Journal: Journal of Inflammation Research

Article Title: Melatonin Ameliorates Axonal Hypomyelination of Periventricular White Matter by Transforming A1 to A2 Astrocyte via JAK2/STAT3 Pathway in Septic Neonatal Rats

doi: 10.2147/JIR.S337499

Figure Lengend Snippet: Melatonin activated MT1/JAK2/STAT3 pathway in primary astrocytes exposed to IL-1α, C1q and TNF-α in vitro. ( A ) Shows optical density changes of p-JAK2 relative to JAK2, p-STAT3 relative to STAT3 and MT1 relative to GAPDH in each group. IL-1α + C1q + TNF-α treatment for 24h significantly decreased the optical density of MT1, P-JAK2 and P-STAT3 proteins when compared with the corresponding controls. Melatonin reversed the changes, but the effect was prevented by luzindole. Additionally, blockage of JAK2 or STAT3 inhibited the effect of melatonin in modulating the A1/A2 astrocyte polarization. ( B ) Shows A1-marker C3, A2-marker S100A10, p-STAT3, STAT3 and GAPDH immunoreactive bands after IL-1α + C1q + TNF-α, IL-1α + C1q + TNF-α + melatonin, and IL-1α + C1q + TNF-α + AG490 (an inhibitor of JAK2 activity) treatment when compared with the corresponding control in primary astrocyte. ( C ) show immunoreactive bands after IL-1α + C1q + TNF-α, IL-1α + C1q + TNF-α + melatonin, IL-1α + C1q + TNF-α + STAT-IN-3 (an inhibitor of STAT3 activity) treatment when compared with the corresponding control in primary astrocyte. Note melatonin treatment activates MT1/JAK2/STAT3 pathway and then promoted A1 to A2 polarization. RT-qPCR shows mRNA expression changes in neurotrophic factors LIF and FGF2. GAPDH was used as the internal control. IL-1α + C1q + TNF-α treatment for 24h significantly decreased the mRNA expression of LIF and FGF2 when compared with the corresponding controls. Melatonin reversed the changes, but the effect of melatonin was prevented by luzindole ( D ). * P < 0.05, ** P < 0.01, *** P <0.001 (n=3 for each group).

Article Snippet: AG490 (10 μM) (the blocker of JAK2, MCE, CAS No.133550-30-5) and STAT-IN-3 (10 μM) (the blocker of STAT3, MCE, CAS No.2361304-26-7) were used along with melatonin to detect their effects on A1 astrocytes.

Techniques: In Vitro, Marker, Activity Assay, Control, Quantitative RT-PCR, Expressing

Journal: Mediators of Inflammation

Article Title: LIF Upregulates Expression of NK-1R in NHBE Cells

doi: 10.1155/MI/2006/84829

Figure Lengend Snippet: Effects of AG-490, PD-98059, or PMA on LIF-induced activation of signal transduction and activation of transcription_(STAT3) and ERK1/2 . Western blot was performed on NHBE cells that had been preincubated with or without AG-490, PD-98059, or PMA and then stimulated with LIF. (a) LIF induced activation of tyrosine phosphorylation of STAT3, and tyrosine phosphorylation of STAT3 was inhibited by AG-490, but not by PD-98059, and not affected by PMA. (b) LIF did not enhance the expression of total-STAT3, and its expression was not affected by AG-490, PD-98059, and PMA. (c) LIF induced activation of phosphorylation of ERK1/2, and ERK1/2 activation was inhibited by PD-98059, but not by AG-490; PMA increased the expression of p-ERK1/2 in NHBE cells, but there were no significant differences between the cells stimulated with LIF and the cells stimulated with LIF in the presence of PMA. (d) and (e) LIF did not enhance the expression of total-ERK1/2, furthermore, AG-490, PD-98059, and PMA also did not affect it. Experiments were repeated three times with similar results, and the data was expressed as the mean ratio (target/GAPDH) ± SD.

