Journal: Frontiers in Pharmacology

Article Title: Andrographolide Protects against Aortic Banding-Induced Experimental Cardiac Hypertrophy by Inhibiting MAPKs Signaling

doi: 10.3389/fphar.2017.00808

Figure Lengend Snippet: Andr blocks MAPKs signaling in vivo and in vitro . (A) Representative blots of phosphorylated (P-) and total (T-) ERK1/2, JNK, and P38 in the heart tissues of mice in the indicated groups ( n = 6). (B) Comparison of expression among the indicated groups. ∗ P < 0.05 compared with the corresponding sham group. # P < 0.05 vs. the veh-AB group. (C) Representative blots of phosphorylated (P-) and total (T-) ERK1/2, JNK, and P38 in the cardiomyocytes in the indicated groups ( n = 6). (D) Comparison of expression among the indicated groups. ∗ P < 0.05 compared with the corresponding PBS group. # P < 0.05 vs. the vehicle-Ang II group.

Article Snippet: Cardiomyocytes were treated with ERK1/2 inhibitor (SCH772984, 5 μM, Selleck), JNK inhibitor (SP600125, 10 μM, Sigma), and/or P38 inhibitor (SB209063, 10 μM, Medchem Express) as well as stimulated with Ang II.

Techniques: In Vivo, In Vitro, Comparison, Expressing

Journal: Frontiers in Pharmacology

Article Title: Andrographolide Protects against Aortic Banding-Induced Experimental Cardiac Hypertrophy by Inhibiting MAPKs Signaling

doi: 10.3389/fphar.2017.00808

Figure Lengend Snippet: Andr-mediated cardioprotection depends on the inhibition of MAPKs in cardiomyocytes. Cardiomyocytes were treated with ERK1/2 inhibitor (SCH7729, 5 μM), JNK inhibitor (SP600125, 10 μM), and/or P38 inhibitor (SB209063, 10 μM) as well as stimulated with Ang II (1 μM) and treated with Andr (50 μM). (A) Cell viability was accessed by the cell counting kit-8 assay ( n = 5). (B–E) Immunofluorescence staining of α-actinin and the cell surface area of cardiomyocytes in the indicated groups ( n = 5 samples and n = 100+ cells per group). (F–J) The mRNA levels of ANP and β-MHC in cardiomyocytes in the indicated groups ( n = 6). The results are presented as a fold change, and the results are normalized to GAPDH gene expression. ∗ P < 0.05 compared with the control group. # P < 0.05 vs. the Ang II group.

Article Snippet: Cardiomyocytes were treated with ERK1/2 inhibitor (SCH772984, 5 μM, Selleck), JNK inhibitor (SP600125, 10 μM, Sigma), and/or P38 inhibitor (SB209063, 10 μM, Medchem Express) as well as stimulated with Ang II.

Techniques: Inhibition, Cell Counting, Immunofluorescence, Staining, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Andrographolide Protects against Aortic Banding-Induced Experimental Cardiac Hypertrophy by Inhibiting MAPKs Signaling

doi: 10.3389/fphar.2017.00808

Figure Lengend Snippet: Andr reduces cardiac fibroblast activation via MAPKs. (A–F) Cardiac fibroblasts were treated with different concentrations of Andr (0, 12.5, 25, or 50 μM) and/or stimulated with Ang II (1 μM). (A) Cell viability was accessed by the cell counting kit-8 assay ( n = 5). (B) Cell proliferation was accessed by the cell counting kit-8 assay ( n = 5). (C) Immunofluorescence staining of α-SMA in the indicated groups. (D) The mRNA levels of collagen I, collagen III, and CTGF in cardiac fibroblasts in the indicated groups ( n = 6). (E) Representative blots of phosphorylated (P-) and total (T-) ERK1/2, JNK, P38, and smad4 in the cardiac fibroblasts in the indicated groups ( n = 6). (F) Comparison of expression among the indicated groups. ∗ P < 0.05 compared with the corresponding PBS group. # P < 0.05 vs. the vehicle-Ang II group. (G–J) Cardiac fibroblasts were treated with ERK1/2 inhibitor (SCH7729, 5 μM), JNK inhibitor (SP600125, 10 μM), and/or P38 inhibitor (SB209063, 10 μM) as well as stimulated with Ang II (1 μM) and treated with Andr (50 μM). (G) Cell viability was accessed by the cell counting kit-8 assay ( n = 5). (H) Cell proliferation was accessed by the cell counting kit-8 assay ( n = 5). (I) Immunofluorescence staining of α-SMA in the indicated groups. (J,K) The mRNA levels of collagen I, collagen III, and CTGF in cardiac fibroblasts in the indicated groups ( n = 6). The results are presented as a fold change, and the results are normalized to GAPDH gene expression. ∗ P < 0.05 compared with the control group. # P < 0.05 vs. the Ang II group.

Article Snippet: Cardiomyocytes were treated with ERK1/2 inhibitor (SCH772984, 5 μM, Selleck), JNK inhibitor (SP600125, 10 μM, Sigma), and/or P38 inhibitor (SB209063, 10 μM, Medchem Express) as well as stimulated with Ang II.

