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    MEK kinase inhibitor
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    98
    Thermo Fisher pd98059
    Pd98059, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 98 stars, based on 287 article reviews
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    Millipore pd98059
    Incubation of HLE cells with MHCC97H-derived exosomes promotes EMT through the MAPK/ERK pathway. a Immunofluorescence microscopy of EMT markers in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. b Western blot analysis of EMT markers in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. c Western blot analysis of EMT promoters (ZEB1, ZEB2 and Slug) and MET promoter OVOL1 in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. d Western blot analysis of EMT markers of HLE cells with exposure to MHCC97H-derived exosomes for different durations. e Western blot analysis of phosphorylated and total Erk1/2, Akt and Smad2/3 in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. f Western blot analysis of EMT markers and phosphorylated and total Erk1/2 in HLE cells, exosome-treated HLE cells and exosome-treated HLE cells when cultured with the ERK inhibitor <t>PD98059.</t> Abbreviation: Exo exosome. All experiments were repeated at least three times
    Pd98059, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 8878 article reviews
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    Cell Signaling Technology Inc pd98059
    O-GlcNAcylation regulated activations of Akt and Erk and expression of FOXO3 and MAN1A1. OGT was suppressed in parental (KKU-213 and KKU-214) and highly metastatic (KKU-213L5 and KKU-214L5) CCA cells using siOGT. a siOGT treatment inactivated the phosphorylation of Akt and Erk but not Ikk. b The modulations of Akt and Erk on FOXO3 expression were revealed using Akt and Erk inhibitors. CCA cells were treated with various concentrations of Akt inhibitor, MK-2206 and ( c ) Erk inhibitor, <t>PD98059,</t> for 24 h. The expression of pAkt/Akt, pErk/Erk, phospho-FOXO3, FOXO3 and MAN1A1 were determined using western blotting. d The MK-2206-treated L5 CCA cells were incubated with 20 µM of cycloheximide (CHX) for 1, 3, and 6 h. FOXO3 levels at each time point were determined using western blotting and compared with those of the untreated control cells. The data are represented as the mean ± SD from three independent experiments. Inhibition of Akt activation significantly reduced ( e ) migration and ( f ) invasion abilities of CCA cells and these effects could be rescued by kifunensine (Kif) treatment. The results are mean ± SEM of one representative from two independent experiments. * P
    Pd98059, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tocris pd98059
    O-GlcNAcylation regulated activations of Akt and Erk and expression of FOXO3 and MAN1A1. OGT was suppressed in parental (KKU-213 and KKU-214) and highly metastatic (KKU-213L5 and KKU-214L5) CCA cells using siOGT. a siOGT treatment inactivated the phosphorylation of Akt and Erk but not Ikk. b The modulations of Akt and Erk on FOXO3 expression were revealed using Akt and Erk inhibitors. CCA cells were treated with various concentrations of Akt inhibitor, MK-2206 and ( c ) Erk inhibitor, <t>PD98059,</t> for 24 h. The expression of pAkt/Akt, pErk/Erk, phospho-FOXO3, FOXO3 and MAN1A1 were determined using western blotting. d The MK-2206-treated L5 CCA cells were incubated with 20 µM of cycloheximide (CHX) for 1, 3, and 6 h. FOXO3 levels at each time point were determined using western blotting and compared with those of the untreated control cells. The data are represented as the mean ± SD from three independent experiments. Inhibition of Akt activation significantly reduced ( e ) migration and ( f ) invasion abilities of CCA cells and these effects could be rescued by kifunensine (Kif) treatment. The results are mean ± SEM of one representative from two independent experiments. * P
    Pd98059, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 464 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Selleck Chemicals pd98059
    O-GlcNAcylation regulated activations of Akt and Erk and expression of FOXO3 and MAN1A1. OGT was suppressed in parental (KKU-213 and KKU-214) and highly metastatic (KKU-213L5 and KKU-214L5) CCA cells using siOGT. a siOGT treatment inactivated the phosphorylation of Akt and Erk but not Ikk. b The modulations of Akt and Erk on FOXO3 expression were revealed using Akt and Erk inhibitors. CCA cells were treated with various concentrations of Akt inhibitor, MK-2206 and ( c ) Erk inhibitor, <t>PD98059,</t> for 24 h. The expression of pAkt/Akt, pErk/Erk, phospho-FOXO3, FOXO3 and MAN1A1 were determined using western blotting. d The MK-2206-treated L5 CCA cells were incubated with 20 µM of cycloheximide (CHX) for 1, 3, and 6 h. FOXO3 levels at each time point were determined using western blotting and compared with those of the untreated control cells. The data are represented as the mean ± SD from three independent experiments. Inhibition of Akt activation significantly reduced ( e ) migration and ( f ) invasion abilities of CCA cells and these effects could be rescued by kifunensine (Kif) treatment. The results are mean ± SEM of one representative from two independent experiments. * P