Article Snippet: After 24 h in serum-free medium, cells were stimulated with recombinant human LIF(Chemicon) (5 ng/ml, 30 min for detecting STAT3 and ERK1/2; 5 ng/ml, 24 h for detecting NK-1R) in pre-exposure or absence of AG-490 (JAK2 inhibitor, Biosource) (50 nmol/mL, 1 h), PD-98059 (MEK inhibitor, Cell signaling technology) (20 nmol/mL, 1 h), PMA(ALEXIS Biochemicals) (10 ng/mL, 4 h), and the small interfering RNA(siRNA) against STAT3(Genesil Biotechnology) (2 μg/mL, 24 h).

Techniques: Activation Assay, Transduction, Western Blot, Expressing

Journal: Mediators of Inflammation

Article Title: LIF Upregulates Expression of NK-1R in NHBE Cells

doi: 10.1155/MI/2006/84829

Figure Lengend Snippet: Effects of AG-490, PD-98059, PMA, or siRNA-1(STAT3) on LIF-induced expression of NK-1R detected by immunocytochemistry_(SABC × 200). Immunocytochemistry was performed on cells that had been preincubated with or without AG-490, PD-98059, PMA, or siRNA-1(STAT3) and then stimulated with LIF ((a) control, (b) PD-98059, (c) AG-490, (d) LIF, (e) PMA, (f) LIF + PD-95059, (g) LIF + PMA, (h) LIF + AG-490, (i) LIF + control siRNA, (j) LIF + sham plasmid, (k) LIF + siRNA-1 against STAT3). LIF induced expression of NK-1R, which was inhibited by AG-490, PD-98059, and siRNA-1 against STAT3, but was affected neither by the control siRNA nor the sham plasmid. Experiments were repeated three times with similar results, and the data was expressed as the mean ratio (positive cells number/total cells number) ± SD.

Article Snippet: After 24 h in serum-free medium, cells were stimulated with recombinant human LIF(Chemicon) (5 ng/ml, 30 min for detecting STAT3 and ERK1/2; 5 ng/ml, 24 h for detecting NK-1R) in pre-exposure or absence of AG-490 (JAK2 inhibitor, Biosource) (50 nmol/mL, 1 h), PD-98059 (MEK inhibitor, Cell signaling technology) (20 nmol/mL, 1 h), PMA(ALEXIS Biochemicals) (10 ng/mL, 4 h), and the small interfering RNA(siRNA) against STAT3(Genesil Biotechnology) (2 μg/mL, 24 h).

Techniques: Expressing, Immunocytochemistry, Plasmid Preparation

Journal: Mediators of Inflammation

Article Title: LIF Upregulates Expression of NK-1R in NHBE Cells

doi: 10.1155/MI/2006/84829

Figure Lengend Snippet: Effects of AG-490, PD-98059, PMA, or siRNA-1(STAT3) on LIF-induced expression of NK-1R detected by RT-PCR . RT-PCR was performed on cells that had been preincubated with or without AG-490, PD-98059, PMA, or siRNA-1(STAT3) and then stimulated with LIF. (a) LIF induced expression of NK-1R mRNA, and that was inhibited by AG-490 and PD-98059; PMA increased the expression of NK-1R mRNA in NHBE cells. (b) LIF-induced expression of NK-1R mRNA was inhibited by siRNA-1 against STAT3, but was affected neither by the control siRNA nor the sham plasmid. Experiments were repeated three times with similar results, and the data was expressed as the mean ratio_(target/β-actin) ± SD.