Techniques: Activation Assay, Cell Counting, Immunofluorescence, Staining, Comparison, Expressing, Control

Journal: Brazilian Journal of Medical and Biological Research

Article Title: miR-92a-3p promotes ox-LDL induced-apoptosis in HUVECs via targeting SIRT6 and activating MAPK signaling pathway

doi: 10.1590/1414-431X20209386

Figure Lengend Snippet: Expression levels of miR-92a-3p and SIRT6 in oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs). A , The relative expression level of miR-92a-3p was evaluated by RT-qPCR assay in ox-LDL-treated HUVECs. B and C , RT-qPCR and western blot assays were utilized to determine the mRNA and protein expression levels of SIRT6. D , Protein expression levels of p-JNK/JNK and p-p38 MAPK/p38 MAPK were analyzed with western blot assays in HUVECs. Data are reported as means±SD with three replicates. *P<0.05 compared to control (ANOVA).

Article Snippet: The membranes were blocked with 5% non-fat milk and probed with antibodies against SIRT6, JNK, p-JNK, p38 MAPK, or p-p38 MAPK (1:1000, Santa Cruz Bio-Technology, USA) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Brazilian Journal of Medical and Biological Research

Article Title: miR-92a-3p promotes ox-LDL induced-apoptosis in HUVECs via targeting SIRT6 and activating MAPK signaling pathway

doi: 10.1590/1414-431X20209386

Figure Lengend Snippet: Functional roles of miR-92a-3p in oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs). HUVECs were treated with 50 µg/mL of ox-LDL for 48 h and transfected with inhibitor-negative control (NC), miR-92a-3p inhibitor, mimics-NC, or miR-92a-3p mimics. A , RT-qPCR assay was carried out to evaluate the expression level of miR-92a-3p in HUVECs. B , Apoptosis of HUVECs was determined by flow cytometry assay. C , MTT analysis was utilized to assess cell viability in HUVECs. D , The expression levels of p-JNK/JNK and p-p38 MAPK/p38 MAPK were measured by western blot assays. Data are reported as means±SD with three replicates. *P<0.05 (ANOVA).

Article Snippet: The membranes were blocked with 5% non-fat milk and probed with antibodies against SIRT6, JNK, p-JNK, p38 MAPK, or p-p38 MAPK (1:1000, Santa Cruz Bio-Technology, USA) overnight at 4°C.

Techniques: Functional Assay, Transfection, Negative Control, Quantitative RT-PCR, Expressing, Flow Cytometry, Western Blot

Journal: Brazilian Journal of Medical and Biological Research

Article Title: miR-92a-3p promotes ox-LDL induced-apoptosis in HUVECs via targeting SIRT6 and activating MAPK signaling pathway

doi: 10.1590/1414-431X20209386

Figure Lengend Snippet: iR-92a-3p was involved in oxidized low-density lipoprotein (ox-LDL)-induced cell apoptosis via targeting SIRT6/MAPK signaling pathway. Human umbilical vein endothelial cells (HUVECs) were treated with 50 µg/mL of ox-LDL for 48 h and transfected with pcDNA3.1, pcDNA3.1-SIRT6, miR-92a-3p inhibitor+anti-negative control (NC), or miR-92a-3p inhibitor+si-SIRT6. A - C , Western blot assays were carried out to evaluate expression levels of SIRT6, p-JNK/JNK, and p-p38 MAPK/p38 MAPK in transfected HUVECs. D , Apoptosis of transfected HUVECs was determined by flow cytometry assay. E , MTT analysis was used to assess cell viability of HUVECs post-transfection. Data are reported as means±SD with three replicates. *P<0.05 (ANOVA).

Article Snippet: The membranes were blocked with 5% non-fat milk and probed with antibodies against SIRT6, JNK, p-JNK, p38 MAPK, or p-p38 MAPK (1:1000, Santa Cruz Bio-Technology, USA) overnight at 4°C.

Techniques: Transfection, Negative Control, Western Blot, Expressing, Flow Cytometry

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of the MAP kinase and NF-κB signaling pathways in mediating ECFP/Ang II-induced NHE3 expression in wild-type and Agtr1a -/- mPCT cells. Panel ( A ) shows that in wild-type mPCT cells, ECFP/Ang II stimulated NHE3 expression significantly, and the response was attenuated by the MEK1/MEK2 kinase inhibitor U0126 and the NF-κB activation inhibitor Ro 106–9920, respectively. However, the MEK inhibitor PD 980659 and the p38 MAP kinase inhibitor SB202196 failed to attenuate the effect of ECFP/Ang II on NHE3 expression. Panel ( B ) shows that in Agtr1a -/- mPCT cells, ECFP/Ang II failed to stimulate NHE3 expression, and the inhibitors of the MAP kinases and NF-κB signaling pathways had no significant effects on NHE3 expression. ** p < 0.01 vs. control WT mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of AT 1 and AT 2 receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced Na + /HCO 3 - cotransporter expression in wild-type mPCT cells. Panel ( A ) shows that ECFP/Ang II significantly increased Na + /HCO 3 - expression, and the response was attenuated by losartan but not by PD123319, suggesting a dominant role of AT 1 receptors in mPCT cells. Panel ( B ) shows that the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659 attenuated the effects of ECFP/Ang II on expression, but the p38 MAP kinase inhibitor SB202196 had no effect on Na + /HCO 3 - expression. ** p < 0.01 vs. control WT mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Cells

