Journal: Frontiers in Nutrition

Article Title: Central Nervous System Inflammation Induced by Lipopolysaccharide Up-Regulates Hepatic Hepcidin Expression by Activating the IL-6/JAK2/STAT3 Pathway in Mice

doi: 10.3389/fnut.2021.649640

Figure Lengend Snippet: AG490 significantly reduced p-STAT3 protein expression and eliminated the changes in Fpn1 and Ft-L expression induced by lipopolysaccharide (LPS) in the liver. C57BL/6 wild-type male mice were pre-treated with AG490 (5 mg/kg, IP injection) in 10% dimethyl sulfoxide (DMSO) or vehicle 30 min before LPS (ICV injection, 5 μg in 2 μL of sterile saline). The expression levels of p-STAT3 (A) , Fpn1 (B) , and Ft-L proteins (C) in the liver were measured using western blot. Data are presented as means ± SEM (% control) ( n = 4). For statistical analysis, one-way ANOVA with Tukey's post hoc test was performed.

Article Snippet: The JAK2 inhibitor AG490 was purchased from Selleckchem (Houston, TX, USA); the other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Injection, Sterility, Saline, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: Interleukin-21 promotes osteoclastogenesis in RAW264.7 cells through the PI3K/AKT signaling pathway independently of RANKL

doi: 10.3892/ijmm.2016.2722

Figure Lengend Snippet: Interleukin-21 (IL-21) promotes osteoclastogenesis in RAW264.7 cells through the PI3K/AKT signaling pathway. (A) The phosphorylation of AKT, STAT3 and ERK were examined by western blot analysis. RAW264.7 cells were stimulated with IL-21 (20 ng/ml) for the indicated times in the presence of 5 ng/ml macrophage colony-stimulating factor (M-CSF), and cells were harvested for western blot analysis. (B) Statistical results of (A). (C) The effect of inhibiting STAT3, ERK1/2 and PI3K/AKT signaling pathways on IL-21-induced osteoclastogenesis. RAW264.7 cells were incubated with specific pathways inhibitors [ERK1/2 pathway inhibitor PD98059 (20 µ M), PI3K/AKT inhibitor LY294002 (10 µ M) and STAT3 pathway inhibitor AG490 (50 µ M)] for 30 min prior to stimulation with IL-21 (20 ng/ml). Osteoclast formation was determined using tartrate-resistant acid phosphatase (TRAP) staining after 5 days of culture. Original magnification, ×100. Data are expressed as the means ± SEM of three samples. * P<0.05, ** P<0.01, *** P<0.001 vs. control or as shown in figure.

Article Snippet: AG490 [Janus kinase 2 (JAK2)/STAT3 inhibitor], LY294002 (PI3K/AKT inhibitor), and PD98059 (ERK inhibitor) were supplied by Selleck Chemicals (Houston, TX, USA).

Techniques: Phospho-proteomics, Western Blot, Protein-Protein interactions, Incubation, Staining, Control

Journal: International Journal of Molecular Medicine

Article Title: Interleukin-21 promotes osteoclastogenesis in RAW264.7 cells through the PI3K/AKT signaling pathway independently of RANKL

doi: 10.3892/ijmm.2016.2722

Figure Lengend Snippet: Effect of signaling pathway inhibitors on the expression of osteoclasts markers induced by interleukin-21 (IL-21). RAW264.7 cells were incubated with specific pathways inhibitors (AG490, LY294002 and PD98059) for 30 min prior to stimulation with IL-21 (20 ng/ml). (A and B) RT-PCR results of calcitonin receptor (CTR), cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression. (C) RT-qPCR results of RANK expression. Data are expressed as the means ± SEM of three samples. ** P<0.01, *** P<0.001.

Article Snippet: AG490 [Janus kinase 2 (JAK2)/STAT3 inhibitor], LY294002 (PI3K/AKT inhibitor), and PD98059 (ERK inhibitor) were supplied by Selleck Chemicals (Houston, TX, USA).

Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Immunology

Article Title: Adipocytokines leptin and adiponectin function as mast cell activity modulators

doi: 10.1111/imm.13090

Figure Lengend Snippet: Effect of leptin and adiponectin on mast cell (MC) histamine release. (a, b) MCs were incubated with different concentrations of leptin or adiponectin, compound 48/80 at 5 μg/ml (positive control) or medium alone for 30 min. (c) MCs were stimulated with leptin at 50 ng/ml for 1, 3, 5, 10 or 30 min. (d) MCs were pretreated with medium alone (none), Janus‐activated kinase (JAK2) inhibitor AG490 (10 μm), phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002 (5 μm), extracellular signal‐regulated kinase 1/2 (ERK1/2) inhibitor PD98059 (5 μm), p38 inhibitor SB203580 (10 μm) or phospholipase C (PLC) inhibitor U‐73122 (1 μm) for 1 hr before stimulation with leptin (50 ng/ml) for 30 min. Results are the mean ± SD of three independent experiments performed in duplicate. *P < 0·05, **P < 0·01, ***P < 0·001.

Article Snippet: Human plasma fibronectin purified protein and JAK2 inhibitor AG490 were purchased from Merck Millipore (Billerica, MA) and ERK1/2 inhibitor PD98059 and p38 kinase inhibitor SB203580 were purchased from InvivoGen (San Diego, CA).

Techniques: Incubation, Positive Control

Journal: Immunology

Article Title: Adipocytokines leptin and adiponectin function as mast cell activity modulators

doi: 10.1111/imm.13090

Figure Lengend Snippet: Effect of leptin and adiponectin on mast cell (MC) cysteinyl leukotriene (cysLT) generation. (a, b) MCs were incubated with different concentrations of leptin or adiponectin, calcium ionophore A23187 at 5 μg/ml (positive control) or medium alone for 1 hr. (c) MCs were pretreated with medium alone (none), Janus‐activated kinase (JAK2) inhibitor AG490 (10 μm), phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002 (5 μm), extracellular signal‐regulated kinase 1/2 (ERK1/2) inhibitor PD98059 (5 μm) or p38 inhibitor SB203580 (10 μm) for 1 hr before stimulation with leptin (50 ng/ml). Results are the mean ± SD of three independent experiments performed in duplicate. *P < 0·05, **P < 0·01, ***P < 0·001.

Article Snippet: Human plasma fibronectin purified protein and JAK2 inhibitor AG490 were purchased from Merck Millipore (Billerica, MA) and ERK1/2 inhibitor PD98059 and p38 kinase inhibitor SB203580 were purchased from InvivoGen (San Diego, CA).

Techniques: Incubation, Positive Control

Journal: Immunology

Article Title: Adipocytokines leptin and adiponectin function as mast cell activity modulators

doi: 10.1111/imm.13090

Figure Lengend Snippet: (a) Leptin‐induced, (b) adiponectin‐induced, and (c) tumor necrosis factor ( TNF) ‐induced mast cell (MC) migration. MCs were incubated with different concentrations of leptin, adiponectin, TNF (positive control) or medium alone (control spontaneous MC migration) for 3 hr at 37° in a Boyden microchamber. Laminin‐coated (○) or fibronectin‐coated (●) filters were used. (d) MCs were pretreated with medium alone (none), Janus‐activated kinase (JAK2) inhibitor AG490 (10 μm), phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002 (5 μm), extracellular signal‐regulated kinase 1/2 (ERK1/2) inhibitor PD98059 (5 μm) or p38 inhibitor SB203580 (10 μm) for 1 hr before stimulation with leptin (0·1 ng/ml). (e) MCs were pretreated with medium alone (none), PI3K inhibitor LY294002 (5 μm), ERK1/2 inhibitor PD98059 (5 μm) or p38 inhibitor SB203580 (10 μm) for 1 hr before stimulation with adiponectin (0·1 μg/ml). Ten high‐power fields were counted in each assay (×250). Spontaneous migration served as a control and was referred to 100%. Each point represents the mean ± SD of three independent experiments performed in duplicate. *P < 0·05, **P < 0·01, ***P < 0·001.

Article Snippet: Human plasma fibronectin purified protein and JAK2 inhibitor AG490 were purchased from Merck Millipore (Billerica, MA) and ERK1/2 inhibitor PD98059 and p38 kinase inhibitor SB203580 were purchased from InvivoGen (San Diego, CA).

