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: iScience

Article Title: Inhibition of CD44 suppresses the formation of fibrotic scar after spinal cord injury via the JAK2/STAT3 signaling pathway

doi: 10.1016/j.isci.2024.108935

Figure Lengend Snippet: STAT3 was the downstream of JAK2 in CD44-mediated fibrotic effect in the inflammatory microenvironment (A) Western blotting analyzed the expression of p -JAK2, p-STAT3, STAT3, and p -Smad2 in the fibroblasts stimulated with IS after inhibition of AG490. (B‒E) Quantification of western blotting results in A. N = 3 of each group by one-way ANOVA. (F) Western blotting analyzed the expression of CD44, α-SMA, collagen-I, FN, and laminin in the fibroblasts stimulated with IS after inhibition of AG490. (G‒K) Quantification of western blotting results in F. N = 3 of each group by one-way ANOVA. (L and M) Quantification analysis of the rate of proliferation (%) (L) per 10 6 μm 2 (n = 5) and cell numbers of migration (M) per 10 6 μm 2 (n = 5) in different groups by one-way ANOVA. All data were presented as mean ± SEM. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (See also Figure S3 ).

Article Snippet: AG490 , Targetmol , Cat#T2600;CAS:133550-30-8;Batch:147766.

Techniques: Western Blot, Expressing, Inhibition, Analysis, Migration

Journal: iScience

Article Title: Inhibition of CD44 suppresses the formation of fibrotic scar after spinal cord injury via the JAK2/STAT3 signaling pathway

doi: 10.1016/j.isci.2024.108935

Figure Lengend Snippet: Diagram of molecule mechanism of CD44-JAK2-STAT3 in the fibroblasts following SCI, macrophages are activated and secrete varieties of cytokines and inflammatory factors, and recruit fibroblasts to participate in the subsequent repair process Then CD44 on the fibroblasts would be activated by these proinflammatory factors, which could be inhibited by IM7. The signal is transferred into cells, and activates the JAK2 and STAT3. Activated STAT3 enters into nucleus and regulates the expression of fibrosis-related genes at the transcriptional level by binding specific DNA promoter sequences, which ultimately promotes the proliferation, differentiation, and migration of fibroblasts, leading to the deposition of excessive ECM proteins and finally forms dense fibrotic scar tissue.

Article Snippet: AG490 , Targetmol , Cat#T2600;CAS:133550-30-8;Batch:147766.

Techniques: Expressing, Binding Assay, Migration, Tissue

Journal: iScience

Article Title: Inhibition of CD44 suppresses the formation of fibrotic scar after spinal cord injury via the JAK2/STAT3 signaling pathway

doi: 10.1016/j.isci.2024.108935

Figure Lengend Snippet:

Article Snippet: AG490 , Targetmol , Cat#T2600;CAS:133550-30-8;Batch:147766.

Techniques: Cell-Signaling, Recombinant, In Vivo, Lysis, Acid Assay, Software

Journal: Frontiers in physiology

Article Title: Acute exercise reduces feeding by activating IL-6/Tubby axis in the mouse hypothalamus.

doi: 10.3389/fphys.2022.956116

Figure Lengend Snippet: FIGURE 3 Hypothalamic IL-6 action through JAK2 to activate TUB. (A) Evaluation of cumulative food intake (g) at 4 h, 12 h, and 24 h (n = 5 in each group); Western blot showing (B) TUB tyrosine phosphorylation and (C) TUB associated with JAK2 from hypothalamic lysates from wt/wt mice (6–8 weeks old) obtained in the colony of B6-tub/tub mice under resting conditions (Sed). The mice were treated with vehicle or AG490 (JAK2inhibitor), IL-6, or IL-6 with AG490 by intracerebroventricular (ICV) injections. We used male mice. All values are expressed as means ± standard deviation (SD). Western blot assays (n = 4 in each group). Two-way ANOVA was used to analyze A, while one-way ANOVA with Tukey’s multiple comparisons tests was used to analyze B and C. *ICV IL-6 vs. Veh, AG490, and IL-6 plus AG490 (p < 0.0078); #ICV IL-6 vs. Veh, AG490 and IL-6 plus AG490 (p < 0.0041); ϕ ICV IL-6 vs. Veh, AG490, and IL-6 plus AG490 (p < 0.0136).