Article Title: Intracellular Angiotensin II Stimulation of Sodium Transporter Expression in Proximal Tubule Cells via AT 1 (AT 1a ) Receptor-Mediated, MAP Kinases ERK1/2- and NF-кB-Dependent Signaling Pathways

doi: 10.3390/cells12111492

Figure Lengend Snippet: The roles of AT 1 and AT 2 receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type and Agtr1a -/- mPCT cells. Panel ( A ) shows that ECFP/Ang II increased NF-κB, p65 expression in wild-type mPCT cells, and the response was attenuated by both losartan and PD123319, supporting an important role of AT 1 and AT 2 receptors in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. Panel ( B ) shows that ECFP/Ang II alone had no significant effect on NF-κB, p65 expression in Agtr1a -/- mPCT cells, but both losartan and PD123319 potentiated this response. Panel ( C ) shows that in wild-type mPCT cells, the effect of ECFP/Ang II on NF-κB, p65 expression was attenuated by the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659, respectively. However, the p38 MAP kinase inhibitor SB202196 had no effect on ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. ** p < 0.01 vs. control WT or Agtr1a -/- mPCT cells. ++ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II, or Agtr1a -/- mPCT cells transfected with ECFP/ANG II.

Article Snippet: To determine the potential signaling mechanisms involved in Ad- Sglt2-ECFP/Ang II -induced biological responses, WT and Agtr1a -/- mPCT cells expressing Ad- Sglt2-ECFP/Ang II were concurrently treated with the AT 1 receptor antagonist losartan (10 μM; Tocris, Minneapolis, MN, USA), the AT 2 receptor antagonist PD 123319 (10 μM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 μM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 μM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 μM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 μM; MCE, Belleville, NJ, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Transfection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production.

doi: 10.4049/jimmunol.171.2.628

Figure Lengend Snippet: FIGURE 3 Effect of SB203580 on LPS-induced IL-12 p40 protein pro- duction and mRNA expression. PMA-treated THP-1 cells were cultured with 0.1% DMSO (control vehicle) and SB203580 (specific inhibitor of p38 MAP kinase) for 30 min before stimulation with LPS (1 g/ml). A, After 24 h, IL-12 p40 protein in culture supernatants was evaluated by ELISA. Values represent mean SEM of four experiments. , p 0.05 compared with LPS-stimulated THP-1 cells. B, After 12 h, total RNA was extracted, and expression of IL-12 p40 mRNA was evaluated by Northern blotting, as described in the legend to Fig. 1.

Article Snippet: To determine the amounts of precipitated JNK, p38 MAP kinase, ERK, and c-Jun, blots were stripped and reprobed using phosphorylation state-independent anti-JNK (SC-571; Santa Cruz Biotechnology, Santa Cruz, CA), anti-p38 MAP kinase (Cell Signaling Technology), anti-ERK (Cell Signaling Technology), and c-Jun (Cell Signaling Technology) Abs.

Techniques: Expressing, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Northern Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production.

doi: 10.4049/jimmunol.171.2.628

Figure Lengend Snippet: FIGURE 2. Effect of LPS on JNK, p38 MAP kinase, and ERK activa- tion in human macrophages. PMA-treated THP-1 cells were stimulated with LPS (1 g/ml) for indicated periods. Activities of JNK, p38 MAP kinase, and ERK were determined by Western blotting whole cell lysates using Abs specific for phosphorylated, activated forms of JNK (A, top), p38 MAP kinase (B, top), and ERK (C, top). Corresponding bottom panels are Western blots using Abs to total JNK (A, bottom), p38 MAP kinase (B, bottom), and ERK (C, bottom), indicating amounts of precipitated enzymes.

Article Snippet: To determine the amounts of precipitated JNK, p38 MAP kinase, ERK, and c-Jun, blots were stripped and reprobed using phosphorylation state-independent anti-JNK (SC-571; Santa Cruz Biotechnology, Santa Cruz, CA), anti-p38 MAP kinase (Cell Signaling Technology), anti-ERK (Cell Signaling Technology), and c-Jun (Cell Signaling Technology) Abs.

Techniques: Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production.

doi: 10.4049/jimmunol.171.2.628

Figure Lengend Snippet: FIGURE 7 Effect of GSH-OEt on LPS induced JNK, p38 MAP kinase, and ERK activation. A–C, PMA-treated THP-1 cells cultured with GSH-OEt for 4 h before stimulation with LPS (1 g/ml) for 30 min. Activities of JNK (A), p38 MAP kinase (B), and ERK (C) were determined by Western blotting, as described in the legend to Fig. 2. D and E, Levels of JNK and p38 MAP kinase phosphorylation were quantified by densitometry. D, OD of p46- and p54- phosphorylated protein bands was corrected for total p46 and p54 protein, respectively. E, OD of p38- phosphorylated protein band was corrected for total p38 MAP kinase protein. Results are expressed as ratio (%) of control value (LPS-stimulated THP-1 cells). Values represent means SEM of four ex- periments. , p 0.05 compared with LPS-stimu- lated THP-1 cells.

Article Snippet: To determine the amounts of precipitated JNK, p38 MAP kinase, ERK, and c-Jun, blots were stripped and reprobed using phosphorylation state-independent anti-JNK (SC-571; Santa Cruz Biotechnology, Santa Cruz, CA), anti-p38 MAP kinase (Cell Signaling Technology), anti-ERK (Cell Signaling Technology), and c-Jun (Cell Signaling Technology) Abs.

