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    p38 MAP kinase inhibitor
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    99
    Millipore sb203580
    Effects of JNK or <t>p38</t> <t>MAPK</t> inhibitor on CRP-induced JNK phosphorylation, p38 MAPK phosphorylation, NF-κB p65 phosphorylation and RAGE expression in HCAECs. (A) Western blot analysis of JNK phosphorylation and p38 MAPK phosphorylation in HCAECs treated with vehicle, CRP, JNK inhibitor (SP600125), p38 MAPK inhibitor (SB203580), JNK inhibitor with CRP or p38 MAPK with CRP. (B) RAGE expression in HCAECs. Data are represented as the mean±SEM ( n =3–6). b P
    Sb203580, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9051 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb203580/product/Millipore
    Average 99 stars, based on 9051 article reviews
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    sb203580 - by Bioz Stars, 2021-01
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    99
    Cell Signaling Technology Inc sb203580
    Ameliorative effects of metformin on testosterone-induced endoplasmic reticulum (ER) stress in cumulus oocyte complexes (COCs) mediated via reduction of <t>p38</t> MAPK phosphorylation . ( A ) Unfolded protein response (UPR) sensor protein levels in COCs treated with 10 μM testosterone (T) and 1 mM metformin (met). ( B ) Changes in ER stress-related protein levels in COCs treated with 10 μM testosterone and 10 μM SB203580 (a p38 MAPK inhibitor) as detected by western blot. ( C ) Western blot results showing the changes in the indicated proteins in COCs treated with 10 μM testosterone after transfection with si-p38 MAPK. Real-time qPCR results showing the effects of SB203580 ( D ) and si-p38 MAPK ( E ) on ER stress-related mRNA transcription levels in COCs treated with 10 μM testosterone; the signals were normalized to those of Gapdh . The data are presented as means ± SEMs. * P
    Sb203580, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb203580/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1159 article reviews
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    sb203580 - by Bioz Stars, 2021-01
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    99
    Selleck Chemicals sb203580
    <t>SB203580</t> inhibits expansion of regulatory T cells (Tregs) in LPS-treated mice. C57BL/6J mice were injected with 200 µg of LPS (i.p.) or PBS, and treated with or without SB203580 (25 mg/kg/day, i.p.) immediately after LPS challenge. All mice were sacrificed 24 h after LPS treatment. Blood, spleen, and lymph nodes were harvested. The proportion of Foxp3 + Tregs in CD4 + T cells and expression of Ki-67 by Tregs were analyzed by FACS, gating on Foxp3 + cells. The absolute number of Tregs was calculated. (A) Expression of Foxp3 by CD4 + T cells. Number shows the proportion of gated cells. (B) Expression of Ki-67 by Foxp3 − and Foxp3 + cells. Number shows the proportion of positive cells in the respective quadrants. (A,B) Typical FACS plots were shown. (C) Summary of proportion of Tregs in CD4 + T cells in the peripheral blood, spleen and LNs. (D) Summary of absolute number of Tregs in the spleen. (E) Ki-67 expression (MFI) by Foxp3 + Tregs. Data [means ± SEM) in (C) were pooled from three separate experiments (spleen and lymph nodes: N = 9, peripheral blood: N = 6), and in (D,E) ( N = 3) were representatives of at least three separate experiments with similar results. By comparison with LPS alone group, * p
    Sb203580, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 703 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb203580/product/Selleck Chemicals
    Average 99 stars, based on 703 article reviews
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    sb203580 - by Bioz Stars, 2021-01
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    99
    Tocris sb203580
    <t>p38</t> <t>MAPK</t> signaling pathway is involved in CXCR3 protein expression in MSCs, CXCR3-mediated chemotaxis invasion, and transendothelial migration induced by IL-1β. a MSCs were pretreated with p38 MAPK inhibitor <t>SB203580,</t> at a concentration of 25 uM, then stimulated with IL-1β for 30 min. The expression of CXCR3 cytosolic and membrane protein were detected by Western blotting. b Quantitative results of the Western blotting of CXCR3 cytosolic and membrane protein expression of a . The data represent mean ± SD ( n = 3). Statistical analysis was determined by Student’s t test and one-way ANOVA. * P
    Sb203580, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 870 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb203580/product/Tocris
    Average 99 stars, based on 870 article reviews
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    sb203580 - by Bioz Stars, 2021-01
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    92
    Merck KGaA sb203580
    Analysis of the Rho signaling activity. (A) Effect of TGF-β2 on RhoA activity in HTM cells. (B) Effects of TGF-β2, Y-27632 and <t>SB203580</t> on myosin light chain (MLC)-2 phosphorylation in HTM cells. Cells with or without 10 μM Y-27632 or 10 μM SB203580 pretreatment for 30 min were stimulated with 2.5 ng/ml TGF-β2 for 30 min. Data shown in upper panels are results of representative Western blot analyses of phosphorylated MLC2 (pMLC2) and total MLC2. Relative changes in the ratio of MLC phosphorylation are shown in the lower graph. Data are shown as means ± SE, n = 10. *P
    Sb203580, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb203580/product/Merck KGaA
    Average 92 stars, based on 287 article reviews
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    sb203580 - by Bioz Stars, 2021-01
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    92
    Biomol GmbH sb203580
    NF-κB translocation is a functional index during Japanese encephalitis virus (JEV)-induced matrix metalloproteinase (MMP)-9 expression in rat brain astrocytes-1 cells. (A) Cells were pretreated with helenalin (HLN) for 1 h and then infected with JEV (moi = 1) for 16 h. (B) Cells were transfected with p65 siRNA, and then infected with JEV for 16 h. (A, B) The conditioned media were assessed for MMP-9 expression by gelatin zymography. The cell lysates were analysed by Western blot using an anti-NF-κB p65 or anti-GAPDH antibody. (C, D) Time dependence of JEV-induced NF-κB translocation and IκB-α degradation. Cells were pretreated with or without 1 µM helenalin, 1 µM U0126, 1 µM SP600125, 1 µM <t>SB203580,</t> 10 mM NAC, 10 µM diphenylene iodonium chloride (DPI) and 100 µM apocynin (APO) for 1 h, and then were stimulated with JEV (moi = 1) for the indicated time intervals. Nuclear and cytosolic fractions were analysed by Western blot using an anti-NF-κB (p65), anti-phospho-IκBα, anti-IκBα antibody or anti-GAPDH antibody. (E) Cells were pretreated with 1 µM helenalin for 1 h, and then infected with JEV (moi = 1) for 6 h. The RNA samples were analysed by real-time PCR for the levels of MMP-9 mRNA. (F) Cells were transiently transfected with an MMP-9-luc reporter gene, pretreated with helenalin for 1 h and then infected with JEV for 6 h. The promoter activity of MMP-9 was measured. Data are expressed as mean ± SEM of three independent experiments. * P
    Sb203580, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb203580/product/Biomol GmbH
    Average 92 stars, based on 224 article reviews
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    sb203580 - by Bioz Stars, 2021-01
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    92
    LC Laboratories sb203580
    Putative close contacts between the mutation sites of p38α kinase and the inhibitor <t>SB203580</t>
    Sb203580, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb203580/product/LC Laboratories
    Average 92 stars, based on 261 article reviews
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    sb203580 - by Bioz Stars, 2021-01
    92/100 stars
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    99
    Beyotime sb203580
    Putative close contacts between the mutation sites of p38α kinase and the inhibitor <t>SB203580</t>
    Sb203580, supplied by Beyotime, used in various techniques. Bioz Stars score: 99/100, based on 306 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    sb203580 - by Bioz Stars, 2021-01
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    92
    Cayman Chemical sb203580
    Optimizing culture conditions for supporting the stemness of pig muscle stem cells in vitro . To find suitable in vitro culture conditions for pig muscle stem cells, various culture components were tested. (A) Comparative analysis of the proliferation rate of pig muscle stem cells cultured with 10% FBS-supplemented MEM and SkGM-2. (B) The effect of basal media on pig muscle stem cells during the in vitro long-term culture. (C) The effect of basal media on the expression of myogenic marker genes during the in vitro long-term culture using qPCR. (D) The effect of <t>SB203580</t> on pig muscle stem cells during the in vitro long-term culture. (E) The effect of SB203580 on the expression of myogenic marker genes during the in vitro long-term culture using qPCR. * p
    Sb203580, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb203580/product/Cayman Chemical
    Average 92 stars, based on 299 article reviews
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    sb203580 - by Bioz Stars, 2021-01
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    96
    InvivoGen sb203580
    P38 and TAK1 inhibitors neutralize the negative effect of TLR8 activation on dendritic growth. (A and D) WT neurons were treated with CL075/poly dT for 30 min at 18 DIV and subjected to immunostaining with phospho-P38, phospho-TAK1, and neuronal marker NeuN antibodies as indicated. Counterstaining with DAPI was performed to label nuclei. (B and E) GFP was transfected at 12 DIV. CL075/poly dT and P38 inhibitor <t>SB203580</t> (B) and three TAK1 inhibitors, Takinib, 5ZO, and NG25 (E), were added at 17 DIV for 1 d in WT cultured neurons. (C) SB203580 treatment did not influence the effect of CL075 on younger WT neurons at 4 DIV. (B–E) Sample size ( n ) indicates the number of examined neurons, which were randomly collected blind from two independent experiments. Data are presented as the mean + SEM (error bars). Bars: (A) 25 µm; (B) 50 µm; (D) 20 µm. **, P
    Sb203580, supplied by InvivoGen, used in various techniques. Bioz Stars score: 96/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    AG Scientific sb203580
    Expression levels of HPGD, COX and mPGES1 following HCQ stimulation by wortmannin, PD98059 and <t>SB203580.</t> The RA-FLS were serum-starved for 2 h and subsequently treated with HCQ (10 µ M) for 24 h. Wortmannin (0.1, 0.5 and 1 nM), PD98059 (10, 50 and 100 µ M) and SB203580 (1, 10 and 50 µ M) were added 0.5 h prior to HCQ stimulation. The expression levels of (A and B) HPGD and (C and D) COX1, COX2 and mPGES1 in RA-FLS following the different treatments. RA-FLS, rheumatoid arthritis fibroblast-like synoviocytes; CON, control; HCQ, hydroxychloroquine; W, wortmannin; PD, PD98059; SB, SB203580; HPGD, 15-hydroxyprosta-glandin dehydrogenase; ACTB, β-actin; COX, cyclooxygenase; mPGES1, microsomal prostaglandin E2 synthase 1.
    Sb203580, supplied by AG Scientific, used in various techniques. Bioz Stars score: 92/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb203580/product/AG Scientific
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    sb203580 - by Bioz Stars, 2021-01
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    92
    Merck & Co sb203580
    Retinoic acid potentiates BMP inhibition of feather patterning. (A) RA administration reduces the density of placodes, which are detected by β - catenin in situ hybridization, completely inhibiting placode formation at high doses. Suppression of BMP signaling with 4 µM dorsomorphin and 5 µM <t>SB203580</t> rescues placode formation in the presence of RA. (B) Quantification of placode density on neck and body upon RA treatment. With increasing doses of RA the feather density on body and neck converges and ultimately all feather placode formation is suppressed. (C) RA sensitizes body skin to BMP-driven inhibition of feather development. The application of 0.1 µM RA has little effect on the placode pattern and application of 40 ng/ml BMP12 permits placode formation on the body. Co-treatment with RA and BMP12 has a synergistic effect, completely suppressing feather development on the body. Conversely, treatment of skin with the RA synthesis inhibitor Citral renders the neck resistant to suppression of placode formation by BMPs.
    Sb203580, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 132 article reviews
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    sb203580 - by Bioz Stars, 2021-01
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    92
    Abcam sb203580
    Co-culture with macrophages increases IL1α and IL1β expression in MDA-MB-231 cells in a TAK1-dependent manner. a qPCR analysis of Il1α , Il1β , Tgfβ , and Tnfα expression in macrophages cultured in the presence or absence of MDA-MB-231 cells for 5 days. b qPCR analysis of IL1α , IL1β , TGFβ , and TNFα expression in MDA-MB-231 cells cultured in the presence or absence of macrophages for 5 days. c Percentage of IL1α-positive (left) and IL1β-positive (right) cells in GFP-tagged MDA-MB-231 monocultures and GFP-tagged MDA-MB-231 cells cultured with macrophages. d Immunofluorescent staining of lungs dissected from mice injected with GFP and luciferase-tagged MDA-MB-231 cells. Representative examples of images taken from three immunostainings of two independent experiments are shown. GFP (yellow), macrophage marker F4/80 (magenta), IL1α or IL1β (white), DAPI (gray in single channel, blue in merge image) are shown. Scale bar = 20 μm. e qPCR analysis of IL1α and IL1β expression in MDA-MB-231 cells stably expressing doxycycline-inducible dominant-negative TAK1 (TAK1-dn), grown in co-culture with macrophages for 3 days in the presence or absence of doxycycline (doxy). f qPCR analysis of IL1α and IL1β expression in MDA-MB-231 cells cultured in the presence or absence of macrophages and treated or not with 1μM <t>SB203580.</t> In a , b , e , and f , GFP-tagged MDA-MB-231 cells were used, and macrophages and MDA-MB-231 cells were separated by FACS for RNA extraction. Graphs show means ± SD of three ( e , f ), four ( a ), five ( c , right panel) and six ( b , c , left panel) independent experiments. Statistical significance analyzed by unpaired two-tailed Student’s t test * p ≤ 0.05; ** p ≤ 0.01
    Sb203580, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of JNK or p38 MAPK inhibitor on CRP-induced JNK phosphorylation, p38 MAPK phosphorylation, NF-κB p65 phosphorylation and RAGE expression in HCAECs. (A) Western blot analysis of JNK phosphorylation and p38 MAPK phosphorylation in HCAECs treated with vehicle, CRP, JNK inhibitor (SP600125), p38 MAPK inhibitor (SB203580), JNK inhibitor with CRP or p38 MAPK with CRP. (B) RAGE expression in HCAECs. Data are represented as the mean±SEM ( n =3–6). b P