    Pd98059, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 394 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH pd98059
    Gelatinolytic activities in bronchoalveolar lavage fluid (A, D) and the supernatants of alveolar macrophages in culture (B, E). MMP-9 expression in bronchoalveolar lavage fluid (C, F). The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125(IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO); <t>LPS-PD98059,</t> LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Alveolar macrophages (10 6 /m1 of RPMI medium) were incubated for 24 hours. BAL fluid and culture supernatants were analyzed by sensitive zymography, followed by scanning densitometry. 92 kD and 66 kD gelatinolytic bands correspond to MMP-9 and MMP-2, respectively. Densitometry of 92 kD bands is expressed in arbitrary densitometric units. Western blots of BAL fluid with anti-MMP-9 antibodywere employed to monitor MMP-9. Values are represented as means ± SEM of results from 5 rats in each group. * Significant differences between saline, p
    Pd98059, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cayman Chemical pd98059
    Dabrafenib reduced the expression of phosphorylated JNK and c-jun in both SH-SY5Y cells and mice ( A ) LDH/cell viability assay under the condition of ERK inhibitor. Neuroprotective effects of dabrafenib (5 μM) were not dependent on the downregulation of activated ERK expression by <t>PD98059</t> (30 μM) or U0126 (3 μM). Neuroprotective effects were assessed by the LDH and cell viability assay in SH-SY5Y cells at 24 h after MPP + (3 mM) exposure. ** , P -value
    Pd98059, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 96/100, based on 318 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA pd98059
    While <t>PD98059</t> or compound C had little effect on influencing the effect of β-elemene in phosphorylation of AMPKα or ERK1/2, metformin reversed the effect of β-elemene on phosphorylation of Akt. A549 (A) and PC9 (B) cells were treated with PD98059 (20 μM) or compound C (20 μM) for 2 hrs before exposure of the cells to β-elemene (20 μg/ml) for an additional 24 hrs. Afterwards, the expression of p-ERK1/2 and p-AMPKα protein and their total forms was detected by Western blot. (C) A549 and PC9 cells were treated with metformin (5 mM) and β-elemene (20 μg/ml) for up to 2 hrs. Afterwards, the expression of p-Akt and p-AMPKα protein and total ones were detected by Western blot. The bar graphs represent the mean ± SD of p-Akt/GAPDH of three independent experiments. * indicates significant difference from untreated control cells. ** indicates significant difference from β-elemene treated alone ( P
    Pd98059, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 264 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pd98059/product/Merck KGaA
    Average 92 stars, based on 264 article reviews
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    pd98059 - by Bioz Stars, 2021-01
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    98
    InvivoGen pd98059
    While <t>PD98059</t> or compound C had little effect on influencing the effect of β-elemene in phosphorylation of AMPKα or ERK1/2, metformin reversed the effect of β-elemene on phosphorylation of Akt. A549 (A) and PC9 (B) cells were treated with PD98059 (20 μM) or compound C (20 μM) for 2 hrs before exposure of the cells to β-elemene (20 μg/ml) for an additional 24 hrs. Afterwards, the expression of p-ERK1/2 and p-AMPKα protein and their total forms was detected by Western blot. (C) A549 and PC9 cells were treated with metformin (5 mM) and β-elemene (20 μg/ml) for up to 2 hrs. Afterwards, the expression of p-Akt and p-AMPKα protein and total ones were detected by Western blot. The bar graphs represent the mean ± SD of p-Akt/GAPDH of three independent experiments. * indicates significant difference from untreated control cells. ** indicates significant difference from β-elemene treated alone ( P
    Pd98059, supplied by InvivoGen, used in various techniques. Bioz Stars score: 98/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam pd98059
    Phosphor-serine activation of m-calpain dominants KCC2 regulation in 0 Mg 2+ seizure model. (A,B) Immunoblotting detection of both plasma membrane (A) and cytoplasm (B) KCC2 expression level change in hippocampal slice 0 Mg 2+ model treated with either μ-calpain or m-calpain selective inhibitor CI I and CI IV, respectively. (C) Expression of either μ- or m-calpain from seizure slices induced by 0 Mg 2+ . (D) Serine phosphorylation level by IP method. Naïve or 0 Mg 2+ treated slices were used as inputs to form immune complex with μ- or m-calpain antibodies and detected by phosphor-serine antibody. Phosphor-serine levels were normalized to corresponded calpain levels. (E) Phosphor-serine level of m-calpain from 0 Mg 2+ , 0 Mg 2+ + <t>PD98059</t> or K252a treated slices. (F) Immunoblotting showed KCC2 level in 0 Mg 2+ + MDL-28170 or PD98059 treated slices, compared with none treated or 0 Mg 2+ treated alone slices. (G) KCC2 level of in vivo rats pre-treated with either MDL-28170 or SL-327 before PTZ kindling, in compared with vehicle PTZ treated rats. CI I, Calpain Inhibitor I; CI IV, Calpain Inhibitor IV; Calp, calpain; p-Ser, Phosphor-serine. * p
    Pd98059, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co pd98059