Techniques: Activation Assay, Cell Culture, Western Blot, Phospho-proteomics, Control

Journal: International Journal of Endocrinology

Article Title: Effect of Gelam Honey on the Oxidative Stress-Induced Signaling Pathways in Pancreatic Hamster Cells

doi: 10.1155/2013/367312

Figure Lengend Snippet: Effect of quercetin and Gelam honey extract on pAkt (Ser473) expression.Quantitative analysis and representative western blot analysis of pAkt (Ser473) in HIT-T15 cells pretreated with quercetin and honey extract in cells cultured in 20 mM ((a), (c), (e)) and 50 mM ((b), (d), (f)) glucose. A sustained increase in the level of pAkt (ser473) was observed after pretreatment with quercetin and honey extract. Akt inhibitor VIII prevented the expression Akt ser473 phosphorylation induced by quercetin and honey extract. The results were normalized with β actin antibody. Data were presented as the mean ± standard deviation. (e) * P < 0.05; # P < 0.005 quercetin and honey extract treated compared to the 20 mM glucose alone. (f) * P < 0.05; # P < 0.005 quercetin and honey extract treated compared to the 50 mM glucose alone.

Article Snippet: To investigate inhibitory effects on Akt signaling pathway, cells were incubated with 5 μ M Akt inhibitor VIII (Santa Cruz Biotechnology sc-203173) for one hour before pretreatment with quercetin and honey extract.

Techniques: Expressing, Western Blot, Cell Culture, Phospho-proteomics, Standard Deviation

Journal: International Journal of Endocrinology

Article Title: Effect of Gelam Honey on the Oxidative Stress-Induced Signaling Pathways in Pancreatic Hamster Cells

doi: 10.1155/2013/367312

Figure Lengend Snippet: The effect of flavonoids and Gelam honey extract on insulin content. (a) Effect of pretreatment with quercetin and Gelam honey extract and the addition of Akt inhibitor VIII on the insulin content in cells cultured in 20 mM glucose. There was a significant increase in insulin content (* P < 0.05) when the cells were pretreated with quercetin and honey. There was a significant decrease in insulin content (* P < 0.05) when the cells were treated with Akt inhibitor VIII, before pretreating with quercetin and Gelam honey extract. (b) Effect of pretreatment with quercetin and Gelam honey and the addition of Akt inhibitor VIII on the insulin content in cells cultured in 50 mM glucose. There was a significant increase in insulin content (* P < 0.05, # P < 0.005) when the cells were pretreated with quercetin and honey. There was a significant decrease in insulin content (* P < 0.05, # P < 0.005) when the cells were treated with Akt inhibitor VIII, before pretreating with quercetin and Gelam honey extract.

Article Snippet: To investigate inhibitory effects on Akt signaling pathway, cells were incubated with 5 μ M Akt inhibitor VIII (Santa Cruz Biotechnology sc-203173) for one hour before pretreatment with quercetin and honey extract.

Techniques: Cell Culture

Journal: Signal Transduction and Targeted Therapy

Article Title: RPS15 interacted with IGF2BP1 to promote esophageal squamous cell carcinoma development via recognizing m 6 A modification

doi: 10.1038/s41392-023-01428-1

Figure Lengend Snippet: RPS15 overexpression promotes the translation of core E2F and p38 MAPK pathway proteins. a Schematic illustrating the ribosome profiling of KYSE30 cells (Con) and RPS15-overexpressing KYSE30 cells (RPS15). b GO analysis (upper) and KEGG-pathway analysis (lower) of transcripts preferentially bound by ribosomes in the RPS15 group. The most enriched ribosomal/translational GO gene sets and associated FDR values are shown, which were mainly enriched in translation and MAPK signaling pathways. c Heat map of the log2 fold-change of the RPS15 group relative to that of the control group for ribosome protein genes for mRNA expression, ribosome-protected fragments (RPF), and translation efficiency (TE). d Heat map of the log2 fold-change of the RPS15 group relative to that of the control for each gene of the E2F pathway and p38 MAPK pathway target-gene set for mRNA expression, RPF, and TE. Genes were categorized according to their GO biological process functions in the relative order of the TE fold-change. e Scatter plot of the translational efficiency of individual RP genes (right panel) and the hallmark p38 MAPK-target gene set transcripts (left panel). The x-axis represents the log2 fold-change in RNA-seq and the y-axis represents the log2 fold-change in ribosome profiling. f Expression of the hallmark p38 MAPK-target gene set in RPS15-overexpressing KYSE30 cells (left) and KYSE450 cells (right) determined by Western blot. g Western blot analysis results of expression of the hallmark p38 MAPK-target gene set in KYSE150 cells (left) and KYSE510 cells (right) with or without RPS15 knockout. h , i Statistical analyses of Boyden Chamber migration and invasion assays for KYSE30 cells ( h ) and KYSE450 cells ( i ) with stably transfected control vector or RPS15-overexpressing vector with or without SB203580 treated for 24 h. The statistical analysis results are shown. j Growth curves measured using Incucyte live-cell analyses of KYSE30 cells stably transfected with control vector (blue) or RPS15-overexpressing vector (red) and treated with SB203580 or SB203580 combined DDP for 72 h. k Growth curves measured using Incucyte live-cell analyses of KYSE450 cells stably transfected with control vector (blue) or RPS15-overexpressing vector (red) and treated with SB203580 or SB203580 combined DDP for 72 h. Data were analyzed using unpaired t -tests. * P < 0.05, ** P < 0.01, and *** P < 0.001. l Western blot analysis to detect expression of the hallmark MAPK-target gene set in KYSE30 cells (upper) and KYSE450 cells (lower) stably transfected with control vector or RPS15-overexpressing vector and treated with SB203580 or DDP for 2 days. Con control, Veh vehicle

Article Snippet: Approximately 24 h later, 10 μM folic acid (T0062, TargetMol), 8 μM p38 MAPK inhibitor SB203580 (T1746, TargetMol), and/or cisplatin were added at the indicated concentrations.