Article Snippet: ICV injection of AG490 (50 mM) (cat #0414, Tocris Bioscience, Bristol, United Kingdom) was used to inhibit JAK2 as previously described (Damm et al., 2013; Quaresma et al., 2017).

Techniques: Western Blot, Phospho-proteomics, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: An Autocrine IL-6/IGF-1R Loop Mediates EMT and Promotes Tumor Growth in Non-small Cell Lung Cancer

doi: 10.7150/ijbs.31999

Figure Lengend Snippet: IL-6 induced EMT through activation of STAT3. Cells were transfected with either STAT3 siRNA (siSTAT3) or negative control siRNA (NC siRNA) and activation of STAT3 was measured by western blot (A) . Cells were incubated with IL-6 (50ng/ml) with or without AG 490 (50uM/L) for 48 hours and harvested for western blot of EMT markers (B) and JAK/STAT3 signaling molecules (D) . Cells were transfected with siSTAT3 and EMT markers (C) and JAK/STAT3 signaling molecules (E) were measured by western blotting. Cells were treated with IL-6 with or without IL-6 antibody and EMT markers were measured (F) . Blots were representatives of three experiments.

Article Snippet: The inhibitors, dissolved in dimethyl sulfoxide (DMSO), were used in the following concentrations: STAT3 inhibitor AG-490 (50uM/L) (Selleck Chemicals, Houston, USA), IGF-1R inhibitor AG-1024 (5uM/L) (Selleck Chemicals), AKT inhibitor MK-2206 2HCl (5uM/L) (Selleck Chemicals, Houston, USA), JAK1 inhibitor ZM 39923 HCl (50uM /L) (Selleck Chemicals, Houston, USA).

Techniques: Activation Assay, Transfection, Negative Control, Western Blot, Incubation

Journal: International Journal of Biological Sciences

Article Title: An Autocrine IL-6/IGF-1R Loop Mediates EMT and Promotes Tumor Growth in Non-small Cell Lung Cancer

doi: 10.7150/ijbs.31999

Figure Lengend Snippet: IL-6 and IGF-1R cross-signal to promote EMT. PC-9 cells were treated with IL-6 with or without IGF-1R inhibitor AG1024 for 48 hours, and IGF-1R pathway (A) and JAK/STAT3 pathway (B) were tested using western blot. Cells were treated with IL-6 with or without STAT3 inhibitor AG490 for 48 hours, and IGF-1R pathway were tested using western blot (C) . STAT3 over-expressed plasmids were constructed and promoter activity of IGF-1R was studied by reporter assay (D) . Blots were representatives of three experiments and results were quantified using Quantity One for comparisons between IL-6 group and control or between IL-6 plus an inhibitor co-treated cells and IL-6 group. *, p<0.05. #, p>0.05 not significant.

Article Snippet: The inhibitors, dissolved in dimethyl sulfoxide (DMSO), were used in the following concentrations: STAT3 inhibitor AG-490 (50uM/L) (Selleck Chemicals, Houston, USA), IGF-1R inhibitor AG-1024 (5uM/L) (Selleck Chemicals), AKT inhibitor MK-2206 2HCl (5uM/L) (Selleck Chemicals, Houston, USA), JAK1 inhibitor ZM 39923 HCl (50uM /L) (Selleck Chemicals, Houston, USA).

Techniques: Western Blot, Construct, Activity Assay, Reporter Assay, Control

Journal: International Journal of Biological Sciences

Article Title: An Autocrine IL-6/IGF-1R Loop Mediates EMT and Promotes Tumor Growth in Non-small Cell Lung Cancer

doi: 10.7150/ijbs.31999

Figure Lengend Snippet: IL-6/ IGF-1R autocrine loop is mediated via STAT3. Cells were treated with IL-6 with or without an inhibitor (AG490, AG1024 or AKT inhibitor MK-2206 2HCl) and RT-PCR analyses were performed after 48 hours. Gene expressions of IL-6 (A and E) , IGF-1 (B and F) and IGF-2 (C and G) were compared between IL-6-treated cells and control group and between IL-6 plus inhibitor co-treated cells and IL-6 group. ELISAs were performed for IGF-1 (D) and IL-6 (H) and results were compared in the same way as the above mentioned RT-PCR analyses. *, p<0.05. **, p<0.01. #, p>0.05 not significant.