Techniques: Migration, Incubation, Positive Control, Control

Journal: International journal of oncology

Article Title: JAK/STAT3 signaling is required for TGF-β-induced epithelial-mesenchymal transition in lung cancer cells.

doi: 10.3892/ijo.2014.2310

Figure Lengend Snippet: Figure 2. AG490 inhibits Smad3 activation and transcriptional responses in lung cancer A549 and H1650 cells. (A) AG490 inhibited phosphorylation of Stat3. After serum starvation overnight, cells were subjected to AG490 treatment (JAK2/STAT3 inhibitor, 50 or 100 µM) for 4 h, and the expression of p-Stat3, total Stat3 was analyzed using western blotting. (B) AG490 suppressed TGF-β-induced Smad3 activation and Snail upregulation. Cells were pretreated with 50 µM AG490 for 4 h and then treated with 5 ng/ml TGF-β1 for 4 h as indicated. Levels of p-Samd3, Smad3 and Snail were monitored by western blotting. (C) AG490 attenuated TGF-β-induced PAI-1 promoter activation. After transient transfection with PAI-1 promoter construct, cells were treated with 50 µM AG490 for 4 h and then incubated for 18 h in the absence or presence of 5 ng/ml TGF-β1. Relative luciferase activity was expressed as the mean fold change from basal level ± SD of three independent experiments. (D and E) AG490 diminished TGF-β-induced increase in Snail and MMP2. Cells were treated with 50 µM AG490 for 4 h and then exposed to 5 ng/ml TGF-β1, and the mRNA levels of Snail (treated with TGF-β1 for 1 h) and MMP2 (24 h) were determined by quantitative RT-PCR. *P<0.05; **P<0.01.

Article Snippet: JAK2 inhibitor AG490 and Smad3 phosphorylation inhibitor SIS3 were purchased from Merck KGaA (Darmstadt, Germany) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively.

Techniques: Activation Assay, Phospho-proteomics, Expressing, Western Blot, Transfection, Construct, Incubation, Luciferase, Activity Assay, Quantitative RT-PCR

Journal: International journal of oncology

Article Title: JAK/STAT3 signaling is required for TGF-β-induced epithelial-mesenchymal transition in lung cancer cells.

doi: 10.3892/ijo.2014.2310

Figure Lengend Snippet: Figure 3. AG490 abrogates TGF-β-induced migration and invasion in A549 and H1650 cells. (A) AG490 inhibited TGF-β-induced cell migration. Cells were treated with 50 µM AG490 for 4 h followed by 5 ng/ml TGF-β1 for 12 h, and allowed to migrate through an 8-µM pore in transwells. Migrated cells through the pores were stained with 1% crystal violet and counted under a light microscope (magnification, x200). (B) AG490 suppressed TGF‑β-induced cell invasion. Cells were treated as above and allowed to pass through Matrigel-coated membrane in transwells. Invaded cells through the filter were stained and counted. The data summarized in the bar charts are presented as mean ± SD of three independent fields. *P<0.05; **P<0.01.

Article Snippet: JAK2 inhibitor AG490 and Smad3 phosphorylation inhibitor SIS3 were purchased from Merck KGaA (Darmstadt, Germany) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively.

Techniques: Migration, Staining, Light Microscopy, Membrane

Journal: JAK-STAT

Article Title: Propofol mediates signal transducer and activator of transcription 3 activation and crosstalk with phosphoinositide 3-kinase/AKT

doi: 10.4161/jkst.29554

Figure Lengend Snippet: Figure 2. Effects of PI3K/AKT or JAK2/STAT3 signaling inhibition on propofol-induced STAT3 and AKT phosphorylation. Cells were pre-incubated with different inhibitors for 30 min, and then treated with propofol for another 30 min. Cell lysates were collected and AKT and STAT3 phosphorylation was detected. ( A ) Pretreatment with wortmannin (Wort) or API-2 reduced STAT3 phosphorylation at tyr705. ( B ) Wortmannin or API-2 pretreatment reduced propofol-induced STAT3 phosphorylation at ser727. ( C and D ) Pretreatment with AG490 or stattic inhibited propofol-induced AKT phosphorylation at ser473 and thr308. No changes in protein expression were observed with total STAT3 and total AKT in all groups ( A–D ).