Techniques: Over Expression, Control, Expressing, RNA Sequencing Assay, Western Blot, Knock-Out, Migration, Stable Transfection, Transfection, Plasmid Preparation

Journal: Signal Transduction and Targeted Therapy

Article Title: RPS15 interacted with IGF2BP1 to promote esophageal squamous cell carcinoma development via recognizing m 6 A modification

doi: 10.1038/s41392-023-01428-1

Figure Lengend Snippet: IGF2BP1 binds the 3′-UTR of MKK6 and MAPK14 in an m6A-dependent manner. a Distribution of m 6 A peaks of IGF2BP1 RIP-seq data across MAPK14 and MKK6 mRNA transcripts. b Enrichment of m6A modification in 3′-UTR region of MKK6 with Flag-tagged IGF2BP1 in KYSE30 cells (left); RIP-qPCR showing the binding of IGF2BP1 to the 3′-UTR region of MKK6 (right). c Enrichment of m6A modification in 3′-UTR region of MAPK14 with Flag-tagged IGF2BP1 in KYSE30 cells (left); RIP-qPCR showing the binding of IGF2BP1 to the 3′-UTR region of MAPK14 (right). d Agarose gel electrophoresis showing the binding of IGF2BP1 to the 3′-UTR region of MKK6 and MAPK14 in KYSE30 cells (left) and KYSE450 cells (right). e Western blot detected protein expression of the hallmark p38 MAPK-target gene set in RPS15-overexpressing KYSE30 cells (left) and KYSE450 cells (right) with or without IGF2BP1 knockdown. f Growth curves measured using Incucyte live-cell analyses of KYSE30 cells (upper) and KYSE450 cells (lower) stably transfected with control vector (blue) or RPS15-overexpressing vector (purple) and treated with IGF2BP1 knockdown or without IGF2BP1 knockdown for 72 h. Data were analyzed using unpaired t-tests. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Approximately 24 h later, 10 μM folic acid (T0062, TargetMol), 8 μM p38 MAPK inhibitor SB203580 (T1746, TargetMol), and/or cisplatin were added at the indicated concentrations.

Techniques: Modification, Binding Assay, Agarose Gel Electrophoresis, Western Blot, Expressing, Knockdown, Stable Transfection, Transfection, Control, Plasmid Preparation

Journal: Signal Transduction and Targeted Therapy

Article Title: RPS15 interacted with IGF2BP1 to promote esophageal squamous cell carcinoma development via recognizing m 6 A modification

doi: 10.1038/s41392-023-01428-1

Figure Lengend Snippet: Schematic depicting the contribution of RPS15 and IGF2BP1 to ESCC progression via the p38 MAPK pathway. a RPS15 overexpression promotes the proliferation and motility of ESCC cells. Mechanistic investigation revealed that RPS15 interacts with the KH domain of IGF2BP1, which directly binds and recognizes the MAPK14 and MKK6 mRNA 3′-UTR, and promotes translation of core p38 MAPK pathway proteins. b , RPS15 inhibition with folic acid treatment, IGF2BP1 ablation, or treatment with SB203580 suppresses ESCC proliferation and metastasis via the p38 MAPK signaling pathway

Article Snippet: Approximately 24 h later, 10 μM folic acid (T0062, TargetMol), 8 μM p38 MAPK inhibitor SB203580 (T1746, TargetMol), and/or cisplatin were added at the indicated concentrations.

Techniques: Over Expression, Inhibition

Journal: International journal of molecular sciences

Article Title: Tumor Necrosis Factor-α-Induced C-C Motif Chemokine Ligand 20 Expression through TNF Receptor 1-Dependent Activation of EGFR/p38 MAPK and JNK1/2/FoxO1 or the NF-κB Pathway in Human Cardiac Fibroblasts.

doi: 10.3390/ijms23169086

Figure Lengend Snippet: Figure 4. Involvement of p38 MAPK in TNF-α-induced CCL20 expression. (A) Cells were pretreated with 3 µM p38 inhibitor VIII (p38i VIII) for 1 h and then incubated with 5 ng/mL TNF-α for 12 h. The conditioned media were utilized to determine the CCL20 level via an ELISA kit. (B) Cells were pretreated with 3 µM p38i VIII for 1 h and then incubated with 5 ng/mL TNF-α for the indicated time. The mRNA levels and promoter activity of CCL20 were determined by real-time PCR (2 h) and promoter assay (4 h), respectively. (C) Cells were transfected with scrambled or p38α siRNA and then incubated with TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. The protein levels of p38α were determined by Western blot with GAPDH as a loading control. (D) Cells were pretreated without or with 10 µM AG1478 or 3 µM p38i VIII for 1 h and then treated with TNF-α for the indicated times (0, 5, 10, 15, 30, and 60 min). The phosphorylation of p38 and EGFR was determined by Western blot with GAPDH as a loading control. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). # p < 0.05, as compared with the cells exposed to vehicle alone; or significantly different as indicated.