Article Snippet: The inhibitors, dissolved in dimethyl sulfoxide (DMSO), were used in the following concentrations: STAT3 inhibitor AG-490 (50uM/L) (Selleck Chemicals, Houston, USA), IGF-1R inhibitor AG-1024 (5uM/L) (Selleck Chemicals), AKT inhibitor MK-2206 2HCl (5uM/L) (Selleck Chemicals, Houston, USA), JAK1 inhibitor ZM 39923 HCl (50uM /L) (Selleck Chemicals, Houston, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control

Journal: International Journal of Biological Sciences

Article Title: An Autocrine IL-6/IGF-1R Loop Mediates EMT and Promotes Tumor Growth in Non-small Cell Lung Cancer

doi: 10.7150/ijbs.31999

Figure Lengend Snippet: Inhibiting STAT3 partly restores gefitinib sensitivity. (A to D) PC-9 cells were treated with gefitinib (50nmol/L) for 48 h. Cells were stained with annexin V-FITC and PI and subsequently analyzed using flow cytometry. (E) Quantification of gefitinib-induced apoptotic cells by annexin/PI double-staining. Apoptosis was evaluated by determining the percentage of annexin V-positive cells. The data are representative of three independent experiments and compared between IL-6 treated cells and gefitinib group, or between IL-6 plus an inhibitor co-treated cells and IL-6 group. *, p<0.05. **, p<0.01. #, p>0.05 not significant.

Article Snippet: The inhibitors, dissolved in dimethyl sulfoxide (DMSO), were used in the following concentrations: STAT3 inhibitor AG-490 (50uM/L) (Selleck Chemicals, Houston, USA), IGF-1R inhibitor AG-1024 (5uM/L) (Selleck Chemicals), AKT inhibitor MK-2206 2HCl (5uM/L) (Selleck Chemicals, Houston, USA), JAK1 inhibitor ZM 39923 HCl (50uM /L) (Selleck Chemicals, Houston, USA).

Techniques: Staining, Flow Cytometry, Double Staining

Journal: International Journal of Biological Sciences

Article Title: An Autocrine IL-6/IGF-1R Loop Mediates EMT and Promotes Tumor Growth in Non-small Cell Lung Cancer

doi: 10.7150/ijbs.31999

Figure Lengend Snippet: Inhibiting either STAT3 or IGF-1R attenuates cell migration. PC-9 cells (A) and A549 cells (B) were seeded and treated either with IL-6 alone or in the presence of both IL-6 and an STAT3 or IGF-1R inhibitor. Migration was shown as photograph and the number of migratory cells was determined using a transwell matrix penetration assay and quantified for analysis of significance. *, p<0.05. #, p>0.05 not significant.

Article Snippet: The inhibitors, dissolved in dimethyl sulfoxide (DMSO), were used in the following concentrations: STAT3 inhibitor AG-490 (50uM/L) (Selleck Chemicals, Houston, USA), IGF-1R inhibitor AG-1024 (5uM/L) (Selleck Chemicals), AKT inhibitor MK-2206 2HCl (5uM/L) (Selleck Chemicals, Houston, USA), JAK1 inhibitor ZM 39923 HCl (50uM /L) (Selleck Chemicals, Houston, USA).

Techniques: Migration

Journal: Journal of molecular and cellular cardiology

Article Title: Differential hypertrophic effects of cardiotrophin-1 on adult cardiomyocytes from normotensive and spontaneously hypertensive rats.

doi: 10.1016/j.yjmcc.2006.03.433

Figure Lengend Snippet: Fig. 5. Cells were pre-incubated with the indicated chemical inhibitors for 1 h and then with CT-1 for 48 h. Cell area (A), length (B) and width (C) were determined by planimetry. Values represent the mean±standard error of a minimum of 120 cardiomyocytes per experimental condition. Three independent experiments performed in three rats yielded identical results. **p<0.01 vs. baseline; *p<0.05 vs. baseline; $$p<0.01 vs. CT-1; $p<0.05 vs. CT-1. W, Wortmannin; PD1, PD98059 at 10−6 mol/L to inhibit p42/44; PD30, PD98059 at 30×10−6 mol/L to inhibit ERK-5; AG, AG490.

Article Snippet: 10− 6 mol/L to inhibit ERK5 (Calbiochem), the STATs inhibitor AG490 at 10−6 mol/L (Calbiochem), antibodies against LIFR (Santa Cruz) and gp130 (Santa Cruz) at 10−6 mol/L.