Article Snippet: After 24 h in serum-free medium, cells were stimulated with recombinant human LIF(Chemicon) (5 ng/ml, 30 min for detecting STAT3 and ERK1/2; 5 ng/ml, 24 h for detecting NK-1R) in pre-exposure or absence of AG-490 (JAK2 inhibitor, Biosource) (50 nmol/mL, 1 h), PD-98059 (MEK inhibitor, Cell signaling technology) (20 nmol/mL, 1 h), PMA(ALEXIS Biochemicals) (10 ng/mL, 4 h), and the small interfering RNA(siRNA) against STAT3(Genesil Biotechnology) (2 μg/mL, 24 h).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

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: 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:

Article Title: Leptin Receptor JAK2/STAT3 Signaling Modulates Expression of Frizzled Receptors in Articular Chondrocytes

doi: 10.1016/j.joca.2010.09.006

Figure Lengend Snippet: Chondrocyte cell lines (C-28/I2 and T/C-28a2) were treated with recombinant leptin (100 nM) and harvested over a time course (0–100 min). (A) C-28/I2 whole cell lysates were separated by SDS-PAGE and analyzed using antibodies specific for JAK2 and pJAK2. (B) T/C-28a2 whole cell lysates were separated by SDS-PAGE and analyzed using antibodies specific for ERK1/2 and pERK1/2. (C) C-28/I2 whole cell lysates were separated by SDS-PAGE and analyzed using antibodies specific for STAT3 and pSTAT3. Band intensities (shown as representative photographs) were quantified using densitometry and in each case t=0 was set to 1 relative unit (r.u.). Tables represent the mean of three or more separate experiments with 95% confidence intervals represented as (upper limit, lower limit) and p-values. Leptin treatment led to significant increases in pJAK2 at 10 and 30 min, pERK1/2 at 30 and 100 min, and pSTAT3 at 10, 30 and 100 min. GAPDH protein levels analyzed as controls remained unchanged in all experiments.

Article Snippet: Cycloheximide (Sigma, 10 mg/ml), JAK2 phosphorylation inhibitor Tyrphostin AG490 (LC Laboratories, 50 nM) and PI3K inhibitors LY294002 (Calbiochem, 25 μM) and wortmannin (Sigma, 50 nM) were added 30 minutes before leptin.

Techniques: Recombinant, SDS Page

Journal:

Article Title: Leptin Receptor JAK2/STAT3 Signaling Modulates Expression of Frizzled Receptors in Articular Chondrocytes

doi: 10.1016/j.joca.2010.09.006

Figure Lengend Snippet: C-28/I2 cells were pre-treated with (A) the JAK2 inhibitor AG490 (50 nM), (B) the PI3K inhibitor LY294002 (25 uM), and (C) the PI3K inhibitor wortmannin (50 nM) followed by treatment with leptin (100 nM). Cells were harvested at 24 hours and whole cell lysates were separated by SDS-PAGE and analyzed using antibodies specific for Fzd1. Leptin-mediated increases in the expression of Fzd1 were inhibited by pre-treatment with AG490 but unchanged by pre-treatment with LY294002 or wortmannin. (D) Replicates were harvested 30 min post-leptin treatment and analyzed using Akt and pAkt-specific antibodies. Leptin-mediated activation of phospho-Akt was inhibited by pre-treatment with either LY294002 or wortmannin. Bands are shown as representative photographs from four or more separate experiments. White lines separate nonadjacent lanes from the same gel. GAPDH protein levels analyzed as controls remained unchanged in all experiments.

Article Snippet: Cycloheximide (Sigma, 10 mg/ml), JAK2 phosphorylation inhibitor Tyrphostin AG490 (LC Laboratories, 50 nM) and PI3K inhibitors LY294002 (Calbiochem, 25 μM) and wortmannin (Sigma, 50 nM) were added 30 minutes before leptin.

