jak2 specific inhibitor ag490  (MedChemExpress)

Journal: Cell Biology and Toxicology

Article Title: METTL3, m6A modification, and EGR1: interplay affecting myocardial I/R injury outcomes

doi: 10.1007/s10565-024-09937-7

Figure Lengend Snippet: The effect of EGR1 on mitophagy through the regulation of the JAK2/STAT3 pathway. Note: ( A ) Western blot analysis of the expression and quantification of JAK2/STAT3 pathway-related proteins in cardiomyocytes from different treatment groups; ( B ) Schematic diagram showing the treatment of AG490 after shEGR1 transfection in the H/R damage model; ( C ) Western blot analysis of the expression and quantification of JAK2/STAT3 pathway-related proteins in cardiomyocytes from different treatment groups; ( D ) TEM to observe the morphology of cardiomyocyte mitochondria in each group, with a scale bar of 500 nm and arrows indicating mitochondria; ( E ) Representative immunofluorescence images showing the co-localization of GFP-LC3B (green) and mitochondria (MTR-Red, red) in cardiomyocytes from different treatment groups, with a scale bar of 25 μm, and quantification of the number of co-localized spots between GFP-LC3B and mitochondria, with DAPI (blue) indicating the nucleus; ( F ) Representative immunofluorescence images showing the co-localization of MTR-Green (green) and lysosomes (LTR, red) in cardiomyocytes from different treatment groups, with a scale bar of 50 μm, and quantification of the number of co-localized spots between lysosomes and mitochondria in each cell; ( G ) Western blot analysis of the expression and quantification of mitophagy-related proteins in cardiomyocytes from different treatment groups; ( H ) Assessment of cell viability of cardiomyocytes in different treatment groups using the CCK-8 method; ( I ) Measurement of the levels of cTnI and CK-MB in the supernatant of cardiomyocytes in different treatment groups using the ELISA method. * indicates a significant difference between two groups with P < 0.05, ** indicates a significant difference between two groups with P < 0.01, *** indicates a significant difference between two groups with P < 0.001, **** indicates a significant difference between two groups with P < 0.0001. All experiments were repeated three times

Article Snippet: Furthermore, the H/R+shEGR1+3-MA and H/R+shEGR1+AG490 groups were transfected with shEGR1 and treated with the mitophagy inhibitor 3-Methyladenine (3-MA, 5 mM; MCE, HY-19312) or the JAK2/STAT3 pathway-specific inhibitor AG490 (50 μM; MCE, HY-12000) during H/R model construction for 36 hours (Chen et al. ; Zeng et al. ; Yin et al. ).

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, CCK-8 Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Biology and Toxicology

Article Title: METTL3, m6A modification, and EGR1: interplay affecting myocardial I/R injury outcomes

doi: 10.1007/s10565-024-09937-7

Figure Lengend Snippet: The Impact of EGR1/JAK2/STAT3 axis-mediated mitophagy dysfunction on pyroptosis. Note: ( A ) Ultrastructural morphology of cardiomyocytes observed under SEM. Scale bar=10 μm; ( B ) Representative images of TUNEL staining in cardiomyocytes from each group (Scale bar=50 μm) and the percentage of TUNEL-positive cells; ( C ) LDH release results in cardiomyocytes from each group measured by ELISA; ( D ) Expression and quantification of pyroptosis-related proteins in myocardial cells from each group detected by Western blot; ( E ) Levels of IL1β and IL18 in the supernatant of cardiomyocytes from each group measured by ELISA; ( F ) Ultrastructural morphology of cardiomyocytes observed under SEM. Scale bar=10 μm; ( G ) Representative images of TUNEL staining in cardiomyocytes from each group (Scale bar=50 μm) and the percentage of TUNEL-positive cells; ( H ) LDH release results in cardiomyocytes from each group measured by ELISA; ( I ) Expression and quantification of pyroptosis-related proteins in myocardial cells from each group detected by Western blot; ( J ) Levels of IL1β and IL18 in the supernatant of cardiomyocytes from each group measured by ELISA; C1-Cas1: Cleaved-Caspase 1; * indicates p < 0.05 compared to the control group, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. All experiments were repeated three times

Article Snippet: Furthermore, the H/R+shEGR1+3-MA and H/R+shEGR1+AG490 groups were transfected with shEGR1 and treated with the mitophagy inhibitor 3-Methyladenine (3-MA, 5 mM; MCE, HY-19312) or the JAK2/STAT3 pathway-specific inhibitor AG490 (50 μM; MCE, HY-12000) during H/R model construction for 36 hours (Chen et al. ; Zeng et al. ; Yin et al. ).