    Journal: Acta Pharmacologica Sinica

    Article Title: C-reactive protein stimulates RAGE expression in human coronary artery endothelial cells in vitro via ROS generation and ERK/NF-κB activation

    doi: 10.1038/aps.2014.163

    Figure Lengend Snippet: Effects of JNK or p38 MAPK inhibitor on CRP-induced JNK phosphorylation, p38 MAPK phosphorylation, NF-κB p65 phosphorylation and RAGE expression in HCAECs. (A) Western blot analysis of JNK phosphorylation and p38 MAPK phosphorylation in HCAECs treated with vehicle, CRP, JNK inhibitor (SP600125), p38 MAPK inhibitor (SB203580), JNK inhibitor with CRP or p38 MAPK with CRP. (B) RAGE expression in HCAECs. Data are represented as the mean±SEM ( n =3–6). b P

    Article Snippet: NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), ERK inhibitor (PD98059), p38 MAPK inhibitor (SB203580) and JNK inhibitor (SP600125) were purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Expressing, Western Blot

    Dose-dependent effects of chemerin on the biological characteristic of EPCs. The control group was not subjected to any drug treatment; the control group received only the same volume of solvent used for the drug treatments in the treatment groups. The chemerin treatment groups included 2.5 ng/mL, 25 ng/mL, 50 ng/mL and 100 ng/mL protein-stimulated groups. EPCs were treated for 24 h. The inhibitor group was stimulated with 50 ng/mL chemerin protein plus 10 μmol/mL SB 203580.  a  Adherence of EPCs, a: VS. the control group,  P 

    Journal: Lipids in Health and Disease

    Article Title: Chemerin enhances the adhesion and migration of human endothelial progenitor cells and increases lipid accumulation in mice with atherosclerosis

    doi: 10.1186/s12944-020-01378-5

    Figure Lengend Snippet: Dose-dependent effects of chemerin on the biological characteristic of EPCs. The control group was not subjected to any drug treatment; the control group received only the same volume of solvent used for the drug treatments in the treatment groups. The chemerin treatment groups included 2.5 ng/mL, 25 ng/mL, 50 ng/mL and 100 ng/mL protein-stimulated groups. EPCs were treated for 24 h. The inhibitor group was stimulated with 50 ng/mL chemerin protein plus 10 μmol/mL SB 203580. a Adherence of EPCs, a: VS. the control group, P 

    Article Snippet: Treatment of EPCsAfter culturing for 7 days, the adherent cells were resuspended and randomly divided into the control group, recombinant human chemerin protein (R & D California, USA, catalogue #: 2324-CM-025/CF) treatment groups and inhibitor group (SB 203580, Sigma, Missouri, USA, catalogue #: S8307).

    Techniques:

    Effects of chemerin on lipid parameters in ApoE−/− mice. WT mice were not treated with any drugs ( n = 10). The control group ( n = 6) was injected with PBS by weight, the chemerin group ( n = 8) was injected with chemerin protein by weight, and the inhibitor group ( n = 8) was injected with chemerin protein and the p38 MAPK-specific inhibitor SB 203580 by weight. The results are presented as the mean ± SD. a Serum T-CHO concentration. The results are expressed as the mean ± SEM. b Serum TG concentration. c Serum LDL concentration. d Serum HDL concentration. e Liver T-CHO concentration. f Liver TG concentration. g Liver LDL concentration. h Liver HDL concentration. a: VS. the WT group, P

    Journal: Lipids in Health and Disease

    Article Title: Chemerin enhances the adhesion and migration of human endothelial progenitor cells and increases lipid accumulation in mice with atherosclerosis

    doi: 10.1186/s12944-020-01378-5

    Figure Lengend Snippet: Effects of chemerin on lipid parameters in ApoE−/− mice. WT mice were not treated with any drugs ( n = 10). The control group ( n = 6) was injected with PBS by weight, the chemerin group ( n = 8) was injected with chemerin protein by weight, and the inhibitor group ( n = 8) was injected with chemerin protein and the p38 MAPK-specific inhibitor SB 203580 by weight. The results are presented as the mean ± SD. a Serum T-CHO concentration. The results are expressed as the mean ± SEM. b Serum TG concentration. c Serum LDL concentration. d Serum HDL concentration. e Liver T-CHO concentration. f Liver TG concentration. g Liver LDL concentration. h Liver HDL concentration. a: VS. the WT group, P

    Article Snippet: Treatment of EPCsAfter culturing for 7 days, the adherent cells were resuspended and randomly divided into the control group, recombinant human chemerin protein (R & D California, USA, catalogue #: 2324-CM-025/CF) treatment groups and inhibitor group (SB 203580, Sigma, Missouri, USA, catalogue #: S8307).

    Techniques: Mouse Assay, Injection, Concentration Assay

    Time-dependent effects of chemerin on the biological characteristic of EPCs. The control group was not subjected to any drug treatment; the control group received only the same volume of solvent used for the drug treatments in the treatment groups. The chemerin treatment groups included 12 h, 24 h, 36 h and 48 h protein-stimulated groups. EPCs were treated with chemerin at a concentration of 50 ng/mL. The inhibitor group was stimulated with 50 ng/mL chemerin protein plus 10 μmol/mL SB 203580 for 24 h. a Adherence of EPCs, a: VS. the control group, P

    Journal: Lipids in Health and Disease

    Article Title: Chemerin enhances the adhesion and migration of human endothelial progenitor cells and increases lipid accumulation in mice with atherosclerosis

    doi: 10.1186/s12944-020-01378-5

    Figure Lengend Snippet: Time-dependent effects of chemerin on the biological characteristic of EPCs. The control group was not subjected to any drug treatment; the control group received only the same volume of solvent used for the drug treatments in the treatment groups. The chemerin treatment groups included 12 h, 24 h, 36 h and 48 h protein-stimulated groups. EPCs were treated with chemerin at a concentration of 50 ng/mL. The inhibitor group was stimulated with 50 ng/mL chemerin protein plus 10 μmol/mL SB 203580 for 24 h. a Adherence of EPCs, a: VS. the control group, P

    Article Snippet: Treatment of EPCsAfter culturing for 7 days, the adherent cells were resuspended and randomly divided into the control group, recombinant human chemerin protein (R & D California, USA, catalogue #: 2324-CM-025/CF) treatment groups and inhibitor group (SB 203580, Sigma, Missouri, USA, catalogue #: S8307).

    Techniques: Concentration Assay

    Role of Fn14 and intracellular signaling pathways in rTWEAK-induced pro-inflammatory cytokine expression. IL-6, IL-8, and MCP-1 protein levels in GO orbital fibroblasts (n = 3) pretreated with ERK kinase (PD98059, 20 μM), JNK (SP600125, 20 μM), p38 MAPK (SB203580, 20 μM), PI3K (LY294002, 20 μM), NF-κB p65 (SC514, 10 μM), and Fn14 (ITEM4, 2.5 μg/ml) inhibitor for 1 h followed by rTWEAK treatment (100 ng/ml, 24 h) were measured by ELISA. Data in columns represent mean ± standard deviation of three experiments. *P

    Journal: PLoS ONE

    Article Title: Tumor necrosis factor-like weak inducer of apoptosis induces inflammation in Graves’ orbital fibroblasts

    doi: 10.1371/journal.pone.0209583

    Figure Lengend Snippet: Role of Fn14 and intracellular signaling pathways in rTWEAK-induced pro-inflammatory cytokine expression. IL-6, IL-8, and MCP-1 protein levels in GO orbital fibroblasts (n = 3) pretreated with ERK kinase (PD98059, 20 μM), JNK (SP600125, 20 μM), p38 MAPK (SB203580, 20 μM), PI3K (LY294002, 20 μM), NF-κB p65 (SC514, 10 μM), and Fn14 (ITEM4, 2.5 μg/ml) inhibitor for 1 h followed by rTWEAK treatment (100 ng/ml, 24 h) were measured by ELISA. Data in columns represent mean ± standard deviation of three experiments. *P

    Article Snippet: Inhibitors of MAPK kinase 1 (PD98059), p38 MAPK (SB203580), JNK (SP600125), phosphoinositide 3-kinase (PI3K; LY294002), and NF-κB p65 (SC514) were purchased from Calbiochem (La Jolla, CA, USA).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Effect of Fn14 inhibitor on rTWEAK-induced hyaluronan production in GO cells. Hyaluronan (ng/ml) level in GO orbital fibroblasts (n = 3) was measured by ELISA after treatment with rTWEAK (10, 100 and 200 ng/ml) for 6 and 24 h. Cells (n = 3) were pretreated with ERK kinase (PD98059, 20 μM), JNK (SP600125, 20 μM), p38 MAPK (SB203580, 20 μM), NF-κB p65 (SC514, 10 μM), and Fn14 (ITEM4, 2.5 μg/ml) inhibitor for 1 h followed by rTWEAK treatment (100 ng/ml, 24 h). Data in columns represent mean ± standard deviation of three experiments. *P

    Journal: PLoS ONE

    Article Title: Tumor necrosis factor-like weak inducer of apoptosis induces inflammation in Graves’ orbital fibroblasts

    doi: 10.1371/journal.pone.0209583

    Figure Lengend Snippet: Effect of Fn14 inhibitor on rTWEAK-induced hyaluronan production in GO cells. Hyaluronan (ng/ml) level in GO orbital fibroblasts (n = 3) was measured by ELISA after treatment with rTWEAK (10, 100 and 200 ng/ml) for 6 and 24 h. Cells (n = 3) were pretreated with ERK kinase (PD98059, 20 μM), JNK (SP600125, 20 μM), p38 MAPK (SB203580, 20 μM), NF-κB p65 (SC514, 10 μM), and Fn14 (ITEM4, 2.5 μg/ml) inhibitor for 1 h followed by rTWEAK treatment (100 ng/ml, 24 h). Data in columns represent mean ± standard deviation of three experiments. *P

    Article Snippet: Inhibitors of MAPK kinase 1 (PD98059), p38 MAPK (SB203580), JNK (SP600125), phosphoinositide 3-kinase (PI3K; LY294002), and NF-κB p65 (SC514) were purchased from Calbiochem (La Jolla, CA, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

    Potential mechanisms of CRP promote AKI CRP activates Smad3 via both TGF-β1 or ERK/p38 MAP kinase-dependent mechanisms, which upregulates p27 to cause TEC growth arrest at the G1 cell cycle by suppressing CDK2/cyclin E activities.

