistically with BH3 Mimetics Preferentially in Ruxolitinib-Persistent JAK2-V617F-Positive Leukemic Cells

doi: 10.3390/cancers12020406

Figure Lengend Snippet: WP1130 causes oxidative stress to activate stress-related p38/JNK MAPKs pathways and DNA damage responses to induce apoptosis. ( A ) HEL cells were treated for 6 h with or without 5 µM WP1130 and subjected to immunoblot analysis with antibodies against indicated proteins. ß-actin was used for a loading control. ( B ) HEL cells were left untreated as control (Cont.) or treated for 3 h with 3 μM WP1130 (WP), 3 μM G9, and 5 mM NAC, as indicated, and analyzed for reactive oxygen species (ROS) by flow cytometry. ( C ) HEL cells left untreated or pretreated with 40 mM NAC for 1 h were further treated with 5 μM WP1130 for indicated times and analyzed by immunoblot analysis. The results obtained from duplicate gels are shown above or below a thin horizontal line. ( D ) Primary post-MPN sAML cells expressing JAK2-V617F were treated with 5 μM WP1130 for indicated times and analyzed. An arrow indicates the position of cleaved PARP. ( E ) HEL cells were left untreated as control or treated for 24 h with 3.5 μM WP1130 and 5 mM NAC, as indicated, and analyzed for DNA content. Percentages of apoptotic cells with the sub-G1 DNA content are indicated. ( F ) HEL cells were left untreated as control or treated for 24 h with 3.5 μM WP1130, 50 μM SB203580, and 50 μM SP600125, as indicated, and analyzed.

Article Snippet: For immunoblot analysis, monoclonal antibodies against phospho-JAK2 (Tyr1007/1008) (#3776), JAK2 (#3230), phospho-STAT5 (Tyr694) (#9359), phospho-MEK1/2 (Ser217/221) (#9121), phospho-p90RSK (Ser380) (#9335), phospho-p70 S6 Kinase (Thr389) (#9234), phospho-4EBP1 (Thr37/46) (#2855), MEK1/2 (#9122), HA-Tag (#3724), phospho-SAPK/JNK (Thr183/Tyr185) (#9251), phospho-p38 MAP Kinase (Thr180/Tyr182) (#9211), phospho-Histone H2A.X (Ser139) (#9718), phospho-ATM (Ser1981) (#4526), phospho-Chk2 (Thr68) (#2661), JAK1 (#3344), JAK3 (#8827), Bcl-xL (#2461), Mcl-1 (#5453), and LC3B (#2775) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Control, Flow Cytometry, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Leptin Receptor Metabolism Disorder in Primary Chondrocytes from Adolescent Idiopathic Scoliosis Girls

doi: 10.3390/ijms17071160

Figure Lengend Snippet: JAK2 and STAT3 phosphorylation status in the AIS and the control groups. ( A ) Protein samples were acquired from the cartilage tissue of the participants in both the AIS and the control groups. The expression of p-JAK2, total JAK2, p-STAT3, and total STAT3 was analyzed by Western blotting. β-actin was used as an internal reference. Representative Western blots from three independent experiments are shown; ( B ) Relative quantitation of the p-JAK2/JAK2 and p-STAT3/STAT3 levels is shown in the graphs, and the mean value for the control groups is 1.0. The Y axis represents the fold change between the AIS and control groups. * p < 0.05 vs. the controls.

Article Snippet: The rabbit monoclonal antibody against JAK2, rabbit monoclonal antibody against p-JAK2 (Tyr1008), mouse monoclonal antibody against STAT3, rabbit monoclonal antibody against p-STAT3 (Ser727) and rabbit monoclonal antibody against CHC were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, Control, Expressing, Western Blot, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: Leptin Receptor Metabolism Disorder in Primary Chondrocytes from Adolescent Idiopathic Scoliosis Girls

doi: 10.3390/ijms17071160

Figure Lengend Snippet: CHC knockdown enhances leptin’s effect on the chondrocytes from AIS patients. Chondrocytes from the AIS group were transfected with the control siRNA or CHC siRNA, followed by treatment with or without leptin (10 ng/mL) for 5 h. ( A ) The GAG content was visualized by Alcian blue staining. Stronger Alcian blue staining was observed in the leptin+CHC siRNA group than in the other group. Scale bars = 50 μm; ( B ) Total proteins were extracted. The expression of p-JAK2, total JAK2, p-STAT3, and total STAT3 was analyzed by Western blotting. β-actin was used as an internal reference. Representative Western blots from three independent experiments are shown; ( C ) The relative quantitation of the p-JAK2/JAK2 and p-STAT3/STAT3 levels is shown in the graphs. * p < 0.05 vs. the group untreated with leptin or CHC siRNA; # p < 0.05 vs. the leptin-treated group; ( D – F ) Total RNAs were extracted from the chondrocytes. The relative mRNA expression of chondrogenic marker genes, including Aggrecan, ColII, and Sox9, was detected by quantitative real-time PCR. The Y axis represents the relative fold change in the transcript levels in the four groups. The untreated control group was set to 1.0. The data are displayed as the means ± SDs from 3 experiments. * p < 0.05 vs. the group untreated with leptin or CHC siRNA; # p < 0.05 vs. the leptin-treated group.

Article Snippet: The rabbit monoclonal antibody against JAK2, rabbit monoclonal antibody against p-JAK2 (Tyr1008), mouse monoclonal antibody against STAT3, rabbit monoclonal antibody against p-STAT3 (Ser727) and rabbit monoclonal antibody against CHC were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knockdown, Transfection, Control, Staining, Expressing, Western Blot, Quantitation Assay, Marker, Real-time Polymerase Chain Reaction