Article Snippet: Antibodies of anti-TNFR1 (Cat#sc-52739), anti-TNFR2 (Cat#sc-8041), and p38 MAPK inhibitor (p38i) VIII were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay, Real-time Polymerase Chain Reaction, Promoter Assay, Transfection, Western Blot, Control, Phospho-proteomics

Journal: International journal of molecular sciences

Article Title: Tumor Necrosis Factor-α-Induced C-C Motif Chemokine Ligand 20 Expression through TNF Receptor 1-Dependent Activation of EGFR/p38 MAPK and JNK1/2/FoxO1 or the NF-κB Pathway in Human Cardiac Fibroblasts.

doi: 10.3390/ijms23169086

Figure Lengend Snippet: Figure 6. Involvement of FoxO1 in TNF-α-induced CCL20 expression. (A) Cells were pretreated with 1 µM AS1842856 for 1 h and then incubated with 5 ng/mL TNF-α for 12 h. The conditioned media were utilized to determine the CCL20 level via an ELISA kit. (B) Cells were pretreated with 1 µM AS1842856 for 1 h and then incubated with 5 ng/mL TNF-α for the indicated time. The mRNA levels and promoter activity of CCL20 were determined by real-time PCR (2 h) and promoter assay (4 h), respectively. (C) Cells were transfected with scrambled or FoxO1 siRNA and then incubated with TNF-α for 2 h. The mRNA levels of CCL20 were determined by real-time PCR. The protein levels of FoxO1 were determined by Western blot with GAPDH as a loading control. (D) Cells were pretreated without or with 1 µM AS1842856, 3 µM p38i VIII, or 3 µM SP600125 for 1 h and then treated with TNF-α for the indicated times (0, 5, 10, 15, 30, and 60 min). The phosphorylation of FoxO1, p38, or JNK1/2 was determined by Western blot with GAPDH as a loading control. (E) Cells were pretreated without or with 3 µM p38i VIII, 3 µM SP600125, or 1 µM AS1842856 for 1 h and then stimulated with TNF-α for the indicated time intervals or 30 min. The binding of FoxO1 to the promoter region of CCL20 was determined with a ChIP assay. Data are expressed as mean ± S.E.M. of three independent experiments (n = 3). # p < 0.05, as compared with the cells exposed to vehicle alone; or significantly different as indicated.

Article Snippet: Antibodies of anti-TNFR1 (Cat#sc-52739), anti-TNFR2 (Cat#sc-8041), and p38 MAPK inhibitor (p38i) VIII were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay, Real-time Polymerase Chain Reaction, Promoter Assay, Transfection, Western Blot, Control, Phospho-proteomics, Binding Assay

Journal: International journal of molecular sciences

Article Title: Tumor Necrosis Factor-α-Induced C-C Motif Chemokine Ligand 20 Expression through TNF Receptor 1-Dependent Activation of EGFR/p38 MAPK and JNK1/2/FoxO1 or the NF-κB Pathway in Human Cardiac Fibroblasts.

doi: 10.3390/ijms23169086

Figure Lengend Snippet: Figure 8. Schematic diagram illustrating the proposed signaling pathway involved in TNF-α-induced CCL20 expression and secretion in HCFs. TNF-α-induced CCL20 expression was, at least partially, mediated through binding to TNFR1 leading to transactivation of EGFR. Activated EGFR promoted the phosphorylation of p38 MAPK- or JNK1/2-dependent FoxO1 activation, which further bound with the FoxO1 response element (FRE) on the CCL20 promoter. In addition, TNF-α also turned on NF-κB transcription factors. Either FoxO1 or NF-κB activation could enhance the expression of CCL20 induced by TNF-α, which may be engaged in the inflammatory responses in HCFs. A better understanding of mechanisms underlying the regulation of the CCL20 gene by TNF-α will support more opportunities to develop anti-inflammatory therapeutic strategies for treating cardiac inflammation.

Article Snippet: Antibodies of anti-TNFR1 (Cat#sc-52739), anti-TNFR2 (Cat#sc-8041), and p38 MAPK inhibitor (p38i) VIII were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Binding Assay, Phospho-proteomics, Activation Assay

Journal: Molecular and Cellular Biology

Article Title: Trophic Factor Withdrawal: p38 Mitogen-Activated Protein Kinase Activates NHE1, Which Induces Intracellular Alkalinization

doi: 10.1128/mcb.21.22.7545-7557.2001

Figure Lengend Snippet: FIG. 4. IL-3 withdrawal and anisomycin treatment both activate p38 MAPK and induce alkalinization. (A) Treatment with anisomycin (a p38 MAPK activator) induces alkalinization in the presence (top) or absence (bottom) of IL-3. FL5.12A cells were treated with 100 g of anisomycin per ml. The intracellular pH was measured as described in Materials and Methods. (B) IL-3 withdrawal activates the phosphorylation of p38 MAPK and ATF-2 but not ERK1/2, p90RSK, or JNK. FL5.12A cells were incubated without IL-3 for 0 to 3 h, as well as stimulated with anisomycin, and levels of phosphorylated p38 and phosphorylated ATF-2 (top panel), phosphorylated ERK1/2 or phosphorylated p90RSK (second panel from bottom), and phosphorylated JNK (bottom panel) were evaluated from whole-cell lysates. Nonphosphorylated p38 and ERK were included as loading controls. In addition, p38 MAPK was immunoprecipitated from protein lysates, made from cells incubated without IL-3 for 0 to 3 h, and used in an in vitro kinase assay to phosphorylate an ATF-2 peptide (second panel from top). SDS-polyacrylamide gel electrophoresis and Western blot analysis were performed as described in Materials and Methods.