    Journal: Kidney international

    Article Title: C-reactive protein promotes acute kidney injury by impairing tubular epithelial cell regeneration via the CD32-Smad3-p27 dependent inhibition of CDK2/cyclin E mechanism

    doi: 10.1016/j.kint.2016.06.010

    Figure Lengend Snippet: Potential mechanisms of CRP promote AKI CRP activates Smad3 via both TGF-β1 or ERK/p38 MAP kinase-dependent mechanisms, which upregulates p27 to cause TEC growth arrest at the G1 cell cycle by suppressing CDK2/cyclin E activities.

    Article Snippet: To define the crosstalk pathway of CRP-induced Smad3 signaling via ERK/p38 MAP kinases, specific inhibitors to ERK1/2 (PD98059, 10 μg/ml) and p38 (SB203580, 10 μg/ml) from Calbiochem (La Jolla, CA, USA) were added into the culture 1 h before CRP (10μg/ml) stimulation.

    Techniques:

    Ameliorative effects of metformin on testosterone-induced endoplasmic reticulum (ER) stress in cumulus oocyte complexes (COCs) mediated via reduction of p38 MAPK phosphorylation . ( A ) Unfolded protein response (UPR) sensor protein levels in COCs treated with 10 μM testosterone (T) and 1 mM metformin (met). ( B ) Changes in ER stress-related protein levels in COCs treated with 10 μM testosterone and 10 μM SB203580 (a p38 MAPK inhibitor) as detected by western blot. ( C ) Western blot results showing the changes in the indicated proteins in COCs treated with 10 μM testosterone after transfection with si-p38 MAPK. Real-time qPCR results showing the effects of SB203580 ( D ) and si-p38 MAPK ( E ) on ER stress-related mRNA transcription levels in COCs treated with 10 μM testosterone; the signals were normalized to those of Gapdh . The data are presented as means ± SEMs. * P

    Journal: Human Reproduction (Oxford, England)

    Article Title: Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK

    doi: 10.1093/humrep/deaa077

    Figure Lengend Snippet: Ameliorative effects of metformin on testosterone-induced endoplasmic reticulum (ER) stress in cumulus oocyte complexes (COCs) mediated via reduction of p38 MAPK phosphorylation . ( A ) Unfolded protein response (UPR) sensor protein levels in COCs treated with 10 μM testosterone (T) and 1 mM metformin (met). ( B ) Changes in ER stress-related protein levels in COCs treated with 10 μM testosterone and 10 μM SB203580 (a p38 MAPK inhibitor) as detected by western blot. ( C ) Western blot results showing the changes in the indicated proteins in COCs treated with 10 μM testosterone after transfection with si-p38 MAPK. Real-time qPCR results showing the effects of SB203580 ( D ) and si-p38 MAPK ( E ) on ER stress-related mRNA transcription levels in COCs treated with 10 μM testosterone; the signals were normalized to those of Gapdh . The data are presented as means ± SEMs. * P

    Article Snippet: For determining the role of p38 MAPK in hyperandrogenism-induced ER stress, the GCs were pre-treated with the p38 MAPK inhibitor SB203580 (10 μM; Cell Signaling Technology, Boston, MA, USA) for 2 h and then incubated with 10 μM testosterone for another 24 h. Forskolin (FSK, 10 μM; Med Chem Express Company, Monmouth Junction, NJ, USA) and phorbol 12-myristate 13-acetate (PMA, 20 nM; Med Chem Express Company, Monmouth Junction, NJ, USA) were used to mimic LH stimulation ( ).

    Techniques: Western Blot, Transfection, Real-time Polymerase Chain Reaction

    Ameliorative effects of metformin on testosterone-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) mediated via reduction of p38 MAPK phosphorylation . ( A ) Unfolded protein response (UPR) sensor protein levels in GCs after treatment with 10 μM testosterone (T) and 1 mM metformin (met). ( B ) Protein changes in GCs treated with 10 μM testosterone and 10 μM SB203580 (a p38 MAPK inhibitor) as detected by western blot. ( C ) Western blot results showing the changes in the indicated proteins in GCs treated with 10 μM testosterone after transfection with si-p38 MAPK. ( D , E ) Effects of SB203580 (D) and si-p38 MAPK (E) on ER stress-related mRNA expression levels in GCs treated with 10 μM testosterone; the signals were normalized to those of Gapdh . The data are presented as means ± SEMs. * P

    Journal: Human Reproduction (Oxford, England)

    Article Title: Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK

    doi: 10.1093/humrep/deaa077

    Figure Lengend Snippet: Ameliorative effects of metformin on testosterone-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) mediated via reduction of p38 MAPK phosphorylation . ( A ) Unfolded protein response (UPR) sensor protein levels in GCs after treatment with 10 μM testosterone (T) and 1 mM metformin (met). ( B ) Protein changes in GCs treated with 10 μM testosterone and 10 μM SB203580 (a p38 MAPK inhibitor) as detected by western blot. ( C ) Western blot results showing the changes in the indicated proteins in GCs treated with 10 μM testosterone after transfection with si-p38 MAPK. ( D , E ) Effects of SB203580 (D) and si-p38 MAPK (E) on ER stress-related mRNA expression levels in GCs treated with 10 μM testosterone; the signals were normalized to those of Gapdh . The data are presented as means ± SEMs. * P

    Article Snippet: For determining the role of p38 MAPK in hyperandrogenism-induced ER stress, the GCs were pre-treated with the p38 MAPK inhibitor SB203580 (10 μM; Cell Signaling Technology, Boston, MA, USA) for 2 h and then incubated with 10 μM testosterone for another 24 h. Forskolin (FSK, 10 μM; Med Chem Express Company, Monmouth Junction, NJ, USA) and phorbol 12-myristate 13-acetate (PMA, 20 nM; Med Chem Express Company, Monmouth Junction, NJ, USA) were used to mimic LH stimulation ( ).

    Techniques: Western Blot, Transfection, Expressing

    SB203580 inhibits expansion of regulatory T cells (Tregs) in LPS-treated mice. C57BL/6J mice were injected with 200 µg of LPS (i.p.) or PBS, and treated with or without SB203580 (25 mg/kg/day, i.p.) immediately after LPS challenge. All mice were sacrificed 24 h after LPS treatment. Blood, spleen, and lymph nodes were harvested. The proportion of Foxp3 + Tregs in CD4 + T cells and expression of Ki-67 by Tregs were analyzed by FACS, gating on Foxp3 + cells. The absolute number of Tregs was calculated. (A) Expression of Foxp3 by CD4 + T cells. Number shows the proportion of gated cells. (B) Expression of Ki-67 by Foxp3 − and Foxp3 + cells. Number shows the proportion of positive cells in the respective quadrants. (A,B) Typical FACS plots were shown. (C) Summary of proportion of Tregs in CD4 + T cells in the peripheral blood, spleen and LNs. (D) Summary of absolute number of Tregs in the spleen. (E) Ki-67 expression (MFI) by Foxp3 + Tregs. Data [means ± SEM) in (C) were pooled from three separate experiments (spleen and lymph nodes: N = 9, peripheral blood: N = 6), and in (D,E) ( N = 3) were representatives of at least three separate experiments with similar results. By comparison with LPS alone group, * p

    Journal: Frontiers in Immunology

    Article Title: The p38 MAPK Inhibitor SB203580 Abrogates Tumor Necrosis Factor-Induced Proliferative Expansion of Mouse CD4+Foxp3+ Regulatory T Cells

    doi: 10.3389/fimmu.2018.01556

    Figure Lengend Snippet: SB203580 inhibits expansion of regulatory T cells (Tregs) in LPS-treated mice. C57BL/6J mice were injected with 200 µg of LPS (i.p.) or PBS, and treated with or without SB203580 (25 mg/kg/day, i.p.) immediately after LPS challenge. All mice were sacrificed 24 h after LPS treatment. Blood, spleen, and lymph nodes were harvested. The proportion of Foxp3 + Tregs in CD4 + T cells and expression of Ki-67 by Tregs were analyzed by FACS, gating on Foxp3 + cells. The absolute number of Tregs was calculated. (A) Expression of Foxp3 by CD4 + T cells. Number shows the proportion of gated cells. (B) Expression of Ki-67 by Foxp3 − and Foxp3 + cells. Number shows the proportion of positive cells in the respective quadrants. (A,B) Typical FACS plots were shown. (C) Summary of proportion of Tregs in CD4 + T cells in the peripheral blood, spleen and LNs. (D) Summary of absolute number of Tregs in the spleen. (E) Ki-67 expression (MFI) by Foxp3 + Tregs. Data [means ± SEM) in (C) were pooled from three separate experiments (spleen and lymph nodes: N = 9, peripheral blood: N = 6), and in (D,E) ( N = 3) were representatives of at least three separate experiments with similar results. By comparison with LPS alone group, * p

    Article Snippet: Sulfasalazine (Cat#: S1576) and SB203580 (Cat#: S1076) was obtained from Selleckchem.