Techniques: TUNEL Assay, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control

Journal: Cell Biology and Toxicology

Article Title: METTL3, m6A modification, and EGR1: interplay affecting myocardial I/R injury outcomes

doi: 10.1007/s10565-024-09937-7

Figure Lengend Snippet: The effect of METTL3 on the characterization of I/R mice through EGR1/JAK2/STAT3 pathway. Note: ( A ) Expression of METTL3 and EGR1 in cardiac tissue of different groups of mice (n=8) as measured by RT-qPCR; ( B ) Protein expression and quantification of METTL3 and EGR1 in cardiac tissue of different groups of mice (n=8) as determined by Western blot; ( C ) Expression and quantification of JAK2/STAT3 pathway-related proteins in cardiac tissue of different groups of mice (n=8) as measured by Western blot; ( D ) Cardiac ultrasound evaluation of heart function-related indices in different groups of mice (n=6); ( E ) Representative images of Evans blue/TTC double staining in cardiac tissue of different groups of mice (n=8), with blue regions representing normal cardiac tissue, red regions representing ischemic myocardium (AAR), and white regions representing the infarct area (INF) of cardiac tissue. Quantification of INF/AAR and AAR/LV percentages, where LV represents the left ventricle; ( F ) Representative images of HE-stained cardiac tissue in different groups of mice (n=8), Scale bar=50 μm; ( G ) Detection of cTnI and CK-MB levels in serum of different groups of mice (n=8) using ELISA; * indicates a significant difference ( p < 0.05) between two groups, ** indicates a significant difference ( p < 0.01) between two groups, *** indicates a highly significant difference ( p < 0.001) between two groups, **** indicates an extremely significant difference ( p < 0.0001) between two groups

Article Snippet: Furthermore, the H/R+shEGR1+3-MA and H/R+shEGR1+AG490 groups were transfected with shEGR1 and treated with the mitophagy inhibitor 3-Methyladenine (3-MA, 5 mM; MCE, HY-19312) or the JAK2/STAT3 pathway-specific inhibitor AG490 (50 μM; MCE, HY-12000) during H/R model construction for 36 hours (Chen et al. ; Zeng et al. ; Yin et al. ).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Double Staining, Staining, Enzyme-linked Immunosorbent Assay

Journal: BMC Cancer

Article Title: CCL18-NIR1 promotes oral cancer cell growth and metastasis by activating the JAK2/STAT3 signaling pathway

doi: 10.1186/s12885-020-07073-z

Figure Lengend Snippet: AG490 inhibited the CCL18-induced OSCC proliferation, migration, invasion, and EMT. a Western blotting showed that AG490 could effectively inhibit the expression of phosphorylation of both JAK2 and STAT3 in HSC6 cells. b, c and d Results of CCK8 and transwell demonstrated AG490 impaired the proliferation, migration, and invasion of rCCL18-stimulated OSCC cells. e The increased expression of E-cadherin and the decreased expression of N-cadherin and ZEB2 were found in the rCCL18 + AG490 group when compared with the rCCL18 + NC control group. ( *P < 0.05, **P < 0.01, ***P < 0.001, vs. control). The full-length blots are presented in Supplementary Fig. S

Article Snippet: The JAK2/STAT3 signaling pathway specific inhibitor AG490 was purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Migration, Western Blot, Expressing, Phospho-proteomics, Control

Journal: Developmental neuroscience

Article Title: Ciliary Neurotrophic Factor Promotes Müller Glia Differentiation from the Postnatal Retinal Progenitor Pool

doi: 10.1159/000082278

Figure Lengend Snippet: Effects of signaling pathway-specific perturbation on CNTF-induced Müller glial genesis. A, B Percentages of GFAP+ cells (A) and cyclin D3+ cells (B) among total cells in P0 retinal explants cultured for 6 DIV in the presence or absence of CNTF (100 ng/ml) and the MEK inhibitor U0126 (10 μM). Results are presented as the mean ± SEM of 6 independent experiments (n = 6). C Western blots showing inhibition of CNTF-induced STAT3 phosphorylation by AG490 in monolayer retinal cultures. AG490 was added to cells 1 h prior to 15 min of CNTF (10 ng/ml) stimulation. Top and bottom panels show phospho-proteins and total proteins, respectively. D Percentages of Müller markers GFAP (gray) and CRALBP (white) in P0 retinal explants cultured for 6 DIV in the presence or absence of CNTF (10 ng/ml) and Jak inhibitor AG490 (10μM). Results are expressed as the mean ± SEM of 3 independent experiments (n = 3). E Percentages of Müller marker cyclin D3+ cells among GFP+ cells in transfected P0 retinal explants cultured for 6 DIV without exogenous ligands. Results are expressed as the mean ± SEM of 3 independent experiments (n = 3). The asterisks * and ** indicate p values <0.05 and <0.01 between conditions, respectively.