    EBV lytic reactivation by C7 requires the activation of ERK and hypoxia signaling pathways. ( a ) Gene sets enriched with a false discovery rate (FDR) smaller than 0.05 were subject to leading edge analysis to obtain a leading edge subset of genes. Clustered map of leading edge subsets upregulated in C7_24 h treatment compared to solvent control. Degree of upregulation is shown with color intensity (darker color indicates greater fold change). ( b ) AGS-BX1 cells were incubated with 10 μM C7 for 1, 2, 4, 8, 16, 24 and 48 h and the expression of Zta, Rta, and HIF-1α was examined by Western blotting. ( c ) SNU-719 cells were incubated with 20 μM C7 for 8, 16, 24, 48 and 72 h and the expression ofZta, Rta, and HIF-1α was examined by Western blotting. ( d ) AGS-BX1 cells were pretreated with 50 μM <t>PD98059</t> (mitogen-activated protein kinase kinase (MEK) inhibitor) for 1 h, and then incubated with 10 μM M C7 or Dp44mT for 48 h. The expression of Zta, Rta, p-ERK1/2, and HIF-1α was analyzed by Western blotting. ( e ) AGS-BX1 cells were treated with either iron-precomplexed C7 or Dp44mT for 48 h. The expression of Zta, Rta, p-ERK1/2 and HIF-1α was analyzed by Western blotting. ( f ) AGS-BDneo cells were either untreated or treated with 1000 μM deferoxamine, 20 μM C7, or 150 μM CoCl 2 for 48 h. The expression of Zta and HIF-1α was analyzed by Western blotting. ( g ) HA cells were untreated, or treated with either 1000 μM deferoxamine, 20 μM C7, or 150 μM CoCl 2 for 48 h. The expression of Zta and HIF-1α was analyzed by Western blotting. ( h ) HA cells were either untreated or treated with either 20 μM C7, 100, 150 or 500 μM CoCl 2 for 48 h. The expression of Zta and HIF-1α was analyzed by Western blotting. ( i ) Wild-type and HIF-1α knockout HA cells were treated with 20 μM C7 for 24 h. The expression of Zta, EA-D, and HIF-1α was analyzed by Western blotting. Cellular α-tubulin was detected as a loading control.
    Pd98059, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FUJIFILM pd98059
    Cell cycle distribution of nucleus pulposus cells . Serum-deprived nucleus pulposus cells were cultured with no supplements for 24 h (a). The cells were treated with 5 ng/mL transforming growth factor β1 (TGFβ1) for 24 h (b). At 2 h before the addition of TGFβ1, the cells were treated with 16 μM 10058-F4 (c), or with 30 μM <t>PD98059</t> (d). The cells were harvested 24 h after the addition of TGFβ1 and the nuclei were stained with propidium iodide. DNA histograms were generated using flow cytometry. Each plot represents the analysis of 10,000 events. The histograms present typical results and the percentage of cells in G 0 /G 1 , S and G 2 /M phases are shown as the average of duplicated measurements.
    Pd98059, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LC Laboratories pd98059
    Corylin reduces VCAM-1 expression in TNF-α-treated VSMCs through ROS/MAP kinase inhibition. VSMCs were treated with 20 µM corylin, 2 mM NAC or 10 µM DPI for 1 h and then incubated with or without 10 ng/mL TNF-α for 15 min. ( A ) Phosphorylated P38, ERK, and JNK levels were examined by Immunoblot analysis. Total ERK (T-ERK), total P38 (T-P38), and total JNK (T-JNK) were used as an internal control for sample loading. ( B ) VSMCs were pretreated (1 h) with corylin (20 µM), a P38 inhibitor (SB203580; SB; 10, 20, or 30 µM), <t>PD98059</t> (PD; 10, 20, or 30 µM), or a JNK inhibitor (SP600125; SP; 10, 20, or 30 nM) and then treated with 10 ng/mL TNF-α for 24 h. Western blot analysis for VCAM-1 and quantification of VCAM-1 to GAPDH in VSMCs. ( C – E ) After P38, ERK and JNK silencing, cells were treated with TNF-α and corylin. VCAM-1 expression was determined by Western blot analysis. Total P38 (T-P38), total ERK (T-ERK), and total JNK (T-JNK) proteins were used to examine the siRNA effects. GAPDH was used as an internal control for sample loading. ( F , G ) Confluent VSMCs were pretreated (1 h) with 20 µM corylin (C20), 30 µM SB203580 (SB), 30 µM PD98059 (PD), 30 nM SP600125 (SP), 10 µM parthenolide (PAR), and 1 µg/mL anti-VCAM-1 antibody or IgG and then treated with 10 ng/mL TNF-α (T) for 24 h. BCECF/AM-labeled U937 cells were added and cocultured for another 1 h. The adherent U937 cells were counted to evaluate the overall VCAM-1 expression level. The data are shown as the mean ± SD. * p
    Pd98059, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Incubation of HLE cells with MHCC97H-derived exosomes promotes EMT through the MAPK/ERK pathway. a Immunofluorescence microscopy of EMT markers in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. b Western blot analysis of EMT markers in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. c Western blot analysis of EMT promoters (ZEB1, ZEB2 and Slug) and MET promoter OVOL1 in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. d Western blot analysis of EMT markers of HLE cells with exposure to MHCC97H-derived exosomes for different durations. e Western blot analysis of phosphorylated and total Erk1/2, Akt and Smad2/3 in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. f Western blot analysis of EMT markers and phosphorylated and total Erk1/2 in HLE cells, exosome-treated HLE cells and exosome-treated HLE cells when cultured with the ERK inhibitor PD98059. Abbreviation: Exo exosome. All experiments were repeated at least three times

