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: Scientific Reports

Article Title: Combinatorial Drug Testing in 3D Microtumors Derived from GBM Patient-Derived Xenografts Reveals Cytotoxic Synergy in Pharmacokinomics-informed Pathway Interactions

doi: 10.1038/s41598-018-26840-4

Figure Lengend Snippet: Dose response of JX10UAB to WP1066, Selumetinib, Crizotinib, and Cediranib at Day 7. Raw MTT absorbance of (A) WP1066, (B) Selumetinib, (C) Crizotinib and (D) Cediranib are shown with DMSO control representing the 0 µM dose with calculated IC50 indicated (N/A = not applicable). (E – I) Calcein-AM imaging of DMSO or highest doses of each drug (30 µM, 27 µM, 27.7 µM, and 50 µM, respectively) at Day 7 at 4x magnification and 250 ms exposure. Scale bar is 500 µm.

Article Snippet: Serial dilutions of four small molecule inhibitors (SMIs), WP1066 (inhibiting JAK2/STAT3), selumetinib (inhibiting MEK1/2), crizotinib (inhibiting c-MET and ALK), and cediranib (inhibiting VEGFR, Flt-1, Flt-4, c-Kit, and PDGFR) (Selleckchem, Houston, TX, Catalog Numbers S2796, S1008, S1068, S1017, respectively), along with the vehicle control (dimethyl sulfoxide or DMSO) were used to establish a dose titration curve for determining appropriate concentrations of drugs required for 50% inhibition in vitro (IC 50 s) in the 3D microtumor model. All dose-finding experiments were performed as 3-fold serial dilutions, 0.5% final DMSO in triplicate.

Techniques: Control, Imaging

Journal: Scientific Reports

Article Title: Combinatorial Drug Testing in 3D Microtumors Derived from GBM Patient-Derived Xenografts Reveals Cytotoxic Synergy in Pharmacokinomics-informed Pathway Interactions

doi: 10.1038/s41598-018-26840-4

Figure Lengend Snippet: Synergistic combination index (CI) values of WP1066, Selumetinib, Crizotinib, and Cediranib SMIs in combination for all xenolines at Day 7.

Article Snippet: Serial dilutions of four small molecule inhibitors (SMIs), WP1066 (inhibiting JAK2/STAT3), selumetinib (inhibiting MEK1/2), crizotinib (inhibiting c-MET and ALK), and cediranib (inhibiting VEGFR, Flt-1, Flt-4, c-Kit, and PDGFR) (Selleckchem, Houston, TX, Catalog Numbers S2796, S1008, S1068, S1017, respectively), along with the vehicle control (dimethyl sulfoxide or DMSO) were used to establish a dose titration curve for determining appropriate concentrations of drugs required for 50% inhibition in vitro (IC 50 s) in the 3D microtumor model. All dose-finding experiments were performed as 3-fold serial dilutions, 0.5% final DMSO in triplicate.

Techniques:

Journal: Pharmacological research

Article Title: Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling

doi: 10.1016/j.phrs.2020.105414

Figure Lengend Snippet: Cardiac fibroblasts were treated with resistin (100ng/ml) and TGFβ1 (10ng/ml) for the indicated times. The phosphorylation of Smad3, JAK2, STAT3, JNK, c-Jun was analyzed by western blotting (A), and densitometry quantifications were performed (B). β-actin was used as internal controls. The data are mean ± SEM of at least three experiments in triplicates. p*< 0.05, **p < 0.01, ***p < 0.001, 0 min vs indicated minutes in figure.

Article Snippet: For JAK and JNK inhibition, cells were incubated with JAK2 inhibitor (WP1066, # sc-203282, Santa Cruz Biotechnology) and JNK inhibitor (SP600125, # sc-200635, Santa Cruz Biotechnology) at the indicated concentrations selected as per earlier studies [ 28 ].

Techniques: Phospho-proteomics, Western Blot

Journal: Pharmacological research

Article Title: Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling

doi: 10.1016/j.phrs.2020.105414

Figure Lengend Snippet: C57B6 adult mice were infected with AAV9-resistin (AAV9-Retn) or AAV9-empty vectors for 10 weeks. The phosphorylation of JAK2, STAT3 (A) and JNK, c-Jun (B) was analyzed by western blotting with densitometry quantification shown below the blots. β-actin was used as internal controls. The data are mean ± SEM of n=5. p*< 0.05, **p < 0.01 vs AAV9-Empty.

Article Snippet: For JAK and JNK inhibition, cells were incubated with JAK2 inhibitor (WP1066, # sc-203282, Santa Cruz Biotechnology) and JNK inhibitor (SP600125, # sc-200635, Santa Cruz Biotechnology) at the indicated concentrations selected as per earlier studies [ 28 ].