    Techniques: Mouse Assay, Injection, Expressing, FACS

    Upregulation of TNFR2 expression on regulatory T cells (Tregs) induced by tumor necrosis factor (TNF) is abrogated by SB203580. MACS-purified CD4 + T cells were cultured in the presence of IL-2 (10 ng/mL), or IL-2 + TNF (10 ng/mL, each), with medium alone or with SB203580 (1–25 µM). The cells were cultured for 72 h. The surface expression of TNFR2 and intracellular expression of Foxp3 were analyzed with FACS. (A) Typical FACS dot plot of TNFR2 and Foxp3 expression. Data shown are representatives of at least three separate experiments with similar results. Number in the FACS plot shows the proportion of cells in the respective quadrants. (B) Summary of mean fluorescence intensity (MFI) of TNFR2 expression on Tregs (by gating on Foxp3 + cells. N = 3, means ± SEM). (C) Percent inhibition of TNFR2 expression on Foxp3 + Tregs ( N = 3, means ± SEM). The formula used to calculate percent inhibition is: (A − B)/A × 100%, A is MFI of TNFR2 expression treated with TNF/IL-2, B is MFI of TNFR2 expression treated with SB203580 (1–25 µM) + TNF/IL-2. By comparison with “TNF + IL-2” group, ** p

    Journal: Frontiers in Immunology

    Article Title: The p38 MAPK Inhibitor SB203580 Abrogates Tumor Necrosis Factor-Induced Proliferative Expansion of Mouse CD4+Foxp3+ Regulatory T Cells

    doi: 10.3389/fimmu.2018.01556

    Figure Lengend Snippet: Upregulation of TNFR2 expression on regulatory T cells (Tregs) induced by tumor necrosis factor (TNF) is abrogated by SB203580. MACS-purified CD4 + T cells were cultured in the presence of IL-2 (10 ng/mL), or IL-2 + TNF (10 ng/mL, each), with medium alone or with SB203580 (1–25 µM). The cells were cultured for 72 h. The surface expression of TNFR2 and intracellular expression of Foxp3 were analyzed with FACS. (A) Typical FACS dot plot of TNFR2 and Foxp3 expression. Data shown are representatives of at least three separate experiments with similar results. Number in the FACS plot shows the proportion of cells in the respective quadrants. (B) Summary of mean fluorescence intensity (MFI) of TNFR2 expression on Tregs (by gating on Foxp3 + cells. N = 3, means ± SEM). (C) Percent inhibition of TNFR2 expression on Foxp3 + Tregs ( N = 3, means ± SEM). The formula used to calculate percent inhibition is: (A − B)/A × 100%, A is MFI of TNFR2 expression treated with TNF/IL-2, B is MFI of TNFR2 expression treated with SB203580 (1–25 µM) + TNF/IL-2. By comparison with “TNF + IL-2” group, ** p

    Article Snippet: Sulfasalazine (Cat#: S1576) and SB203580 (Cat#: S1076) was obtained from Selleckchem.

    Techniques: Expressing, Magnetic Cell Separation, Purification, Cell Culture, FACS, Fluorescence, Inhibition

    SB203580 inhibits Foxp3 expression in tumor necrosis factor (TNF)-treated regulatory T cells. FACS-sorted CD4 + CD25 + T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 Abs, in the presence or absence of TNF (10 ng/mL), with or without 25 µM SB203580 for 3 days. Foxp3 expression and ratio of Foxp3 + cells were analyzed by FACS. (A) Typical histograms of Foxp3 expression. Number in the histogram indicates the proportion of gated cells. (B) Summary of Foxp3 expression (MFI. N = 3, means ± SEM). (C) Summary of proportion of Foxp3-expressing cells ( N = 3, means ± SEM). By comparison with TNF group (without SB203580), * p

    Journal: Frontiers in Immunology

    Article Title: The p38 MAPK Inhibitor SB203580 Abrogates Tumor Necrosis Factor-Induced Proliferative Expansion of Mouse CD4+Foxp3+ Regulatory T Cells

    doi: 10.3389/fimmu.2018.01556

    Figure Lengend Snippet: SB203580 inhibits Foxp3 expression in tumor necrosis factor (TNF)-treated regulatory T cells. FACS-sorted CD4 + CD25 + T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 Abs, in the presence or absence of TNF (10 ng/mL), with or without 25 µM SB203580 for 3 days. Foxp3 expression and ratio of Foxp3 + cells were analyzed by FACS. (A) Typical histograms of Foxp3 expression. Number in the histogram indicates the proportion of gated cells. (B) Summary of Foxp3 expression (MFI. N = 3, means ± SEM). (C) Summary of proportion of Foxp3-expressing cells ( N = 3, means ± SEM). By comparison with TNF group (without SB203580), * p

    Article Snippet: Sulfasalazine (Cat#: S1576) and SB203580 (Cat#: S1076) was obtained from Selleckchem.

    Techniques: Expressing, FACS

    SB203580 inhibits the upregulation of TNFR2 expression and CD152 expression on regulatory T cells (Tregs) in LPS-treated mice. C57BL/6J mice were injected with 200 µg of LPS (i.p.) or PBS, and treated with or without SB203580 (25 mg/kg/day, i.p.). Mouse spleen were harvested at 24 h after injection for the FACS analysis of CD152 and TNFR2 expression, gating on Foxp3 + cells. (A,B) Typical FACS histograms were shown. Black solid line: vehicle control; gray-filled histogram: LPS treatment; hair line: LPS + SB203580; Dot histogram: isotype control. Summary TNFR2 expression [MFI. (C) ] and CD152 expression [MFI, (D) ] by Foxp3 + Tregs ( N = 3, means ± SEM). Data shown are representatives of at least three separate experiments with similar results. By comparison with LPS alone group, * p

    Journal: Frontiers in Immunology

    Article Title: The p38 MAPK Inhibitor SB203580 Abrogates Tumor Necrosis Factor-Induced Proliferative Expansion of Mouse CD4+Foxp3+ Regulatory T Cells

    doi: 10.3389/fimmu.2018.01556

    Figure Lengend Snippet: SB203580 inhibits the upregulation of TNFR2 expression and CD152 expression on regulatory T cells (Tregs) in LPS-treated mice. C57BL/6J mice were injected with 200 µg of LPS (i.p.) or PBS, and treated with or without SB203580 (25 mg/kg/day, i.p.). Mouse spleen were harvested at 24 h after injection for the FACS analysis of CD152 and TNFR2 expression, gating on Foxp3 + cells. (A,B) Typical FACS histograms were shown. Black solid line: vehicle control; gray-filled histogram: LPS treatment; hair line: LPS + SB203580; Dot histogram: isotype control. Summary TNFR2 expression [MFI. (C) ] and CD152 expression [MFI, (D) ] by Foxp3 + Tregs ( N = 3, means ± SEM). Data shown are representatives of at least three separate experiments with similar results. By comparison with LPS alone group, * p

    Article Snippet: Sulfasalazine (Cat#: S1576) and SB203580 (Cat#: S1076) was obtained from Selleckchem.

    Techniques: Expressing, Mouse Assay, Injection, FACS

    SB203580 (SB) inhibits tumor necrosis factor (TNF)-mediated expansion of regulatory T cells (Tregs) in vitro . CD4 + T cells were purified from LNs and spleen of normal C57BL/6J mice by MACS. The cells were labeled with CFSE and cultured in the presence of IL-2 (10 ng/mL), or IL-2 + TNF (10 ng/mL, each), with medium alone or with different concentrations of SB203580 (SB, 1, 5, 10, and 25 µM). After 72 h, the proliferation of Tregs and the proportion of Foxp3 + cells were analyzed by FACS, based on CFSE expression and Foxp3 expression. The absolute number of Foxp3-expressing Tregs was calculated. (A) In the presence of IL-2, TNF preferentially stimulated the proliferation of Tregs. (B,C) SB203580 blocked TNF-mediated proliferation of Tregs. Analysis was gated on Foxp3 + Tregs. (D) SB203580 decreased the proportion of Foxp3 + Tregs in the cultured CD4 + T cells. (E) SB203580 reduced the absolute number of Tregs in the cultured CD4 + T cells. (A,B) Show the typical FACS plots. The number in the histogram indicates the proportion of gated cells (%). (C,D) Show the summary of results ( N = 3, means ± SEM). By comparison with “TNF + IL-2” group, * p

    Journal: Frontiers in Immunology

    Article Title: The p38 MAPK Inhibitor SB203580 Abrogates Tumor Necrosis Factor-Induced Proliferative Expansion of Mouse CD4+Foxp3+ Regulatory T Cells

    doi: 10.3389/fimmu.2018.01556

    Figure Lengend Snippet: SB203580 (SB) inhibits tumor necrosis factor (TNF)-mediated expansion of regulatory T cells (Tregs) in vitro . CD4 + T cells were purified from LNs and spleen of normal C57BL/6J mice by MACS. The cells were labeled with CFSE and cultured in the presence of IL-2 (10 ng/mL), or IL-2 + TNF (10 ng/mL, each), with medium alone or with different concentrations of SB203580 (SB, 1, 5, 10, and 25 µM). After 72 h, the proliferation of Tregs and the proportion of Foxp3 + cells were analyzed by FACS, based on CFSE expression and Foxp3 expression. The absolute number of Foxp3-expressing Tregs was calculated. (A) In the presence of IL-2, TNF preferentially stimulated the proliferation of Tregs. (B,C) SB203580 blocked TNF-mediated proliferation of Tregs. Analysis was gated on Foxp3 + Tregs. (D) SB203580 decreased the proportion of Foxp3 + Tregs in the cultured CD4 + T cells. (E) SB203580 reduced the absolute number of Tregs in the cultured CD4 + T cells. (A,B) Show the typical FACS plots. The number in the histogram indicates the proportion of gated cells (%). (C,D) Show the summary of results ( N = 3, means ± SEM). By comparison with “TNF + IL-2” group, * p

    Article Snippet: Sulfasalazine (Cat#: S1576) and SB203580 (Cat#: S1076) was obtained from Selleckchem.

    Techniques: In Vitro, Purification, Mouse Assay, Magnetic Cell Separation, Labeling, Cell Culture, FACS, Expressing

    p38 MAPK signaling pathway is involved in CXCR3 protein expression in MSCs, CXCR3-mediated chemotaxis invasion, and transendothelial migration induced by IL-1β. a MSCs were pretreated with p38 MAPK inhibitor SB203580, at a concentration of 25 uM, then stimulated with IL-1β for 30 min. The expression of CXCR3 cytosolic and membrane protein were detected by Western blotting. b Quantitative results of the Western blotting of CXCR3 cytosolic and membrane protein expression of a . The data represent mean ± SD ( n = 3). Statistical analysis was determined by Student’s t test and one-way ANOVA. * P

    Journal: Stem Cell Research & Therapy

    Article Title: Interleukin-1β induces CXCR3-mediated chemotaxis to promote umbilical cord mesenchymal stem cell transendothelial migration

    doi: 10.1186/s13287-018-1032-9

    Figure Lengend Snippet: p38 MAPK signaling pathway is involved in CXCR3 protein expression in MSCs, CXCR3-mediated chemotaxis invasion, and transendothelial migration induced by IL-1β. a MSCs were pretreated with p38 MAPK inhibitor SB203580, at a concentration of 25 uM, then stimulated with IL-1β for 30 min. The expression of CXCR3 cytosolic and membrane protein were detected by Western blotting. b Quantitative results of the Western blotting of CXCR3 cytosolic and membrane protein expression of a . The data represent mean ± SD ( n = 3). Statistical analysis was determined by Student’s t test and one-way ANOVA. * P

    Article Snippet: The p38 MAPK inhibitor SB203580 (Tocris, UK) was added to MSCs 2 h prior to stimulation at concentrations of 25 μM, 10 μM, and 20 μM, respectively.

