Journal: Advanced Science

Article Title: Ion‐Driven Regulation of Ca 2 + Flow by Ti/Zn Composite Activates ERK/MAPK Signaling and Maintains Mitochondrial Homeostasis to Promote Macrophage‐Mediated Bone Regeneration

doi: 10.1002/advs.76941

Figure Lengend Snippet: RNA‐seq and mechanistic analyses showing Ti/Zn‐induced M2 polarization via ERK/MAPK signaling. Ti/Zn represents the Ti/Zn‐1500 composite, which is a Zn‐infiltrated Ti scaffold with a pore size of 1500 µm. (A) Principle component analysis of all genes in macrophage cultured in Ti/Zn composite extract versus Ti extract. (B) Enriched KEGG pathways of Ti/Zn composite versus Ti extract. (C) Volcano plot of transcriptomic analysis of differentially expressed genes. (D) GO analysis of Ti/Zn composite versus Ti extract. (E) Heatmap analysis of differential expression genes of Zn receptors. ( n = 3). (F) The immunoblots show the p‐ERK and ERK in macrophage after cultured in extract with or without TPEN or PD98059 administration. (G) Quantitative analyses of the immunoblots in (F). (H) The immunoblots show the p‐p38, p38, p‐cJUN, and cJUN in macrophage after cultured in extract with or without TPEN or PD98059 administration. (I) The secretion of TNF‐α from macrophage after cultured in extract with or without TPEN or PD98059 administration. ( n = 3, ***represents p < 0.001, t ‐test).

Article Snippet: TPEN, a high‐affinity zinc chelator, and PD98059, an ERK1/2 signaling inhibitor, were purchased from MedChem Express (New Jersey, USA).

Techniques: RNA Sequencing, Pore Size, Cell Culture, Quantitative Proteomics, Western Blot

Journal: PLoS ONE

Article Title: Disassembly of Shank and Homer Synaptic Clusters Is Driven by Soluble β-Amyloid 1-40 through Divergent NMDAR-Dependent Signalling Pathways

doi: 10.1371/journal.pone.0006011

Figure Lengend Snippet: Cultured cortical neurons were pre-treated for 45 min with the indicated pharmacological inhibitors before being treated for 1 h with Aβ (1 µM). Inhibitors were present in the medium during Aβ treatment. (A) PI-3K/mTOR pathway is required for Homer1b declustering. Pre-treatment with wortmannin (Wort, 2 µM) abolished the ability of Aβ to disperse Homer1b clusters (101.1±15.3%, cf. Aβ+Wort and Wort alone). Likewise, inhibition of the PI-3K downstream kinases PKB (with API-2, 30 µM) or mTor (with rapamycin, Rapa, 5 µM) prevented Aβ-induced declustering of Homer1b (90.6±8.4%, cf. Aβ+AP2 and API-2 alone; 95±8.2%, cf. Aβ+Rapa and rapamycin alone). Either the blockade of MEK/ERK pathway by pre-treatment with UO126 (10 µM; 70.9±4.8% UO126+Aβ vs UO126 alone) or cdk-5 inhibition by roscovitine (10 µM;63.7±5.8%, cf. Aβ+Rosco and Rosco alone) pre-treatment did affect Aβ-induced Homer declustering. (B) MEK/ERK pathway, but not PI-3K pathway, is required for Aβ-induced dispersal of Shank1. Pre-treatment with the PI-3K inhibitor wortmannin before Aβ treatment did not affect Aβ-induced Shank cluster dispersal (64.7±10.3%, cf. Wort+Aβ and Wort alone; p<0.05). Notably, wortmannin itself decreased Shank1 (but not Homer1b) cluster size (74.3±10.3%, cf. Wort and vehicle; p<0.05). Two structurally unrelated MEK inhibitors, UO126 (10 µM) and PD98059 (25 µM) prevented Aβ-induced declustering of Shank1 (96.8±12.1%, cf. PD98059+Aβ and PD98059 alone; 92.2±15.4%, cf. UO126+Aβ and UO126 alone). Likewise, the blockade of the Erk targeted kinase RSK by pre-treatment with SL0101-1 blocked Aβ effects (100.4±9.8%, p>0.05). Pre-treatment of neurons with the specific cdk-5 inhibitor roscovitine (Rosco, 10 µM) did not interfere with the ability of Aβ to reduce the size of Shank1 (72.6±10.8%, cf. Rosco+Aβ and Aβ alone) clusters. (C,D) PP2B is required for the dispersal of Homer1b, but not Shank1, clusters by Aβ. Pre-treatment with structurally unrelated PP2B inhibitors FK506 (10 µM) or cyclosporin (Cyclo, 100 µM) significantly blocked (p<0.05) Aβ-induced Homer1b declustering (98.8±10.4%, comparing FK506+Aβ vs. Aβ alone; 83.4±5.8%, comparing Aβ+Cyclo and Cyclo alone; and 61±7.9% in vehicle+Aβ-treated cells; see panel C ); however, all PP2B inhibitors were ineffective in preventing Shank1 declustering after Aβ exposure (61.3±4.5%, cf. FK506+Aβ and FK506 alone; 90.8±12.4%, cf. Cyclo+Aβ and Cyclo alone, see panel D ). For each condition, 8–12 neurons from 3–4 replicate experiments were considered (N = 8–12), and at least 50 synapses on each neuron were evaluated (on average, n = 500 puncta/condition).