Article Snippet: AG490 (selective JAK2 inhibitor) was purchased from Cayman Chemical.

Techniques: Inhibition, Incubation, Expressing

Journal: bioRxiv

Article Title: Tau accumulation activates STAT1 triggering memory deficits via suppressing NMDA receptor expression

doi: 10.1101/437814

Figure Lengend Snippet: (A, B) Overexpression of hTau in HEK293 cells for 48 h increased the activity-dependent phosphorylation of JAK2, JNK1 and ERK1 compared with the empty vector control (Ctrl) measured by Western blotting (n=3). (C-F) Pharmacological inhibition of ERK1 (C, D) or JNK1 (E, F) for 24 h did not significantly affect the hTau-induced STAT1 phosphorylation at pY-STAT1 (Tyr701) in total extracts (C, E) and the nuclear fraction (D, F) measured by Western blotting (n=3). The alteration of pS-STAT1 (Ser727) confirms the efficacy of JNK1 inhibitors. (G-J) Pharmacological inhibition of JAK2 (G, H) or knockdown JAK2 by siRNA (I, J) abolished hTau-induced STAT1 phosphorylation at Tyr701 in total extracts (G, I) and the nuclear fraction (H, J) (n=3). (K, L) The phosphorylated JAK2 level increased in the hippocampus of 12 m-old hTau transgenic mice (K), and the hippocampus of C57 mice infected with AAV-hTau (1.13×10 13 v.g./ml) (L). Data were presented as mean ± SD (student’s unpaired t-test); *, p <0.05, **, p <0.01 vs Ctrl, eGFP or WT.

Article Snippet: Itacitinib (JAK1 inhibitor, from MCE), TG-101348 (special JAK2 inhibitor, from MCE), JAK2 siRNA (sc-39099, from Santa Cruz), AG490 (JAK2 inhibitor, from Santa Cruz), SP600125 (the inhibitor of JNK1, from Santa Cruz) and FR180204 (the inhibitor of ERK1, from Santa Cruz) were purchased.

Techniques: Over Expression, Activity Assay, Plasmid Preparation, Western Blot, Inhibition, Transgenic Assay, Infection

Journal: International Journal of Medical Sciences

Article Title: Estrogen receptor α/prolactin receptor bilateral crosstalk promotes bromocriptine resistance in prolactinomas

doi: 10.7150/ijms.51176

Figure Lengend Snippet: PRL exposure induces ERα phosphorylation via the JAK2-PI3K/Akt-MEK/ERK pathway in primary cultured HPA cells. ( A ) PRLR-knockdown abolishes PRL-induced JAK2-PI3K/Akt-MEK/ERK signaling. After overnight serum-starvation, HPA cells transiently transfected with control or PRLR-specific shRNA (shPRLR) were stimulated with PRL (20 ng/ml) for 4 h and the cells were harvested for western blotting using the indicated antibodies. ( B ) MMQ cells were treated with PRL (20 ng/ml) in the absence and presence of the JAK2 inhibitor AG-490 (50 μM), the PI3K inhibitor wortmannin (0.5 µM), the MEK inhibitor U0126 (10 µM) or the STAT5 inhibitor pimozide (5 µM). AG-490, wortmannin and U0126 partially blocked PRL-induced ERα phosphorylation, with the most profound effect in the AG-490 group; pimozide had no effect on PRL-induced ERα phosphorylation. PRL, prolactin; ERα, estrogen receptor α; HPA, human pituitary adenoma; PRLR, prolactin receptor; sh, short hairpin (RNA); CON. Control; Pim, pimozide; Wort, wortmannin.

Article Snippet: PRL, estradiol (E2), fulvestrant, the JAK2 inhibitor AG-490, the PI3K inhibitor Wortmannin, the ERK inhibitor U0126, and the STAT5 inhibitor pimozide were purchased from Selleck Chemicals.

Techniques: Phospho-proteomics, Cell Culture, Knockdown, Transfection, Control, shRNA, Western Blot