    Techniques: Expressing, Chemotaxis Assay, Migration, Concentration Assay, Western Blot

    Schematic diagram of IL-1β signaling pathway in CXCR3-mediated MSC transendothelial migration. A schematic diagram depicts the proposed role of IL-1β signaling pathway in MSC transendothelial migration. The process of cell migration is initiated by IL-1β through p38 MAPK-induced expression of CXCR3 in MSCs in response to CXCL9

    Journal: Stem Cell Research & Therapy

    Article Title: Interleukin-1β induces CXCR3-mediated chemotaxis to promote umbilical cord mesenchymal stem cell transendothelial migration

    doi: 10.1186/s13287-018-1032-9

    Figure Lengend Snippet: Schematic diagram of IL-1β signaling pathway in CXCR3-mediated MSC transendothelial migration. A schematic diagram depicts the proposed role of IL-1β signaling pathway in MSC transendothelial migration. The process of cell migration is initiated by IL-1β through p38 MAPK-induced expression of CXCR3 in MSCs in response to CXCL9

    Article Snippet: The p38 MAPK inhibitor SB203580 (Tocris, UK) was added to MSCs 2 h prior to stimulation at concentrations of 25 μM, 10 μM, and 20 μM, respectively.

    Techniques: Migration, Expressing

    Analysis of the Rho signaling activity. (A) Effect of TGF-β2 on RhoA activity in HTM cells. (B) Effects of TGF-β2, Y-27632 and SB203580 on myosin light chain (MLC)-2 phosphorylation in HTM cells. Cells with or without 10 μM Y-27632 or 10 μM SB203580 pretreatment for 30 min were stimulated with 2.5 ng/ml TGF-β2 for 30 min. Data shown in upper panels are results of representative Western blot analyses of phosphorylated MLC2 (pMLC2) and total MLC2. Relative changes in the ratio of MLC phosphorylation are shown in the lower graph. Data are shown as means ± SE, n = 10. *P

    Journal: PLoS ONE

    Article Title: p38 MAP Kinase Inhibitor Suppresses Transforming Growth Factor-β2–Induced Type 1 Collagen Production in Trabecular Meshwork Cells

    doi: 10.1371/journal.pone.0120774

    Figure Lengend Snippet: Analysis of the Rho signaling activity. (A) Effect of TGF-β2 on RhoA activity in HTM cells. (B) Effects of TGF-β2, Y-27632 and SB203580 on myosin light chain (MLC)-2 phosphorylation in HTM cells. Cells with or without 10 μM Y-27632 or 10 μM SB203580 pretreatment for 30 min were stimulated with 2.5 ng/ml TGF-β2 for 30 min. Data shown in upper panels are results of representative Western blot analyses of phosphorylated MLC2 (pMLC2) and total MLC2. Relative changes in the ratio of MLC phosphorylation are shown in the lower graph. Data are shown as means ± SE, n = 10. *P

    Article Snippet: Y-27632 and SB203580 were purchased from Merck Millipore (Darmstadt, Germany).

    Techniques: Activity Assay, Western Blot

    Effects of TGF-β2, Y-27632 and SB203580 on actin stress fibers in HTM cells. HTM cells were pretreated with 10 μM Y-27632 (B, E) or 10 μM SB203580 (C, F) for 30 min, and then stimulated with 2.5 ng/ml TGF-β2 (D-F) for 24 h, stained with phalloidin-FITC, and observed by fluorescence microscopy. Scale bar: 50 μm.

    Journal: PLoS ONE

    Article Title: p38 MAP Kinase Inhibitor Suppresses Transforming Growth Factor-β2–Induced Type 1 Collagen Production in Trabecular Meshwork Cells

    doi: 10.1371/journal.pone.0120774

    Figure Lengend Snippet: Effects of TGF-β2, Y-27632 and SB203580 on actin stress fibers in HTM cells. HTM cells were pretreated with 10 μM Y-27632 (B, E) or 10 μM SB203580 (C, F) for 30 min, and then stimulated with 2.5 ng/ml TGF-β2 (D-F) for 24 h, stained with phalloidin-FITC, and observed by fluorescence microscopy. Scale bar: 50 μm.

    Article Snippet: Y-27632 and SB203580 were purchased from Merck Millipore (Darmstadt, Germany).

    Techniques: Staining, Fluorescence, Microscopy

    Effects of TGF-β2, Y-27632 and SB203580 on transactivation of Smad complexes in HTM cells. HTM cells were pretreated with 10 μM Y-27632 or 10 μM SB203580 for 30 min, and then stimulated with 2.5 ng/ml TGF-β2 for 24 h. Relative signals from a luciferase reporter gene fused with 12 repeats of the Smad-complex binding element (CAGA) were compared among samples. Signals from a plasmid containing Renilla luciferase were used as an internal control. Data are shown as means ± SE, n = 9. *P

    Journal: PLoS ONE

    Article Title: p38 MAP Kinase Inhibitor Suppresses Transforming Growth Factor-β2–Induced Type 1 Collagen Production in Trabecular Meshwork Cells

    doi: 10.1371/journal.pone.0120774

    Figure Lengend Snippet: Effects of TGF-β2, Y-27632 and SB203580 on transactivation of Smad complexes in HTM cells. HTM cells were pretreated with 10 μM Y-27632 or 10 μM SB203580 for 30 min, and then stimulated with 2.5 ng/ml TGF-β2 for 24 h. Relative signals from a luciferase reporter gene fused with 12 repeats of the Smad-complex binding element (CAGA) were compared among samples. Signals from a plasmid containing Renilla luciferase were used as an internal control. Data are shown as means ± SE, n = 9. *P

    Article Snippet: Y-27632 and SB203580 were purchased from Merck Millipore (Darmstadt, Germany).

    Techniques: Luciferase, Binding Assay, Plasmid Preparation

    Effects of TGF-β2, Y-27632 and SB203580 on phosphorylation of Smad2 in HTM cells. HTM cells were pretreated with 10 μM Y-27632 (A) or 10 μM SB203580 (B) for 30 min, and then stimulated with 2.5 ng/ml TGF-β2 for 24 h. Data shown in upper panels are results of representative Western blot analyses of phosphorylated Smad2 (pSmad2) and total Smad2/3. Relative changes in the pSmad2 to total Smad2 expression level are shown in the lower graph. Data are shown as means ± SE, n = 6. **P

    Journal: PLoS ONE

    Article Title: p38 MAP Kinase Inhibitor Suppresses Transforming Growth Factor-β2–Induced Type 1 Collagen Production in Trabecular Meshwork Cells

    doi: 10.1371/journal.pone.0120774

    Figure Lengend Snippet: Effects of TGF-β2, Y-27632 and SB203580 on phosphorylation of Smad2 in HTM cells. HTM cells were pretreated with 10 μM Y-27632 (A) or 10 μM SB203580 (B) for 30 min, and then stimulated with 2.5 ng/ml TGF-β2 for 24 h. Data shown in upper panels are results of representative Western blot analyses of phosphorylated Smad2 (pSmad2) and total Smad2/3. Relative changes in the pSmad2 to total Smad2 expression level are shown in the lower graph. Data are shown as means ± SE, n = 6. **P

    Article Snippet: Y-27632 and SB203580 were purchased from Merck Millipore (Darmstadt, Germany).

    Techniques: Western Blot, Expressing

    Effects of TGF-β2, Y-27632 and SB203580 on COL1A2 induction in HTM cells. HTM cells were pretreated with 10 μM Y-27632 or 10 μM SB203580 for 30 min, and then stimulated with 2.5 ng/ml TGF-β2 for 24 h. Data are shown as means ± SE. *P

    Journal: PLoS ONE

    Article Title: p38 MAP Kinase Inhibitor Suppresses Transforming Growth Factor-β2–Induced Type 1 Collagen Production in Trabecular Meshwork Cells

    doi: 10.1371/journal.pone.0120774

    Figure Lengend Snippet: Effects of TGF-β2, Y-27632 and SB203580 on COL1A2 induction in HTM cells. HTM cells were pretreated with 10 μM Y-27632 or 10 μM SB203580 for 30 min, and then stimulated with 2.5 ng/ml TGF-β2 for 24 h. Data are shown as means ± SE. *P

    Article Snippet: Y-27632 and SB203580 were purchased from Merck Millipore (Darmstadt, Germany).

    Techniques:

    Effects of TGF-β2, Y-27632 and SB203580 on nuclear translocation of Smad2/3 in HTM cells. HTM cells were pretreated with 10 μM Y-27632 (B, E) or 10 μM SB203580 (C, F) for 30 min, and then stimulated with 2.5 ng/ml TGF-β2 (D-F) for 24 h, immunolabeled with anti-Smad2/3 antibody (red), and observed by fluorescence microscopy. Cell nuclei were counterstained by DAPI (blue). Scale bar: 50 μm. (G) Relative expressions of nucleic Smad2 to its cytoplasmic expression are shown in the graph. Nucleic and cytoplasmic proteins were extracted, and the expression level of Smad2 was assessed separately. Data are shown as means ± SE, n = 5.

    Journal: PLoS ONE

    Article Title: p38 MAP Kinase Inhibitor Suppresses Transforming Growth Factor-β2–Induced Type 1 Collagen Production in Trabecular Meshwork Cells

    doi: 10.1371/journal.pone.0120774

    Figure Lengend Snippet: Effects of TGF-β2, Y-27632 and SB203580 on nuclear translocation of Smad2/3 in HTM cells. HTM cells were pretreated with 10 μM Y-27632 (B, E) or 10 μM SB203580 (C, F) for 30 min, and then stimulated with 2.5 ng/ml TGF-β2 (D-F) for 24 h, immunolabeled with anti-Smad2/3 antibody (red), and observed by fluorescence microscopy. Cell nuclei were counterstained by DAPI (blue). Scale bar: 50 μm. (G) Relative expressions of nucleic Smad2 to its cytoplasmic expression are shown in the graph. Nucleic and cytoplasmic proteins were extracted, and the expression level of Smad2 was assessed separately. Data are shown as means ± SE, n = 5.

    Article Snippet: Y-27632 and SB203580 were purchased from Merck Millipore (Darmstadt, Germany).

    Techniques: Translocation Assay, Immunolabeling, Fluorescence, Microscopy, Expressing

    NF-κB translocation is a functional index during Japanese encephalitis virus (JEV)-induced matrix metalloproteinase (MMP)-9 expression in rat brain astrocytes-1 cells. (A) Cells were pretreated with helenalin (HLN) for 1 h and then infected with JEV (moi = 1) for 16 h. (B) Cells were transfected with p65 siRNA, and then infected with JEV for 16 h. (A, B) The conditioned media were assessed for MMP-9 expression by gelatin zymography. The cell lysates were analysed by Western blot using an anti-NF-κB p65 or anti-GAPDH antibody. (C, D) Time dependence of JEV-induced NF-κB translocation and IκB-α degradation. Cells were pretreated with or without 1 µM helenalin, 1 µM U0126, 1 µM SP600125, 1 µM SB203580, 10 mM NAC, 10 µM diphenylene iodonium chloride (DPI) and 100 µM apocynin (APO) for 1 h, and then were stimulated with JEV (moi = 1) for the indicated time intervals. Nuclear and cytosolic fractions were analysed by Western blot using an anti-NF-κB (p65), anti-phospho-IκBα, anti-IκBα antibody or anti-GAPDH antibody. (E) Cells were pretreated with 1 µM helenalin for 1 h, and then infected with JEV (moi = 1) for 6 h. The RNA samples were analysed by real-time PCR for the levels of MMP-9 mRNA. (F) Cells were transiently transfected with an MMP-9-luc reporter gene, pretreated with helenalin for 1 h and then infected with JEV for 6 h. The promoter activity of MMP-9 was measured. Data are expressed as mean ± SEM of three independent experiments. * P