Techniques: Incubation

Journal: Journal of molecular and cellular cardiology

Article Title: Differential hypertrophic effects of cardiotrophin-1 on adult cardiomyocytes from normotensive and spontaneously hypertensive rats.

doi: 10.1016/j.yjmcc.2006.03.433

Figure Lengend Snippet: Fig. 6. (A) SHR cardiomyocytes were pre-incubated for 1 h with the STAT3 blocker AG490 and then with CT-1 for 24 h. A representative Western blot and the histogram with bars representing the mean±standard error of three independent experiments are shown for each protein. *p<0.01 vs. baseline and CT-1+AG. AG, AG490 (B) Angiotensinogen mRNA was assayed in cardiomyocytes from Wistar rats and SHR before and after 3 h incubation with CT-1. Bars represent mean±standard error of triplicates in three independent experiments. $p<0.01 vs. baseline; *p<0.01 vs. CT-1.

Article Snippet: 10− 6 mol/L to inhibit ERK5 (Calbiochem), the STATs inhibitor AG490 at 10−6 mol/L (Calbiochem), antibodies against LIFR (Santa Cruz) and gp130 (Santa Cruz) at 10−6 mol/L.

Techniques: Incubation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Prolactin Regulates Tuberoinfundibular Dopamine Neuron Discharge Pattern: Novel Feedback Control Mechanisms in the Lactotrophic Axis

doi: 10.1523/JNEUROSCI.0129-12.2012

Figure Lengend Snippet: The LV and HV components of IPrl are activated by distinct signal transduction pathways. A, I–V relationship of IPrl in control (green; n = 14), wortmannin (200 nm; red; n = 8), and AG490 (10 μm; blue; n = 3). Wortmannin, but not AG490, significantly modifies IPrl at VHold = +40 mV. Values expressed as mean ± SEM. ***p < 0.005. B, I–V relationship of IPrl at a concentration of 500 nm Prl (green; n = 14), 40 nm Prl in which both the LV and HV components were present (red; n = 13 of 19), and 40 nm Prl in which only the LV component was present (blue; n = 6 of 19). Values expressed as mean ± SEM. ***p < 0.005.

Article Snippet: TTX was purchased from Alomone Labs. AG490 [( E )-2-cyano-3-(3,4-dihydrophenyl)- N -(phenylmethyl)-2-propenamide], nimodipine, paxilline, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}} SKF96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1 H -imidazole hydrochloride), UCL1648 [6,10-diaza-3(1,3)8,(1,4)-dibenzena-1,5(1,4)-diquinolinacy clodecaphane], and wortmannin were purchased from Tocris Bioscience. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 6. caption a7 Prl broadens TIDA APs.

Techniques: Transduction, Concentration Assay

Journal: Biochimica et biophysica acta

Article Title: Prolactin and glucocorticoid signaling induces lactation-specific tight junctions concurrent with β-casein expression in mammary epithelial cells.

doi: 10.1016/j.bbamcr.2016.04.023

Figure Lengend Snippet: Fig. 4. Prl and Dex influence the expression patterns of Cldn3 and Cldn4 in MECs (A) Immunostaining images of Cldn3 (green) and Cldn4 (green) with Ocln (red) in MECs cultured with or without Prl (0.5 U/mL) and Dex (1 μM) for 5 days. Nuclei stained with DAPI are blue. The scale bars represent 10 μm. (B–F) The results of western blotting and densitometry analysis of Cldn3 and Cldn4 with β-actin used as an internal control. MECs were cultured for 5 days with or without Prl (0.5 U/mL or 0–2.5 U/mL) and Dex (1 μM). MECs were pretreated with 10 μM AG490 (JAK inhibitor) and 5 μM RU486 (glucocorticoid receptor antagonist) 1 h before Prl (0.5 U/mL) and Dex (100 nM) treatment. The data are presented as the mean (SEM); n = 5; *p b 0.05 and **p b 0.005. (G) The expression of Cldn3 and Cldn4, determined by qRT-PCR. The data are presented as the mean (SEM); n = 6; *p b 0.05 and **p b 0.005.

Article Snippet: AG490 (JAK inhibitor; cat. no. T9969) and RU486 (GR antagonist; cat. no. M3321)were purchased from LKT Laboratories, Inc. (St. Paul, MN).

Techniques: Expressing, Immunostaining, Cell Culture, Staining, Western Blot, Control, Quantitative RT-PCR