Article Snippet: The blots were probed with a rabbit polyclonal antiserum specific for phospho-p38 MAPK protein (D-8; Santa Cruz) and p38 MAPK protein (Santa Cruz), phospho-ATF-2 (Cell Signaling Technologies), phospho-ERK1/2 and ERK1/2 (Cell Signaling Technologies), phosphoJNK/SAPK (Cell Signaling Technologies), and phospho-p90RSK (Cell Signaling Technologies), as specified by the manufacturers, followed by anti-rabbit secondary antibodies conjugated to horseradish peroxidase (HRP) (Santa Cruz), and then developed by enhanced chemiluminescence (ECL) (Pierce) and visualized on BioMax ML film (Kodak).

Techniques: Phospho-proteomics, Incubation, Immunoprecipitation, In Vitro, Kinase Assay, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Trophic Factor Withdrawal: p38 Mitogen-Activated Protein Kinase Activates NHE1, Which Induces Intracellular Alkalinization

doi: 10.1128/mcb.21.22.7545-7557.2001

Figure Lengend Snippet: FIG. 5. Inhibitors of p38 MAPK block alkalinization induced by IL-7 or IL-3 withdrawal and enhance survival. (A) D1 cells were cul- tured in the presence or absence of IL-7 for 5 h and either not treated (None) or treated with 20 M PD169316, 20 M SB202190 (inhibitors of p38 MAPK), or 20 M PD98059 (inhibitor of MEK1). (B) FL5.12A cells were incubated with or without IL-3 for 3 h and either not treated (None) or treated with 20 M PD169316, 20 M SB202190 (inhibitors of p38 MAPK), or 20 M PD98059 (inhibitor of MEK1). Intracellular pH was measured as described in Materials and Methods. (C) The efficacy of the different MAPK inhibitors (20 M) was tested in a p38 MAPK in vitro kinase assay using p38 MAPK immunoprecipiatated from anisomycintreated FL5.12A cells. Of the three p38 MAPK inhib- itors assayed, PD169316 was the most effective in blocking p38 MAPK activity (shown at two concentrations, 20 and 10 M), SB202190 was the second best, and SB203580 had minimal effect; the ERK inhibitor (PD98059) or the nonspecific inhibitor (SB202474) had no effect at the 20 M dose. (D) GFP cells or GFP cells cotransfected with a DN-p38 MAPK were cultured in the absence of IL-3 for 48 h, and the percentage of cells with DNA fragmentation was measured by pro- pidium iodide staining and flow cytometry analysis. Expression of the DN-p38 MAPK decreased DNA fragmentation by 20% during IL-3 withdrawal. GFP cells cultured in the presence of IL-3 were included as a positive control for survival. (E) Loss of NHE or expression of a DN-p38 MAPK in CHO cells prevents alkalinization after anisomycin treatment. CHO cells were made deficient for NHE as previously described (39) and/or transfected with DN-p38 MAPK and evaluated 24 h later for pH changes after 1 h of anisomycin treatment by incor- poration of BCECF as described in Materials and Methods.

Article Snippet: The blots were probed with a rabbit polyclonal antiserum specific for phospho-p38 MAPK protein (D-8; Santa Cruz) and p38 MAPK protein (Santa Cruz), phospho-ATF-2 (Cell Signaling Technologies), phospho-ERK1/2 and ERK1/2 (Cell Signaling Technologies), phosphoJNK/SAPK (Cell Signaling Technologies), and phospho-p90RSK (Cell Signaling Technologies), as specified by the manufacturers, followed by anti-rabbit secondary antibodies conjugated to horseradish peroxidase (HRP) (Santa Cruz), and then developed by enhanced chemiluminescence (ECL) (Pierce) and visualized on BioMax ML film (Kodak).

Techniques: Blocking Assay, Incubation, In Vitro, Kinase Assay, Inhibition, Activity Assay, Cell Culture, Staining, Cytometry, Expressing, Positive Control, Transfection

Journal: Molecular and Cellular Biology

Article Title: Trophic Factor Withdrawal: p38 Mitogen-Activated Protein Kinase Activates NHE1, Which Induces Intracellular Alkalinization

doi: 10.1128/mcb.21.22.7545-7557.2001

Figure Lengend Snippet: FIG. 6. Phosphorylation of NHE1 occurs via different MAPKs in the presence and absence of IL-3. (A) FL5.12A cells were incubated for 3 h with [32P]orthophosphate in the presence () or absence () of IL-3. (B) 32P-labeled FL5.12A cells were incubated for 3 h with PD169316 with () or without () IL-3. (C) 32P-labeled FL5.12A cells were incubated with or without PD98059 in the presence () of IL-3. Cells were lysed, and NHE1 was immunoprecipitated and electrophoresed on SDS–8% polyacrylamide gels and autoradiography was performed as described in Materials and Methods. To confirm the specificity of the NHE antibody (A7) used for immunoprecipitation, parallel cell lysates (not labeled with [32P]orthophosphate) were immunoprecipitated with the anti-NHE antibody (A7), electrophoresed on SDS–8% polyacrylamide gels, analyzed by Western bloting using a different anti-NHE1 antibody, and shown in the lower panels. (D) In vivo coimmunoprecipitation of NHE and p38 MAPK. Protein lysates extracted from cells cultured with or without IL-3, and chemically cross-linked with DSS, were immunoprecipitated with either an antibody specific for NHE1, an antibody for Mapkap2 (positive control), or rabbit Ig (negative control). The lysates were blotted for p38 MAPK as described in Materials and Methods.