    Journal: British Journal of Pharmacology

    Article Title: Japanese encephalitis virus induces matrix metalloproteinase-9 in rat brain astrocytes via NF-?B signalling dependent on MAPKs and reactive oxygen species

    doi: 10.1111/j.1476-5381.2010.00982.x

    Figure Lengend Snippet: NF-κB translocation is a functional index during Japanese encephalitis virus (JEV)-induced matrix metalloproteinase (MMP)-9 expression in rat brain astrocytes-1 cells. (A) Cells were pretreated with helenalin (HLN) for 1 h and then infected with JEV (moi = 1) for 16 h. (B) Cells were transfected with p65 siRNA, and then infected with JEV for 16 h. (A, B) The conditioned media were assessed for MMP-9 expression by gelatin zymography. The cell lysates were analysed by Western blot using an anti-NF-κB p65 or anti-GAPDH antibody. (C, D) Time dependence of JEV-induced NF-κB translocation and IκB-α degradation. Cells were pretreated with or without 1 µM helenalin, 1 µM U0126, 1 µM SP600125, 1 µM SB203580, 10 mM NAC, 10 µM diphenylene iodonium chloride (DPI) and 100 µM apocynin (APO) for 1 h, and then were stimulated with JEV (moi = 1) for the indicated time intervals. Nuclear and cytosolic fractions were analysed by Western blot using an anti-NF-κB (p65), anti-phospho-IκBα, anti-IκBα antibody or anti-GAPDH antibody. (E) Cells were pretreated with 1 µM helenalin for 1 h, and then infected with JEV (moi = 1) for 6 h. The RNA samples were analysed by real-time PCR for the levels of MMP-9 mRNA. (F) Cells were transiently transfected with an MMP-9-luc reporter gene, pretreated with helenalin for 1 h and then infected with JEV for 6 h. The promoter activity of MMP-9 was measured. Data are expressed as mean ± SEM of three independent experiments. * P

    Article Snippet: U0126, SB203580, SP600125 and helenalin (HLN) were from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Translocation Assay, Functional Assay, Expressing, Infection, Transfection, Zymography, Western Blot, Real-time Polymerase Chain Reaction, Activity Assay

    Involvement of MAPKs in matrix metalloproteinase (MMP)-9 mRNA expression induced by Japanese encephalitis virus (JEV) in rat brain astrocytes-1 cells. (A) Cells were pretreated with 1 µM of U0126 (U0), SP600125 (SP) or SB203580 (SB) for 1 h, and then infected with JEV (moi = 1) for 6 h. The RNA samples were analysed by real-time PCR for the levels of MMP-9 mRNA. (B) Cells were transiently transfected with an MMP-9-luc reporter gene, pretreated with 1 µM of U0126, SB203580 or SP600125 for 1 h, and then infected with JEV for 6 h. The promoter activity of MMP-9 was measured. Data are expressed as mean ± SEM of three independent experiments. * P

    Journal: British Journal of Pharmacology

    Article Title: Japanese encephalitis virus induces matrix metalloproteinase-9 in rat brain astrocytes via NF-?B signalling dependent on MAPKs and reactive oxygen species

    doi: 10.1111/j.1476-5381.2010.00982.x

    Figure Lengend Snippet: Involvement of MAPKs in matrix metalloproteinase (MMP)-9 mRNA expression induced by Japanese encephalitis virus (JEV) in rat brain astrocytes-1 cells. (A) Cells were pretreated with 1 µM of U0126 (U0), SP600125 (SP) or SB203580 (SB) for 1 h, and then infected with JEV (moi = 1) for 6 h. The RNA samples were analysed by real-time PCR for the levels of MMP-9 mRNA. (B) Cells were transiently transfected with an MMP-9-luc reporter gene, pretreated with 1 µM of U0126, SB203580 or SP600125 for 1 h, and then infected with JEV for 6 h. The promoter activity of MMP-9 was measured. Data are expressed as mean ± SEM of three independent experiments. * P

    Article Snippet: U0126, SB203580, SP600125 and helenalin (HLN) were from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Transfection, Activity Assay

    Involvement of p38 MAPK in Japanese encephalitis virus (JEV)-induced matrix metalloproteinase (MMP)-9 expression in rat brain astrocytes-1 cells. (A) Cells were pretreated with SB203580 for 1 h and then infected with JEV (moi = 1) for 16 h. (B) Cells were transfected with p38 siRNA, and then infected with JEV for 16 h. (A, B) The conditioned media were assessed for MMP-9 expression by gelatin zymography. The cell lysates were analysed by Western blot using an anti-p38 or anti-GAPDH antibody. (C) Cells were pretreated with 1 µM SB203580 for 1 h and then infected with JEV (moi = 1) for the indicated time intervals. (D) Cells were pretreated with 10 mM NAC, 10 µM diphenylene iodonium chloride (DPI), or 100 µM apocynin (APO) for 1 h and then infected with JEV (moi = 1) for 10 min. The cell lysates were analysed by Western blot using an anti-phospho-p38 MAPK or anti-GAPDH antibody. Data are expressed as mean ± SEM of three independent experiments. * P

    Journal: British Journal of Pharmacology

    Article Title: Japanese encephalitis virus induces matrix metalloproteinase-9 in rat brain astrocytes via NF-?B signalling dependent on MAPKs and reactive oxygen species

    doi: 10.1111/j.1476-5381.2010.00982.x

    Figure Lengend Snippet: Involvement of p38 MAPK in Japanese encephalitis virus (JEV)-induced matrix metalloproteinase (MMP)-9 expression in rat brain astrocytes-1 cells. (A) Cells were pretreated with SB203580 for 1 h and then infected with JEV (moi = 1) for 16 h. (B) Cells were transfected with p38 siRNA, and then infected with JEV for 16 h. (A, B) The conditioned media were assessed for MMP-9 expression by gelatin zymography. The cell lysates were analysed by Western blot using an anti-p38 or anti-GAPDH antibody. (C) Cells were pretreated with 1 µM SB203580 for 1 h and then infected with JEV (moi = 1) for the indicated time intervals. (D) Cells were pretreated with 10 mM NAC, 10 µM diphenylene iodonium chloride (DPI), or 100 µM apocynin (APO) for 1 h and then infected with JEV (moi = 1) for 10 min. The cell lysates were analysed by Western blot using an anti-phospho-p38 MAPK or anti-GAPDH antibody. Data are expressed as mean ± SEM of three independent experiments. * P

    Article Snippet: U0126, SB203580, SP600125 and helenalin (HLN) were from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Expressing, Infection, Transfection, Zymography, Western Blot

    Putative close contacts between the mutation sites of p38α kinase and the inhibitor SB203580

    Journal: Journal of chemical theory and computation

    Article Title: Accurate calculation of mutational effects on the thermodynamics of inhibitor binding to p38? MAP kinase: a combined computational and experimental study

    doi: 10.1021/ct400104x

    Figure Lengend Snippet: Putative close contacts between the mutation sites of p38α kinase and the inhibitor SB203580

    Article Snippet: Supporting Information Principal moieties of the inhibitor SB203580; probability distribution function of the key hydrogen bond observed in simulations of the wild-type p38α-SB203580 complex; scheme for performing alchemical transformations of p38α mutants; quadratic fits of the normalized fluorescence data obtained from titrations of p38α protein with SB203580; time dependence of root mean square deviations (RMSDs) for selected groups of atoms in harmonically restrained simulations of wild-type p38α in complex with SB203580; plots of dH/dλ versus λ for unrestrained and restrained simulations of p38α mutants using the OPLS-AA/L force field; root mean square deviation (RMSD) versus force constant for 12 ns restrained simulations of wild-type p38α kinase in complex with SB203580 using the OPLS-AA/L force field; time dependence of ΔΔG values calculated from simulations using the OPLS-AA/L force field; time dependence of ΔΔG values calculated from simulations using the Amber ff99SB-ILDN force field; time dependence of χ1 angles at residue 38 in simulations of the A51V mutant; time dependence of χ1 angles at residue 38 in simulations of the V38I/A51V mutant; time dependence of χ1 angles at residue 51 in simulations of the A51V mutant; time dependence of χ1 angles at residue 51 in simulations of the V38I/A51V mutant; dihedral distributions for inter-ring angles in the inhibitor sampled during unrestrained simulations using the OPLS-AA/L and Amber ff99SB-ILDN force fields; three partial charge sets for the inhibitor SB203580; additional dihedral restraints applied to SB203580 to maintain its “propeller-like” shape.

    Techniques: Mutagenesis

    Fluorescence binding assay to measure the binding affinity of wild type p38α kinase to the inhibitor SB203580

    Journal: Journal of chemical theory and computation

    Article Title: Accurate calculation of mutational effects on the thermodynamics of inhibitor binding to p38? MAP kinase: a combined computational and experimental study

    doi: 10.1021/ct400104x

    Figure Lengend Snippet: Fluorescence binding assay to measure the binding affinity of wild type p38α kinase to the inhibitor SB203580

    Article Snippet: Supporting Information Principal moieties of the inhibitor SB203580; probability distribution function of the key hydrogen bond observed in simulations of the wild-type p38α-SB203580 complex; scheme for performing alchemical transformations of p38α mutants; quadratic fits of the normalized fluorescence data obtained from titrations of p38α protein with SB203580; time dependence of root mean square deviations (RMSDs) for selected groups of atoms in harmonically restrained simulations of wild-type p38α in complex with SB203580; plots of dH/dλ versus λ for unrestrained and restrained simulations of p38α mutants using the OPLS-AA/L force field; root mean square deviation (RMSD) versus force constant for 12 ns restrained simulations of wild-type p38α kinase in complex with SB203580 using the OPLS-AA/L force field; time dependence of ΔΔG values calculated from simulations using the OPLS-AA/L force field; time dependence of ΔΔG values calculated from simulations using the Amber ff99SB-ILDN force field; time dependence of χ1 angles at residue 38 in simulations of the A51V mutant; time dependence of χ1 angles at residue 38 in simulations of the V38I/A51V mutant; time dependence of χ1 angles at residue 51 in simulations of the A51V mutant; time dependence of χ1 angles at residue 51 in simulations of the V38I/A51V mutant; dihedral distributions for inter-ring angles in the inhibitor sampled during unrestrained simulations using the OPLS-AA/L and Amber ff99SB-ILDN force fields; three partial charge sets for the inhibitor SB203580; additional dihedral restraints applied to SB203580 to maintain its “propeller-like” shape.

    Techniques: Fluorescence, Binding Assay

    A close-up view of the p38α-SB203580 binding site

    Journal: Journal of chemical theory and computation

    Article Title: Accurate calculation of mutational effects on the thermodynamics of inhibitor binding to p38? MAP kinase: a combined computational and experimental study

    doi: 10.1021/ct400104x

    Figure Lengend Snippet: A close-up view of the p38α-SB203580 binding site

    Article Snippet: Supporting Information Principal moieties of the inhibitor SB203580; probability distribution function of the key hydrogen bond observed in simulations of the wild-type p38α-SB203580 complex; scheme for performing alchemical transformations of p38α mutants; quadratic fits of the normalized fluorescence data obtained from titrations of p38α protein with SB203580; time dependence of root mean square deviations (RMSDs) for selected groups of atoms in harmonically restrained simulations of wild-type p38α in complex with SB203580; plots of dH/dλ versus λ for unrestrained and restrained simulations of p38α mutants using the OPLS-AA/L force field; root mean square deviation (RMSD) versus force constant for 12 ns restrained simulations of wild-type p38α kinase in complex with SB203580 using the OPLS-AA/L force field; time dependence of ΔΔG values calculated from simulations using the OPLS-AA/L force field; time dependence of ΔΔG values calculated from simulations using the Amber ff99SB-ILDN force field; time dependence of χ1 angles at residue 38 in simulations of the A51V mutant; time dependence of χ1 angles at residue 38 in simulations of the V38I/A51V mutant; time dependence of χ1 angles at residue 51 in simulations of the A51V mutant; time dependence of χ1 angles at residue 51 in simulations of the V38I/A51V mutant; dihedral distributions for inter-ring angles in the inhibitor sampled during unrestrained simulations using the OPLS-AA/L and Amber ff99SB-ILDN force fields; three partial charge sets for the inhibitor SB203580; additional dihedral restraints applied to SB203580 to maintain its “propeller-like” shape.