    Journal: Cell Death & Disease

    Article Title: HCC-derived exosomes elicit HCC progression and recurrence by epithelial-mesenchymal transition through MAPK/ERK signalling pathway

    doi: 10.1038/s41419-018-0534-9

    Figure Lengend Snippet: Incubation of HLE cells with MHCC97H-derived exosomes promotes EMT through the MAPK/ERK pathway. a Immunofluorescence microscopy of EMT markers in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. b Western blot analysis of EMT markers in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. c Western blot analysis of EMT promoters (ZEB1, ZEB2 and Slug) and MET promoter OVOL1 in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. d Western blot analysis of EMT markers of HLE cells with exposure to MHCC97H-derived exosomes for different durations. e Western blot analysis of phosphorylated and total Erk1/2, Akt and Smad2/3 in HLE cells treated with or without MHCC97H-derived exosomes for 24 h. f Western blot analysis of EMT markers and phosphorylated and total Erk1/2 in HLE cells, exosome-treated HLE cells and exosome-treated HLE cells when cultured with the ERK inhibitor PD98059. Abbreviation: Exo exosome. All experiments were repeated at least three times

    Article Snippet: PD98059 (25 μM, Sigma-Aldrich, St. Louis, MO, USA) was an inhibitor of ERK.

    Techniques: Incubation, Derivative Assay, Immunofluorescence, Microscopy, Western Blot, Cell Culture

    Inhibition of self-derived exosome secretion by Rab27a blockade induces EMT through the MAPK/ERK pathway. Analysis of the expression level of EMT markers in MHCC97H (WT), siSCR/MHCC97H and siRab27a/MHCC97H cells by immunofluorescence microscopy ( a ) and Western blot ( b ). c Analysis of the expression pattern of EMT-associated and MET-associated transcriptional regulators in MHCC97H (WT), siSCR/MHCC97H and siRab27a/MHCC97H cells at the protein level. d Western blot analysis of phosphorylated and total Erk1/2, Akt and Smad2/3 in MHCC97H (WT), siSCR/MHCC97H and siRab27a/MHCC97H cells. e Western blot analysis of phosphorylated and total Erk1/2 and EMT markers in MHCC97H (WT), siSCR/MHCC97H, and siRab27a/MHCC97H cells treated with or without the ERK inhibitor PD98059 for 24 h. All experiments were repeated at least three times