Article Snippet: The blots were probed with a rabbit polyclonal antiserum specific for phospho-p38 MAPK protein (D-8; Santa Cruz) and p38 MAPK protein (Santa Cruz), phospho-ATF-2 (Cell Signaling Technologies), phospho-ERK1/2 and ERK1/2 (Cell Signaling Technologies), phosphoJNK/SAPK (Cell Signaling Technologies), and phospho-p90RSK (Cell Signaling Technologies), as specified by the manufacturers, followed by anti-rabbit secondary antibodies conjugated to horseradish peroxidase (HRP) (Santa Cruz), and then developed by enhanced chemiluminescence (ECL) (Pierce) and visualized on BioMax ML film (Kodak).

Techniques: Phospho-proteomics, Incubation, Labeling, Immunoprecipitation, Autoradiography, Western Blot, In Vivo, Cell Culture, Positive Control, Negative Control

Journal: Molecular and Cellular Biology

Article Title: Trophic Factor Withdrawal: p38 Mitogen-Activated Protein Kinase Activates NHE1, Which Induces Intracellular Alkalinization

doi: 10.1128/mcb.21.22.7545-7557.2001

Figure Lengend Snippet: FIG. 7. Kinase assays demonstrate that p38 MAPK directly phosphorylates NHE1 at a site(s) between amino acids 703 and 793. The numbering system used corresponds to the rabbit NHE1 sequence. (A) Protein lysates from FL5.12A cells cultured with or without IL-3 for 2 h were immunoprecipitated with either an antibody specific for phospho-p38 MAPK (p38) or an antibody specific for the motif, p-TP, and the ability of these kinases to phosphorylated the WT NHE-GST fusion protein was evaluated by an in-gel kinase assay as described in Materials and Methods. Only p38 phosphorylated the WT NHE-GST fusion protein, an activity which increased 1.8-fold during IL-3 withdrawal (left panel). Protein lysates from FL5.12A cells were also immunoprecipitated with c-Jun fusion beads to capture JNK, which was tested in an in vitro kinase assay for its ability to phosphorylate the WT NHEGST fusion protein. Unlike p38 MAPK, JNK did not phosphorylate NHE (right panel). (B) Protein lysates extracted from anisomycin-activated FL5.12A cells were immunoprecipitated with antibody to phospho-p38 MAPK and a kinase assay performed as described in Materials and Methods using the WT NHE-GST fusion protein and seven mutant NHE-GST fusion proteins as substrates. GST alone was included as a negative control. (C) The results of the kinase assay from panel B are summarized in table form. In addition, phospho-p38 MAPK did not phosphorylate NHE-GST fusion proteins containing amino acids 635 to 663. Possible phosphorylation sites by p38 MAPK on NHE are located in the region from amino acids 703 to 743, which excludes the proposed p90RSK phosphorylation site (Serine 703, numbering from human NHE1 sequence). (D) FL5.12A cells, cultured without IL-3 for 0, 1, 2, and 3 h or treated with anisomycin (), were lysed, and the precleared protein extracts were immunoprecipitated with the A7 antibody specific for NHE1 and then run on an 8% Tris-glycine SDS gel and analyzed by Western blotting with the p-TP-specific antibody (left panel). The same blots were then reprobed with a commercially available anti-NHE1 antibody (right panel). NHE1 did not contain an activated p-TP motif. (E) Mass spectrometry analysis, as described in Materials and Methods, identified four sites at Thr 717, Ser 722, Ser 725, and Ser 728 as in vitro targets for p38 MAPK activity. Phosphorylation of one or more of these sites by p38 MAPK modulates alkalinization mediated by NHE during trophic factor withdrawal.

Article Snippet: The blots were probed with a rabbit polyclonal antiserum specific for phospho-p38 MAPK protein (D-8; Santa Cruz) and p38 MAPK protein (Santa Cruz), phospho-ATF-2 (Cell Signaling Technologies), phospho-ERK1/2 and ERK1/2 (Cell Signaling Technologies), phosphoJNK/SAPK (Cell Signaling Technologies), and phospho-p90RSK (Cell Signaling Technologies), as specified by the manufacturers, followed by anti-rabbit secondary antibodies conjugated to horseradish peroxidase (HRP) (Santa Cruz), and then developed by enhanced chemiluminescence (ECL) (Pierce) and visualized on BioMax ML film (Kodak).

Techniques: Sequencing, Cell Culture, Immunoprecipitation, Kinase Assay, Activity Assay, In Vitro, Mutagenesis, Negative Control, Phospho-proteomics, SDS-Gel, Western Blot, Mass Spectrometry