Article Snippet: The specific inhibitor of Jak2 (AG490) was purchased from Calbiochem (San Diego, Calif., USA).

Techniques: Cell Culture, Western Blot, Inhibition, Phospho-proteomics, Marker, Transfection

Journal: Scientific Reports

Article Title: The novel application of cordycepin in maintaining stem cell pluripotency and increasing iPS cell generation efficiency

doi: 10.1038/s41598-020-59154-5

Figure Lengend Snippet: Cordycepin-mediated signaling pathway in mouse ES cells. ( A , B ) Western blot analyses of Jak2, Stat3 and their phosphorylated forms in mouse ES cells following either LIF (1000 units/ml) or cordycepin treatment (1.25, 2.5, 5, 10 μM). Jak2 inhibitor (AG490) was used to assess the effects of cordycepin on Jak2 and Stat3 activation. ( C , D ) Immunofluorescent staining results of Nanog and SSEA1 protein in cordycepin-treated mouse ES cells. AG490 was added to verify the effects of cordycepin on maintaining stem cell properties. Digital images were taken at a magnification of 200X (scale bar: 100 μm). Bars represent mean and SD. Differences between the control group (Ctrl) and experimental groups (LIF and cordycepin) were assessed by two-tailed Student’s t test. *P < 0.05 indicates statistical significance (*P = 0.01–0.05).

Article Snippet: The Jak2 specific inhibitor, AG490 (Sigma-Aldrich), was dissolved in ethanol at a concentration of 5 mM and stored at −20 °C.

Techniques: Western Blot, Activation Assay, Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: The novel application of cordycepin in maintaining stem cell pluripotency and increasing iPS cell generation efficiency

doi: 10.1038/s41598-020-59154-5

Figure Lengend Snippet: Cordycepin induced the expression of IL-6 family proteins and EGF in ES cells. ( A ) Real-time PCR assay was performed to determine the expression of LIF, IL-6, IL-11, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and epidermal growth factor (EGF) in cordycepin-treated mouse ES cells. AG490 was used to validate the role of cordycepin in promotion of expressions of LIF-receptor-related cytokines. ( B , C ) The cordycepin-mediated expressions of LIF and IL-6 in mouse ES cells were examined by ELISA. AG490 was used to evaluate the relationship between cordycepin and Jak2. Bars represent mean and SD. The statistical analysis was conducted using two-tailed Student’s t test ( # p = 0.001–0.01 versus AG negative control).

Article Snippet: The Jak2 specific inhibitor, AG490 (Sigma-Aldrich), was dissolved in ethanol at a concentration of 5 mM and stored at −20 °C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Negative Control

Journal: BioMed Research International

Article Title: α-Hederin Arrests Cell Cycle at G2/M Checkpoint and Promotes Mitochondrial Apoptosis by Blocking Nuclear Factor-κB Signaling in Colon Cancer Cells

doi: 10.1155/2018/2548378

Figure Lengend Snippet: Blockade of NF- κ B signaling is required for α -hederin induction of mitochondrial apoptosis in IL-6-stimulated SW620 cells. SW620 cells were treated with vehicle, IL-6, and/or α -hederin or PDTC at indicated concentrations for 24 h. (a) Hoechst 33258 fluorescence staining. Morphologic changes of apoptotic cells were visualized under a fluorescence microscope (200 x magnification). (b) Western blot analysis of protein abundance of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP with quantification. Significance: ∗∗P <0.01 versus control, # P <0.05 versus IL-6, ## P <0.01 versus IL-6.

Article Snippet: NF- κ B specific inhibitor pyrrolidine dithiocarbamate (PDTC), JAK2/STAT3 signaling specific inhibitor AG490, and ERK specific inhibitor U0126 were obtained from Selleck Chemicals (Houston, TX, USA).

Techniques: Fluorescence, Staining, Microscopy, Western Blot, Quantitative Proteomics, Control