Article Snippet: Nifedipine, (±)-verapamil, roscovitine, NiCl 2 , cycloheximide, cyclosporin and FK506 were purchased from Sigma Chemicals (Deisenhofen, Germany); NMDA, Bay-K4688, SL0101-1, MK801, UO126, PD98059, API-2, wortmannin, cantharidine were from Tocris (Bristol, UK); and sodium orthovanadate, rapamycin, TDZT, SU6656 and MG132 were from Calbiochem (La Jolla, CA).

Techniques: Cell Culture, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Reactive Oxygen Species-generating Mitochondrial DNA Mutation Up-regulates Hypoxia-inducible Factor-1α Gene Transcription via Phosphatidylinositol 3-Kinase-Akt/Protein Kinase C/Histone Deacetylase Pathway

doi: 10.1074/jbc.m109.054221

Figure Lengend Snippet: FIGURE 5. PI3K-Akt and PKC pathways are involved in the ROS-mediated HIF-1 mRNA overexpression. A, P29 and A11 cells were treated with dimethyl sulfoxide, PD98059, SB203580, SP600125, and LY294002 at the indicated concentrations for 18 h. Total RNA was extracted and subjected to Northern blot analysis. The blots were hybridized with a 32P-labeled HIF-1 cDNA. Ethidium bromide staining of the gel is also shown. B, cell lysates prepared from P29, P34, D6, and A11 cells were dissolved by SDS-PAGE. Proteins and phosphorylated proteins and -actin, which served as a loading control, were detected by immunoblotting. C, cell lysates prepared from P29, A11, and the cybrids were subjected to immunoblotting to detect Akt and phosphorylated Akt. -Actin served as a loading control. D, P29 cells were treated with 25 M H2O2 for up to 80 min. Cell lysates were prepared and subjected to immunoblotting as in C. E, A11 cells were treated with ebselene (20 M) for 18 h. Cell lysates were prepared and subjected to immunoblotting as in C. F, P29 and A11 cells were treated with Ro31-8220 at the indicated concentrations for 18 h. Total RNA was analyzed as in A.

Article Snippet: SP600125 was obtained fromTOCRIS Cookson, Ellisville, MO, and PD98059 was from Cell Signaling Technology, Beverly, MA.

Techniques: Over Expression, Northern Blot, Labeling, Staining, SDS Page, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Novel Osteoblastogenic Activity of Magnolia kobus : The Pharmacological Potential for Osteoporosis

doi: 10.3390/ijms27052472

Figure Lengend Snippet: Involvement of p38 MAPK in ME- and magnolin-induced mineralization in MC3T3-E1 cells. ( A ) ARS staining and ( B ) mineralization assays were performed as described in Materials and Methods. Cells were cultured in DM and treated with ME (100 μg/mL) or magnolin (10 μM) for 21 days in the presence or absence of PD98059 (25 μM) and SB203580 (5 μM). Untreated cells were cultured in 10% FBS-MEM alpha. ( A ) ARS staining images were photographed using a microscope (original magnification ×40). Results are presented as the fold-increase of untreated controls. Statistical significance is indicated ( ### p < 0.001, compared with untreated cells; *** p < 0.001, compared with DM-treated cells).

Article Snippet: The following agents were obtained from commercial sources: anti-DLX5 (sc-398150), anti-cathepsin K (sc-48353), and anti-osterix (sc-393325) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-RUNX2 (12556), anti-NFAT2 (NFATc1, 8032), anti-c-Fos (2250), anti-phospho-ERK (T202/Y204) (9101), anti-phospho-p38 MAPK (T180/Y182) (9215), anti-phospho-JNK (T183/Y185) (9251), anti-ERK (9102), anti-p38 MAPK (9212), anti-JNK (9252), anti-phospho-IKKα/β (2697), anti-IKKβ (8943), anti-phospho-IκBα (2859), anti-IκBα (4814), anti-actin (4970) antibodies, and rabbit and mouse IgG-horseradish peroxidase conjugates (Cell Signaling Technology, Beverly, MA, USA); and PD98059 (S1177) and SB203580 (S1076) (Selleck Chemicals, Houston, TX, USA).