    Techniques: Binding Assay

    Optimizing culture conditions for supporting the stemness of pig muscle stem cells in vitro . To find suitable in vitro culture conditions for pig muscle stem cells, various culture components were tested. (A) Comparative analysis of the proliferation rate of pig muscle stem cells cultured with 10% FBS-supplemented MEM and SkGM-2. (B) The effect of basal media on pig muscle stem cells during the in vitro long-term culture. (C) The effect of basal media on the expression of myogenic marker genes during the in vitro long-term culture using qPCR. (D) The effect of SB203580 on pig muscle stem cells during the in vitro long-term culture. (E) The effect of SB203580 on the expression of myogenic marker genes during the in vitro long-term culture using qPCR. * p

    Journal: Food Science of Animal Resources

    Article Title: Optimization of Culture Conditions for Maintaining Pig Muscle Stem Cells In Vitro

    doi: 10.5851/kosfa.2020.e39

    Figure Lengend Snippet: Optimizing culture conditions for supporting the stemness of pig muscle stem cells in vitro . To find suitable in vitro culture conditions for pig muscle stem cells, various culture components were tested. (A) Comparative analysis of the proliferation rate of pig muscle stem cells cultured with 10% FBS-supplemented MEM and SkGM-2. (B) The effect of basal media on pig muscle stem cells during the in vitro long-term culture. (C) The effect of basal media on the expression of myogenic marker genes during the in vitro long-term culture using qPCR. (D) The effect of SB203580 on pig muscle stem cells during the in vitro long-term culture. (E) The effect of SB203580 on the expression of myogenic marker genes during the in vitro long-term culture using qPCR. * p

    Article Snippet: The isolated muscle stem cells were cultured on gelatin-coated dishes in basic GM, which consisted of MEM containing 10% (v/v) FBS, 1× glutamax, 1× AA, and 0.1 mM β-mercaptoethanol (All from Gibco) or Skeletal Muscle Cell Growth Medium-2 BulletKit™ (SkGM-2; Lonza, Basel, Switzerland) supplemented with 20 μM SB203580 (Cayman Chemical, Ann Arbor, USA) according to manufacturer’s instructions.

    Techniques: In Vitro, Cell Culture, Expressing, Marker, Real-time Polymerase Chain Reaction

    The myogenic potential of pig muscle stem cells cultured in SkGM-2-supplemented SB203580. The myogenic ability of pig muscle stem cells cultured in SkGM-2-supplemented SB203580 was examined and defined using immunostaining of myosin heavy chain (MHC). Red and green fluorescence represent nuclei and MHC, respectively. Scale bar=400 μm.

    Journal: Food Science of Animal Resources

    Article Title: Optimization of Culture Conditions for Maintaining Pig Muscle Stem Cells In Vitro

    doi: 10.5851/kosfa.2020.e39

    Figure Lengend Snippet: The myogenic potential of pig muscle stem cells cultured in SkGM-2-supplemented SB203580. The myogenic ability of pig muscle stem cells cultured in SkGM-2-supplemented SB203580 was examined and defined using immunostaining of myosin heavy chain (MHC). Red and green fluorescence represent nuclei and MHC, respectively. Scale bar=400 μm.

    Article Snippet: The isolated muscle stem cells were cultured on gelatin-coated dishes in basic GM, which consisted of MEM containing 10% (v/v) FBS, 1× glutamax, 1× AA, and 0.1 mM β-mercaptoethanol (All from Gibco) or Skeletal Muscle Cell Growth Medium-2 BulletKit™ (SkGM-2; Lonza, Basel, Switzerland) supplemented with 20 μM SB203580 (Cayman Chemical, Ann Arbor, USA) according to manufacturer’s instructions.

    Techniques: Cell Culture, Immunostaining, Fluorescence

    P38 and TAK1 inhibitors neutralize the negative effect of TLR8 activation on dendritic growth. (A and D) WT neurons were treated with CL075/poly dT for 30 min at 18 DIV and subjected to immunostaining with phospho-P38, phospho-TAK1, and neuronal marker NeuN antibodies as indicated. Counterstaining with DAPI was performed to label nuclei. (B and E) GFP was transfected at 12 DIV. CL075/poly dT and P38 inhibitor SB203580 (B) and three TAK1 inhibitors, Takinib, 5ZO, and NG25 (E), were added at 17 DIV for 1 d in WT cultured neurons. (C) SB203580 treatment did not influence the effect of CL075 on younger WT neurons at 4 DIV. (B–E) Sample size ( n ) indicates the number of examined neurons, which were randomly collected blind from two independent experiments. Data are presented as the mean + SEM (error bars). Bars: (A) 25 µm; (B) 50 µm; (D) 20 µm. **, P

    Journal: The Journal of Cell Biology

    Article Title: Endosomal TLR3, TLR7, and TLR8 control neuronal morphology through different transcriptional programs

    doi: 10.1083/jcb.201712113

    Figure Lengend Snippet: P38 and TAK1 inhibitors neutralize the negative effect of TLR8 activation on dendritic growth. (A and D) WT neurons were treated with CL075/poly dT for 30 min at 18 DIV and subjected to immunostaining with phospho-P38, phospho-TAK1, and neuronal marker NeuN antibodies as indicated. Counterstaining with DAPI was performed to label nuclei. (B and E) GFP was transfected at 12 DIV. CL075/poly dT and P38 inhibitor SB203580 (B) and three TAK1 inhibitors, Takinib, 5ZO, and NG25 (E), were added at 17 DIV for 1 d in WT cultured neurons. (C) SB203580 treatment did not influence the effect of CL075 on younger WT neurons at 4 DIV. (B–E) Sample size ( n ) indicates the number of examined neurons, which were randomly collected blind from two independent experiments. Data are presented as the mean + SEM (error bars). Bars: (A) 25 µm; (B) 50 µm; (D) 20 µm. **, P

    Article Snippet: CL075, poly dT, poly(I:C) high molecular weight, and SB203580 were all purchased from InvivoGen.

    Techniques: Activation Assay, Immunostaining, Marker, Transfection, Cell Culture

    Expression levels of HPGD, COX and mPGES1 following HCQ stimulation by wortmannin, PD98059 and SB203580. The RA-FLS were serum-starved for 2 h and subsequently treated with HCQ (10 µ M) for 24 h. Wortmannin (0.1, 0.5 and 1 nM), PD98059 (10, 50 and 100 µ M) and SB203580 (1, 10 and 50 µ M) were added 0.5 h prior to HCQ stimulation. The expression levels of (A and B) HPGD and (C and D) COX1, COX2 and mPGES1 in RA-FLS following the different treatments. RA-FLS, rheumatoid arthritis fibroblast-like synoviocytes; CON, control; HCQ, hydroxychloroquine; W, wortmannin; PD, PD98059; SB, SB203580; HPGD, 15-hydroxyprosta-glandin dehydrogenase; ACTB, β-actin; COX, cyclooxygenase; mPGES1, microsomal prostaglandin E2 synthase 1.

    Journal: Molecular Medicine Reports

    Article Title: 15-Hydroxyprostaglandin dehydrogenase is upregulated by hydroxychloroquine in rheumatoid arthritis fibroblast-like synoviocytes

    doi: 10.3892/mmr.2015.3931

    Figure Lengend Snippet: Expression levels of HPGD, COX and mPGES1 following HCQ stimulation by wortmannin, PD98059 and SB203580. The RA-FLS were serum-starved for 2 h and subsequently treated with HCQ (10 µ M) for 24 h. Wortmannin (0.1, 0.5 and 1 nM), PD98059 (10, 50 and 100 µ M) and SB203580 (1, 10 and 50 µ M) were added 0.5 h prior to HCQ stimulation. The expression levels of (A and B) HPGD and (C and D) COX1, COX2 and mPGES1 in RA-FLS following the different treatments. RA-FLS, rheumatoid arthritis fibroblast-like synoviocytes; CON, control; HCQ, hydroxychloroquine; W, wortmannin; PD, PD98059; SB, SB203580; HPGD, 15-hydroxyprosta-glandin dehydrogenase; ACTB, β-actin; COX, cyclooxygenase; mPGES1, microsomal prostaglandin E2 synthase 1.

    Article Snippet: Wortmannin, PD98059 and SB203580 (A.G. Scientific, Inc., San Diego, CA, USA) were added to the RA-FLS 30 min prior to HCQ stimulation.

    Techniques: Expressing

    Levels of PGE2 release upon hydroxychloroquine stimulation by wortmannin, PD98059 and SB203580. The quantities of PGE2 and 6-keto-PGF1α were measured by enzyme immunoassay. Wortmannin (1 nM), PD98059 (10 µ M) and SB203580 (50 µ M) were added to the cells 0.5 h prior to HCQ (10 µ M) stimulation. The data are expressed as the mean ± standard deviation (1.78±0.18, * P= 0.02; 1.63±0.15, ** P= 0.031, compared with HCQ and 1.00±0.17, † P=0.050, compared with the CON). PGE2, prostaglandin E2; CON, control; HCQ, hydroxychloroquine; W, wortmannin; PD, PD98059; SB, SB203580.

    Journal: Molecular Medicine Reports

    Article Title: 15-Hydroxyprostaglandin dehydrogenase is upregulated by hydroxychloroquine in rheumatoid arthritis fibroblast-like synoviocytes

    doi: 10.3892/mmr.2015.3931

    Figure Lengend Snippet: Levels of PGE2 release upon hydroxychloroquine stimulation by wortmannin, PD98059 and SB203580. The quantities of PGE2 and 6-keto-PGF1α were measured by enzyme immunoassay. Wortmannin (1 nM), PD98059 (10 µ M) and SB203580 (50 µ M) were added to the cells 0.5 h prior to HCQ (10 µ M) stimulation. The data are expressed as the mean ± standard deviation (1.78±0.18, * P= 0.02; 1.63±0.15, ** P= 0.031, compared with HCQ and 1.00±0.17, † P=0.050, compared with the CON). PGE2, prostaglandin E2; CON, control; HCQ, hydroxychloroquine; W, wortmannin; PD, PD98059; SB, SB203580.

    Article Snippet: Wortmannin, PD98059 and SB203580 (A.G. Scientific, Inc., San Diego, CA, USA) were added to the RA-FLS 30 min prior to HCQ stimulation.

    Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

    Retinoic acid potentiates BMP inhibition of feather patterning. (A) RA administration reduces the density of placodes, which are detected by β - catenin in situ hybridization, completely inhibiting placode formation at high doses. Suppression of BMP signaling with 4 µM dorsomorphin and 5 µM SB203580 rescues placode formation in the presence of RA. (B) Quantification of placode density on neck and body upon RA treatment. With increasing doses of RA the feather density on body and neck converges and ultimately all feather placode formation is suppressed. (C) RA sensitizes body skin to BMP-driven inhibition of feather development. The application of 0.1 µM RA has little effect on the placode pattern and application of 40 ng/ml BMP12 permits placode formation on the body. Co-treatment with RA and BMP12 has a synergistic effect, completely suppressing feather development on the body. Conversely, treatment of skin with the RA synthesis inhibitor Citral renders the neck resistant to suppression of placode formation by BMPs.

    Journal: PLoS Biology

    Article Title: Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck FeatheringHow Bird Necks Get Naked

    doi: 10.1371/journal.pbio.1001028

    Figure Lengend Snippet: Retinoic acid potentiates BMP inhibition of feather patterning. (A) RA administration reduces the density of placodes, which are detected by β - catenin in situ hybridization, completely inhibiting placode formation at high doses. Suppression of BMP signaling with 4 µM dorsomorphin and 5 µM SB203580 rescues placode formation in the presence of RA. (B) Quantification of placode density on neck and body upon RA treatment. With increasing doses of RA the feather density on body and neck converges and ultimately all feather placode formation is suppressed. (C) RA sensitizes body skin to BMP-driven inhibition of feather development. The application of 0.1 µM RA has little effect on the placode pattern and application of 40 ng/ml BMP12 permits placode formation on the body. Co-treatment with RA and BMP12 has a synergistic effect, completely suppressing feather development on the body. Conversely, treatment of skin with the RA synthesis inhibitor Citral renders the neck resistant to suppression of placode formation by BMPs.

    Article Snippet: Dorsomorphin, Citral, and all-trans retinoic acid were supplied by Sigma-Aldrich and SB203580 by Merck.

    Techniques: Inhibition, In Situ Hybridization

    Regional disparity in pattern behavior upon suppression of BMP signal transduction. (A) Simulated pattern outcomes upon reduction of Inhibitor potency. Transition from production of circular foci to a striped pattern occurs, first on the less sensitive (simulated body) region, followed by stripe formation on the more sensitive domain (simulated neck) at higher levels of signal suppression. (B) Quantification of pattern characteristics from simulation of diminished Inhibitor potency. The proportion of total Activator positive area that is represented by circular foci is plotted. (C) β - catenin in situ hybridization detecting placode pattern upon suppression of BMP signal transduction in cultured E7.0 chicken skin. SB, p38 MAPK inhibitor SB203580; DM, Smad1/5/8 inhibitor dorsomorphin. Inhibition of p38 MAPK has little effect on the placode pattern, but yielded a robust effect in concert with suppression of Smad function. At low doses of DM stripes begin to form first on the body, then at higher doses on the neck. High doses cause β - catenin expression throughout the skin. (D) Quantification of the proportion of total β - catenin positive area that is represented by circular placodes in cultured skin treated with BMP inhibitors. Statistically significant p values are indicated above data points.

    Journal: PLoS Biology

    Article Title: Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck FeatheringHow Bird Necks Get Naked

    doi: 10.1371/journal.pbio.1001028

    Figure Lengend Snippet: Regional disparity in pattern behavior upon suppression of BMP signal transduction. (A) Simulated pattern outcomes upon reduction of Inhibitor potency. Transition from production of circular foci to a striped pattern occurs, first on the less sensitive (simulated body) region, followed by stripe formation on the more sensitive domain (simulated neck) at higher levels of signal suppression. (B) Quantification of pattern characteristics from simulation of diminished Inhibitor potency. The proportion of total Activator positive area that is represented by circular foci is plotted. (C) β - catenin in situ hybridization detecting placode pattern upon suppression of BMP signal transduction in cultured E7.0 chicken skin. SB, p38 MAPK inhibitor SB203580; DM, Smad1/5/8 inhibitor dorsomorphin. Inhibition of p38 MAPK has little effect on the placode pattern, but yielded a robust effect in concert with suppression of Smad function. At low doses of DM stripes begin to form first on the body, then at higher doses on the neck. High doses cause β - catenin expression throughout the skin. (D) Quantification of the proportion of total β - catenin positive area that is represented by circular placodes in cultured skin treated with BMP inhibitors. Statistically significant p values are indicated above data points.

    Article Snippet: Dorsomorphin, Citral, and all-trans retinoic acid were supplied by Sigma-Aldrich and SB203580 by Merck.

    Techniques: Transduction, In Situ Hybridization, Cell Culture, Inhibition, Expressing

    Naked neck skin displays elevated BMP signaling. (A) Application of recombinant BMP12 to cultured skin for 15 h leads to elevation of SOSTDC1 expression, determined by quantitative RT-PCR. (B–E) Detection of SOSTDC1 expression by in situ hybridization. (B) At E7.5 wild type embryos have two rows of feather placodes running up the neck. SOSTDC1 is expressed at the periphery of the placodes and is not detected in the medial region between the lateral rows of placodes. (C) By E8.5 the medial region of the neck is populated by feather placodes. (D) E7.5 Na/Na embryos have placodes on the dorsum, but widespread SOSTDC1 expression on the neck, including the medial region. (E) At E8.5 the Naked neck skin maintains a high level of widespread SOSTDC1 expression, with peri-placode expression visible on the body. (F) Ex vivo rescue of the Naked neck phenotype by suppression of BMP signaling. E7.0 Na/Na skin was cultured in the presence of dorsomorphin (DM, used at 8 µM) and SB203580 (SB, used at 5 µM), pharmacological inhibitors of BMP signal transduction, for 48 h. This permitted feather development across most of the mutant neck skin.

    Journal: PLoS Biology

    Article Title: Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck FeatheringHow Bird Necks Get Naked

    doi: 10.1371/journal.pbio.1001028

    Figure Lengend Snippet: Naked neck skin displays elevated BMP signaling. (A) Application of recombinant BMP12 to cultured skin for 15 h leads to elevation of SOSTDC1 expression, determined by quantitative RT-PCR. (B–E) Detection of SOSTDC1 expression by in situ hybridization. (B) At E7.5 wild type embryos have two rows of feather placodes running up the neck. SOSTDC1 is expressed at the periphery of the placodes and is not detected in the medial region between the lateral rows of placodes. (C) By E8.5 the medial region of the neck is populated by feather placodes. (D) E7.5 Na/Na embryos have placodes on the dorsum, but widespread SOSTDC1 expression on the neck, including the medial region. (E) At E8.5 the Naked neck skin maintains a high level of widespread SOSTDC1 expression, with peri-placode expression visible on the body. (F) Ex vivo rescue of the Naked neck phenotype by suppression of BMP signaling. E7.0 Na/Na skin was cultured in the presence of dorsomorphin (DM, used at 8 µM) and SB203580 (SB, used at 5 µM), pharmacological inhibitors of BMP signal transduction, for 48 h. This permitted feather development across most of the mutant neck skin.

    Article Snippet: Dorsomorphin, Citral, and all-trans retinoic acid were supplied by Sigma-Aldrich and SB203580 by Merck.

    Techniques: Recombinant, Cell Culture, Expressing, Quantitative RT-PCR, In Situ Hybridization, Ex Vivo, Transduction, Mutagenesis

    Co-culture with macrophages increases IL1α and IL1β expression in MDA-MB-231 cells in a TAK1-dependent manner. a qPCR analysis of Il1α , Il1β , Tgfβ , and Tnfα expression in macrophages cultured in the presence or absence of MDA-MB-231 cells for 5 days. b qPCR analysis of IL1α , IL1β , TGFβ , and TNFα expression in MDA-MB-231 cells cultured in the presence or absence of macrophages for 5 days. c Percentage of IL1α-positive (left) and IL1β-positive (right) cells in GFP-tagged MDA-MB-231 monocultures and GFP-tagged MDA-MB-231 cells cultured with macrophages. d Immunofluorescent staining of lungs dissected from mice injected with GFP and luciferase-tagged MDA-MB-231 cells. Representative examples of images taken from three immunostainings of two independent experiments are shown. GFP (yellow), macrophage marker F4/80 (magenta), IL1α or IL1β (white), DAPI (gray in single channel, blue in merge image) are shown. Scale bar = 20 μm. e qPCR analysis of IL1α and IL1β expression in MDA-MB-231 cells stably expressing doxycycline-inducible dominant-negative TAK1 (TAK1-dn), grown in co-culture with macrophages for 3 days in the presence or absence of doxycycline (doxy). f qPCR analysis of IL1α and IL1β expression in MDA-MB-231 cells cultured in the presence or absence of macrophages and treated or not with 1μM SB203580. In a , b , e , and f , GFP-tagged MDA-MB-231 cells were used, and macrophages and MDA-MB-231 cells were separated by FACS for RNA extraction. Graphs show means ± SD of three ( e , f ), four ( a ), five ( c , right panel) and six ( b , c , left panel) independent experiments. Statistical significance analyzed by unpaired two-tailed Student’s t test * p ≤ 0.05; ** p ≤ 0.01

    Journal: Nature Communications

    Article Title: TAK1 mediates microenvironment-triggered autocrine signals and promotes triple-negative breast cancer lung metastasis

    doi: 10.1038/s41467-018-04460-w

    Figure Lengend Snippet: Co-culture with macrophages increases IL1α and IL1β expression in MDA-MB-231 cells in a TAK1-dependent manner. a qPCR analysis of Il1α , Il1β , Tgfβ , and Tnfα expression in macrophages cultured in the presence or absence of MDA-MB-231 cells for 5 days. b qPCR analysis of IL1α , IL1β , TGFβ , and TNFα expression in MDA-MB-231 cells cultured in the presence or absence of macrophages for 5 days. c Percentage of IL1α-positive (left) and IL1β-positive (right) cells in GFP-tagged MDA-MB-231 monocultures and GFP-tagged MDA-MB-231 cells cultured with macrophages. d Immunofluorescent staining of lungs dissected from mice injected with GFP and luciferase-tagged MDA-MB-231 cells. Representative examples of images taken from three immunostainings of two independent experiments are shown. GFP (yellow), macrophage marker F4/80 (magenta), IL1α or IL1β (white), DAPI (gray in single channel, blue in merge image) are shown. Scale bar = 20 μm. e qPCR analysis of IL1α and IL1β expression in MDA-MB-231 cells stably expressing doxycycline-inducible dominant-negative TAK1 (TAK1-dn), grown in co-culture with macrophages for 3 days in the presence or absence of doxycycline (doxy). f qPCR analysis of IL1α and IL1β expression in MDA-MB-231 cells cultured in the presence or absence of macrophages and treated or not with 1μM SB203580. In a , b , e , and f , GFP-tagged MDA-MB-231 cells were used, and macrophages and MDA-MB-231 cells were separated by FACS for RNA extraction. Graphs show means ± SD of three ( e , f ), four ( a ), five ( c , right panel) and six ( b , c , left panel) independent experiments. Statistical significance analyzed by unpaired two-tailed Student’s t test * p ≤ 0.05; ** p ≤ 0.01

    Article Snippet: In some cases, cells were treated with 1 μg/ml doxycycline, 2 μM PS1145 (R & D System, 975108), or 1 μM SB203580 (Abcam, ab146589) from the beginning of the experiment.

    Techniques: Co-Culture Assay, Expressing, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Cell Culture, Staining, Mouse Assay, Injection, Luciferase, Marker, Stable Transfection, Dominant Negative Mutation, FACS, RNA Extraction, Two Tailed Test