Techniques: Infection, Phospho-proteomics, Western Blot

Journal: Pharmacological research

Article Title: Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling

doi: 10.1016/j.phrs.2020.105414

Figure Lengend Snippet: A) Cardiac fibroblasts were treated with JAK inhibitor WP1066 (5μM) for 30 min and stimulated with resistin (100ng/ml) for an additional 2 hours. The phosphorylation of JAK2 and STAT3 was analyzed by western blotting. B) Representative fluorescence microscopic images of cardiac fibroblasts treated with JAK2 inhibitor for 2 hours and stimulated with resistin in presence of JAK2 inhibitor for an additional 48 hours and then stained for F actin to visualize stress fibers. C) mRNA expression of αSma, Col1a1, Ccn2, Fn, Mmp9, and Timp1 was analyzed by q-PCR in cardiac fibroblasts treated with JAK2 inhibitor for 2 hours and stimulated with resistin for an additional 48 hours. D) Cardiac fibroblasts were treated with JNK inhibitor SP600125 (25μM) for 30 min and stimulated with resistin (100ng/ml) in the presence of JAK2 inhibitor for an additional 2 hours. The phosphorylation of JNK and c-Jun was analyzed by western blotting. E) Representative fluorescence microscopic images of cardiac fibroblasts treated with JNK inhibitor for 2 hours and stimulated with resistin for an additional 48 hours and then stained for F actin to visualize stress fibers; bar size = 200μM. F) mRNA expression of αSma, Col1a1, Ccn2, Fn, Mmp9, and Timp1 was analyzed by q-PCR in cardiac fibroblasts treated with JAK2 inhibitor for 2 hours and stimulated with resistin for additional 48 hours. 18S rRNA was used as an internal control. The data are mean ± SEM of three experiments in triplicates, p*< 0.05, ***p < 0.001.

Article Snippet: For JAK and JNK inhibition, cells were incubated with JAK2 inhibitor (WP1066, # sc-203282, Santa Cruz Biotechnology) and JNK inhibitor (SP600125, # sc-200635, Santa Cruz Biotechnology) at the indicated concentrations selected as per earlier studies [ 28 ].

Techniques: Phospho-proteomics, Western Blot, Fluorescence, Staining, Expressing, Control

Journal: Pharmacological research

Article Title: Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling

doi: 10.1016/j.phrs.2020.105414

Figure Lengend Snippet: Mice were treated as in Figure 7. A) Protein samples were extracted from heart muscles and analyzed for the total and phosphorylated forms of JAK2, STAT3, JNK, and c-Jun by western blotting. B) Quantification of band densities in (A). β-actin was used as internal control. The data are mean ± SEM of n=5. p*< 0.05; **p < 0.01. C) Schematic diagram summarizing the role of JAK2 and JNK signaling pathways in resistin-driven regulation of fibroblast-myofibroblast conversion. Resistin potentially binds to TLR4 and activates JAK2 which in turn phosphorylates STAT3 causing it to translocate into the nucleus and activate pro-fibrotic target genes (i.e. Mmp9, Ccn2, Col1a1, vimentin and Tgfβ1). Resistin also activates JNK, potentially through ASK as we demonstrated previously [26], phosphorylating c-Jun causing it to translocate into the nucleus and activate pro-fibrotic target genes (i.e. Ccn2, Col1a1, Fn, Lox and Tgfβ1). TGFβ1 binds to TGFβ1R and activates the profibrotic genes by phosphorylating its downstream targets like Smad3, and JNK.

Article Snippet: For JAK and JNK inhibition, cells were incubated with JAK2 inhibitor (WP1066, # sc-203282, Santa Cruz Biotechnology) and JNK inhibitor (SP600125, # sc-200635, Santa Cruz Biotechnology) at the indicated concentrations selected as per earlier studies [ 28 ].

Techniques: Muscles, Western Blot, Control, Protein-Protein interactions

Journal: Advances in Bioscience and Biotechnology

Article Title: CXCL10 Induces Lytic Reactivation of EBV through <i>EXTL</i>1

doi: 10.4236/abb.2024.1510039

Figure Lengend Snippet: Figure 3. Activation of the JAK2/STAT3 signaling pathway in CXCL10-induced EBV lytic reactivation is inhibited by JAK2 inhib- itor ruxolitinib

Article Snippet: Recombinant human CXCL10 (IP-10) was purchased from PeproTech (Catalog #: 300-12); CXCR3 inhibitor AMG487 was acquired from MCE (Catalog #: HY-15319); antibodies against BZLF1 and glycoprotein (gp) 350 were obtained from Santa Cruz (Catalog #: sc-53904, sc-56981); antibodies against BRLF1 were sourced from Boster Bio (Catalog #: bs-4542R); antibodies against Extl1 were procured from ImmunoWay Biotechnology (Catalog #: YN5348); antibodies against phosphorylated Janus kinase 2 (p-JAK2) and phosphorylated Signal Transducer and Activator of Transcription 3 (p-STAT3) were purchased from CST (Catalog #: 4406T; Catalog #: 9145T); antibodies against JAK2 were obtained from Affinity (Catalog #: AF6002); antibodies against STAT3 were sourced from Proteintech (Catalog #: 60199-1-Ig); JAK2/STAT3 inhibitor ruxolitinib was acquired from MCE (Catalog #: HY-50856); whole protein extraction kits were purchased from KeyGen Biotech (Catalog #: KGP250); RNA extraction kits were obtained from Shanghai Yishan Biotech (Catalog #: RN001).

Techniques: Activation Assay, Inhibition

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