Techniques: Staining, Cell Culture, Microscopy

Journal: Shock

Article Title: Inhibition of PAR-2 Attenuates Neuroinflammation and Improves Short-Term Neurocognitive Functions Via ERK1/2 Signaling Following Asphyxia-Induced Cardiac Arrest in Rats

doi: 10.1097/shk.0000000000001516

Figure Lengend Snippet: FIG. 5. Inhibition of ERK1/2 abolished the neuroinflammatory effect of PAR-2 activation at 24 h after ACA. Representative Western blot images (A) and quantitative analysis of PAR-2 (B), ERK1/2 (C), p-ERK1/2 (D), IL-6 (E), and TNF- a (F) in the brain revealed increased protein levels at 24 h following ACA except for ERK1/2 compared to the Sham group. Treatment with FSLLRY-NH2 significantly reduced p-ERK1/2 and proinflammatory cytokine levels compared to the ACA þ vehicle group. Further activation of PAR-2 with AC55541 only aggravated the neuroinflammatory response by increasing p-ERK1/2 expression. Potent ERK1/2 inhibitor PD98059 reversed the neuroinflammatory effect of AC55541. Neurologic outcome assessment with NDS at 24 h following ACA (G) revealed that the inhibition of PAR-2 significantly improved neurologic function while AC55541 alone significantly worsened performance compared to the ACA þ vehicle group. This detrimental effect of AC55541 on NDS was reversed by the ERK1/2 inhibitor, PD98059. Data are expressed as mean SD. n ¼ 6/group. ANOVA, Tukey. **P < 0.001 vs. Sham group, *P < 0.05 vs. Sham group, #P < 0.05 vs. ACA þvehicle group, &&P < 0.001 compared to ACA þ AC55541 group, &P < 0.05 compared to ACA þ AC55541 group. ACA indicates asphyxial cardiac arrest.

Article Snippet: Selective PAR-2 activator AC55541 (30 mg/rat) and ERK1/2 inhibitor PD98059 (Santa Cruz Biotechnology, Dallas, Tex; 2 mL of 2 mmol/L) were used for intervention (25).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

doi: 10.3389/fimmu.2018.01517

Figure Lengend Snippet: Impact of coagulation proteases on myofibrocyte phenotype. In (A,B) , responses of wild-type (WT) CD34+ cells are shown as white bars, whereas isolated CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. (A) Cells were incubated with FX in presence or absence of FVIIa and FII (prothrombin) plus FVa. Functional tissue factor on WT cells is illustrated by thrombin generation, angiopoietin-2 (Ang-2) secretion, and CXCL-12 secretion. The presence of human tissue factor pathway inhibitor on purified CD31+ myofibrocytes from CD31-TFPI-Tg mice significantly inhibits all three phenotype changes. (B) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with FX and FII in presence of FVIIa. (C) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with either PAR-1 antagonist (black bars), PAR-2 antagonist (white bars), or PAR-4 antagonist (gray bars) at the indicated concentrations for 30 min before addition of FVIIa with FX (both 10 nM) with or without prothrombin (4 nM) and FVa (6 nM) as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. In comparison of increasing concentrations of antagonists with FVIIa + FX, p = 0.027 for PAR2, but p = NS for PAR1 and PAR4. In comparison of increasing concentrations of antagonists with FVIIa + FX + FII + FVa, p = 0.05 for PAR1, but p = not significant (NS) for PAR2 and PAR4. Analysis by one-way ANOVA Kruskal–Wallis test. (D) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with PAR-1, -2, or -4 agonists at the indicated concentrations. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.017 for comparisons of increasing concentrations of PAR1 agonist, p = 0.012 for PAR2, but p = NS for PAR4 agonist. Analysis by one-way ANOVA Kruskal–Wallis test. (E) Dissection of signaling pathways involved in angiopoietin-2 secretion by WT CD34+ cells induced by 24 h incubation with 10 mM PAR-1 or -2 agonists. Cells were incubated with the agonists with or without 50 mM mitogen-activated protein kinase inhibitor PD98059, 10 mM p38-MAPK inhibitor SB203580, 20 mM NF-kB inhibitor SN50, or 1 mM of the S6K1 inhibitor as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.05 for PAR-1 agonist without inhibitor vs. +PD98509 and vs. +SB203580. p = > 0.05 all other comparisons. Analysis by Mann–Whitney T test. All experiments repeated at least twice.

Article Snippet: To assess PAR-induced cell signaling, starved cells were treated with 0–20 µM of either PAR-1, PAR-2, or PAR-4 antagonists (Peptides International, Louisville, KY, USA) for 30 min before stimulation with FVIIa + FX or FVIIa + FX + FII at indicated doses; or cells were stimulated with 0–100 µM of PAR-1, PAR-2, or PAR-4 agonist (Peptides International); or cells were treated first with or without 50 µM mitogen-activated protein kinase inhibitor PD98059, 10 µM p38-MAPK inhibitor SB203580, 20 µM NF-kB inhibitor SN50, and 1 µM of the S6K1 inhibitor (All from Merck Millipore, Hertfordshire, UK) for 30 min and then stimulated with 10 µM of PAR-1, PAR-2, or PAR-4 agonist.

Techniques: Coagulation, Isolation, Incubation, Functional Assay, Purification, Dissection, MANN-WHITNEY

Journal: iScience

Article Title: Mycobacterial Rv1804c binds to the PEST domain of IκBα and activates macrophage-mediated proinflammatory responses

doi: 10.1016/j.isci.2024.109101

Figure Lengend Snippet:

Article Snippet: PD98059 , MCE , Cat# HY-12028.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Reporter Assay, Transfection, Lysis, Western Blot