    Journal: Cell Death & Disease

    Article Title: HCC-derived exosomes elicit HCC progression and recurrence by epithelial-mesenchymal transition through MAPK/ERK signalling pathway

    doi: 10.1038/s41419-018-0534-9

    Figure Lengend Snippet: Inhibition of self-derived exosome secretion by Rab27a blockade induces EMT through the MAPK/ERK pathway. Analysis of the expression level of EMT markers in MHCC97H (WT), siSCR/MHCC97H and siRab27a/MHCC97H cells by immunofluorescence microscopy ( a ) and Western blot ( b ). c Analysis of the expression pattern of EMT-associated and MET-associated transcriptional regulators in MHCC97H (WT), siSCR/MHCC97H and siRab27a/MHCC97H cells at the protein level. d Western blot analysis of phosphorylated and total Erk1/2, Akt and Smad2/3 in MHCC97H (WT), siSCR/MHCC97H and siRab27a/MHCC97H cells. e Western blot analysis of phosphorylated and total Erk1/2 and EMT markers in MHCC97H (WT), siSCR/MHCC97H, and siRab27a/MHCC97H cells treated with or without the ERK inhibitor PD98059 for 24 h. All experiments were repeated at least three times

    Article Snippet: PD98059 (25 μM, Sigma-Aldrich, St. Louis, MO, USA) was an inhibitor of ERK.

    Techniques: Inhibition, Derivative Assay, Expressing, Immunofluorescence, Microscopy, Western Blot

    EPAH7 promotion of KLF4 expression via C/EBPβ, leading to induction of ADAMTS1 expression in KGN cells. (A) mRNA and protein abundances of KLF4 and ADAMTS1 after KLF4 knockdown in KGN cells detected by western blot analysis and qPCR analysis. The panel (left-to-right) shows representative images of western blot assays, we quantified protein abundances by measuring the densitometry of the immunoreactive bands. (B) mRNA and protein abundances of KLF4 and ADAMTS1 after KLF4 overexpression in KGN cells. (C) mRNA and protein abundances of C/EBPβ, KLF4 and ADAMTS1 after C/EBPβ knockdown in KGN cells. Left, a representative western blot is shown. Right, the immunoreactive bands were densitometrically quantified (above); and mRNA abundance is presented (below). (D) mRNA and protein abundances of C/EBPβ, KLF4 and ADAMTS1 after LAP overexpression in KGN cells. (E) mRNA and protein abundances of EPHA7, C/EBPβ, KLF4 and ADAMTS1 after EPHA7 knockdown in KGN cells. (F) mRNA and protein abundances of EPHA7, C/EBPβ, KLF4 and ADAMTS1 after EPHA7 overexpression and further incubation with PD98059 (ERK1/2 inhibitor) in KGN cells. Above, a representative western blot is shown (left), and the immunoreactive bands for ERK1/2 phosphorylation were quantified densitometrically (right). Middle, the immunoreactive bands for other proteins were also quantified densitometrically. Below, mRNA abundance is presented. (G) Above, we used a ChIP assay to detect the enrichment of C/EBPβ at the KLF4 promoter in KGN cells in response to EPHA7 overexpression. IgG served as the negative control. Below, sequence of the KLF4 promoter spanning–879 to–865 base pairs (bp). Boxed letters indicate putative transcription factor binding sites. TSS, transcription start site. β-actin or GAPDH were used as loading controls for western blot and for qPCR analyses. Blots are representative and data are presented as means ± SEM from 3 to 5 experiments. * P

    Journal: EBioMedicine

    Article Title: Erythropoietin-producing hepatocellular A7 triggering ovulation indicates a potential beneficial role for polycystic ovary syndrome

