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: Oncology Reports

Article Title: α7 nicotinic acetylcholine receptor in tumor-associated macrophages inhibits colorectal cancer metastasis through the JAK2/STAT3 signaling pathway

doi: 10.3892/or.2017.5935

Figure Lengend Snippet: α7nAChR expressed in THP-1 derived macrophages (TMs) inhibits the migration of CRC cells through the JAK2/STAT3 signaling pathway. TMs were treated with or without α-bungarotoxin (α-Btx) for 6 h and then with DMSO (control), AG490, LY294002 and Bay 11–7082 for 1 h, respectively. Subsequently, LoVo cells were co-cultured with TM in 24-well Transwell plates with 8 µm polycarbonate membrane filters for 36 h for the migration assay. (A) Upper panel: Representative images show the migration of LoVo cells under the condition that the TMs did not have the α-Btx treatment and were treated with AG490, LY294002 and Bay 11–7082 only, respectively. Lower panel: Representative images show the migration of LoVo cells under the condition that the TMs were treated with α-Btx only, or dually treated with AG490, LY294002 and Bay 11–7082, respectively. (B) Number of migrated LoVo cells are presented as the mean ± SD of six images (n=6) of three separate experiments. Significance of differences between groups was determined by Student's t-test. P-value <0.05 indicates that there was a significant difference between treatments.

Article Snippet: To investigate the effects of signaling pathway inhibitors on TMα7 −/− -enhanced migration of LoVo cells: TM cells (2×10 5 ) in 500 µl of medium in 24-well tissue culture plates were treated with or without α-bungarotoxin (α-Btx, a potent nicotinicα7 receptor antagonist; Sigma-Aldrich) at 100 nM for 6 h. After being washed with PBS three times, the TM cells were further treated with or without three signaling pathway inhibitors: AG490 (JAK2/STAT3 inhibitor, 20 µM), LY294002 (PI3K inhibitor, 5 µM) and Bay 11–7082 (NF-κB inhibitor, 2 µg/ml) for 1 h. AG490, LY294002, and Bay 11–7082 were purchased from Beyotime Institute of Biotechnology (Shanghai, China).

Techniques: Derivative Assay, Migration, Cell Culture

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

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