Techniques: SDS Page, Expressing, Activation Assay

Journal:

Article Title: Leptin Receptor JAK2/STAT3 Signaling Modulates Expression of Frizzled Receptors in Articular Chondrocytes

doi: 10.1016/j.joca.2010.09.006

Figure Lengend Snippet: (A) Schematic representation of the hybrid receptor ELR consisting of the extracellular domain of the erythropoietin (Epo) receptor and the intracellular domain of LRb. Indicated are JAK2, JAK2-induced phosphorylated tyrosine residues (Tyr985, Tyr1077 and Tyr1138) and respective downstream signaling pathways. C-28/I2 cells were transfected with ELR expression plasmid (2 μg), treated with recombinant Epo (10 mU/ml) and harvested over a time course (0–48 hours). Whole cell lysates were analyzed using antibodies specific for pSTAT3 (B) and Fzd1 (C). Epo treatment of ELR transfectants induced phosphorylation of STAT3 (30 min) and increased expression of Fzd1 at both 24 and 48 hours. Bands are shown as representative photographs from three or more separate experiments. White lines separate nonadjacent lanes from the same gel. GAPDH protein levels analyzed as controls remained unchanged during Epo treatment.

Article Snippet: Cycloheximide (Sigma, 10 mg/ml), JAK2 phosphorylation inhibitor Tyrphostin AG490 (LC Laboratories, 50 nM) and PI3K inhibitors LY294002 (Calbiochem, 25 μM) and wortmannin (Sigma, 50 nM) were added 30 minutes before leptin.

Techniques: Protein-Protein interactions, Transfection, Expressing, Plasmid Preparation, Recombinant, Phospho-proteomics

Journal: Oncology Reports

Article Title: CAFs enhance paclitaxel resistance by inducing EMT through the IL-6/JAK2/STAT3 pathway

doi: 10.3892/or.2018.6311

Figure Lengend Snippet: The JAK2/STAT3 pathway is required for regulating EMT in ovarian cancer cells induced by IL-6. (A) The culture supernatants of CAFs and NFs were applied to OVCAR3 cells. The phosphorylation levels of JAK2 and STAT3 in OVCAR3 cells treated with CAF supernatant were significantly higher than those in cells treated with NF supernatant. After the addition of IL-6 mAb, the phosphorylation levels of JAK2 and STAT3 were decreased. (B) After the JAK2/STAT3-signaling-pathway-specific inhibitor AG490 was added, the expression of the interstitial markers N-cadherin and Vimentin was decreased and the expression of the epithelium marker E-cadherin was increased. These results indicated that CAF-derived IL-6 could mediate EMT in OVCAR3 cells via the JAK2/STAT3 pathway.

Article Snippet: The JAK2/STAT3 pathway inhibitor AG490 was purchased from APExBIO (Apexbio Technology LLC, Houston, TX, USA) and the β-TGF inhibitor SB431542 was obtained from Selleck Chemicals (Houston, TX, USA).

Techniques: Phospho-proteomics, Expressing, Marker, Derivative Assay

Journal: Oncology Reports

Article Title: CAFs enhance paclitaxel resistance by inducing EMT through the IL-6/JAK2/STAT3 pathway

doi: 10.3892/or.2018.6311

Figure Lengend Snippet: CAF-derived IL-6 enhances paclitaxel resistance of ovarian cancer cells through cellular EMT. (A) The culture supernatants of CAFs and NFs were applied to OVCAR3 cells. The number of apoptotic cells was decreased in OVCAR3 cells treated with CAF supernatant. After the addition of IL-6 mAb, paclitaxel resistance was reduced and paclitaxel-induced apoptosis was promoted. (B) The expression of pro-apoptotic protein Bax and caspase-3-p17 was decreased, and the expression of apoptosis-suppressing protein Bcl-2 was enhanced in cells treated with CAF supernatant compared with NF supernatant. (C) The number of apoptotic cells treated with paclitaxel was increased after the addition of SB431542 and AG490.

Article Snippet: The JAK2/STAT3 pathway inhibitor AG490 was purchased from APExBIO (Apexbio Technology LLC, Houston, TX, USA) and the β-TGF inhibitor SB431542 was obtained from Selleck Chemicals (Houston, TX, USA).

Techniques: Derivative Assay, Expressing