    doi: 10.1016/j.ebiom.2018.09.046

    Figure Lengend Snippet: EPAH7 promotion of KLF4 expression via C/EBPβ, leading to induction of ADAMTS1 expression in KGN cells. (A) mRNA and protein abundances of KLF4 and ADAMTS1 after KLF4 knockdown in KGN cells detected by western blot analysis and qPCR analysis. The panel (left-to-right) shows representative images of western blot assays, we quantified protein abundances by measuring the densitometry of the immunoreactive bands. (B) mRNA and protein abundances of KLF4 and ADAMTS1 after KLF4 overexpression in KGN cells. (C) mRNA and protein abundances of C/EBPβ, KLF4 and ADAMTS1 after C/EBPβ knockdown in KGN cells. Left, a representative western blot is shown. Right, the immunoreactive bands were densitometrically quantified (above); and mRNA abundance is presented (below). (D) mRNA and protein abundances of C/EBPβ, KLF4 and ADAMTS1 after LAP overexpression in KGN cells. (E) mRNA and protein abundances of EPHA7, C/EBPβ, KLF4 and ADAMTS1 after EPHA7 knockdown in KGN cells. (F) mRNA and protein abundances of EPHA7, C/EBPβ, KLF4 and ADAMTS1 after EPHA7 overexpression and further incubation with PD98059 (ERK1/2 inhibitor) in KGN cells. Above, a representative western blot is shown (left), and the immunoreactive bands for ERK1/2 phosphorylation were quantified densitometrically (right). Middle, the immunoreactive bands for other proteins were also quantified densitometrically. Below, mRNA abundance is presented. (G) Above, we used a ChIP assay to detect the enrichment of C/EBPβ at the KLF4 promoter in KGN cells in response to EPHA7 overexpression. IgG served as the negative control. Below, sequence of the KLF4 promoter spanning–879 to–865 base pairs (bp). Boxed letters indicate putative transcription factor binding sites. TSS, transcription start site. β-actin or GAPDH were used as loading controls for western blot and for qPCR analyses. Blots are representative and data are presented as means ± SEM from 3 to 5 experiments. * P

    Article Snippet: Concurrently, treatment of cells with the ERK1/2 inhibitor PD98059 (Sigma Chemical, St. Louis, MO) for 24 h hindered the expression of downstream factors after overexpressing EPHA7 ( F).

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Over Expression, Incubation, Chromatin Immunoprecipitation, Negative Control, Sequencing, Binding Assay

    Extracellular signal-regulated kinase (ERK) pathway regulates the neurogenic differentiation 1 (NeuroD1) protein stability via ubiquitination. (A) Western blot analysis of NeuroD1 protein in the absence and presence of PD 98059, a mitogen-activated protein kinase (MEK) inhibitor. Cycloheximide (CHX) was added 30 min prior to the addition of PD 98059. (B) The NeuroD1 protein intensity shown in (a) was presented with respect to the value at t=0 (n=3). (C) Western blot analysis of Myc-tagged mutants of NeuroD1 protein at ERK phosphorylation sites with and without N-acetyl-Leu-Leu-norleucinal (ALLN), a proteasome inhibitor. CHX was added 30 min prior to adding ALLN. (D and E) NeuroD1 protein intensity shown in (c) was presented with respect to the value at t=0 (n=3). Note that the S274A mutant exhibited the highest half-life and ALLN increased the half-lives of the wild type (WT) as well as all mutants. (F) Western blot analysis showing the ubiquitination of NeuroD1 WT in the presence of ALLN. PD 98059 partially attenuated ubiquitination. (G) Myc-tagged mutants of S259A, S266A, S274A, or Triple-A were analyzed for ubiquitination. The experiments were repeated 3 times and the most representative results are shown in Fig. 3A, C, F and G. Note that S274A abolished ubiquitination, whereas S266A showed a lower level of ubiquitination compared to the WT.

    Journal: Experimental Neurobiology

    Article Title: ERK Regulates NeuroD1-mediated Neurite Outgrowth via Proteasomal Degradation

    doi: 10.5607/en20021

    Figure Lengend Snippet: Extracellular signal-regulated kinase (ERK) pathway regulates the neurogenic differentiation 1 (NeuroD1) protein stability via ubiquitination. (A) Western blot analysis of NeuroD1 protein in the absence and presence of PD 98059, a mitogen-activated protein kinase (MEK) inhibitor. Cycloheximide (CHX) was added 30 min prior to the addition of PD 98059. (B) The NeuroD1 protein intensity shown in (a) was presented with respect to the value at t=0 (n=3). (C) Western blot analysis of Myc-tagged mutants of NeuroD1 protein at ERK phosphorylation sites with and without N-acetyl-Leu-Leu-norleucinal (ALLN), a proteasome inhibitor. CHX was added 30 min prior to adding ALLN. (D and E) NeuroD1 protein intensity shown in (c) was presented with respect to the value at t=0 (n=3). Note that the S274A mutant exhibited the highest half-life and ALLN increased the half-lives of the wild type (WT) as well as all mutants. (F) Western blot analysis showing the ubiquitination of NeuroD1 WT in the presence of ALLN. PD 98059 partially attenuated ubiquitination. (G) Myc-tagged mutants of S259A, S266A, S274A, or Triple-A were analyzed for ubiquitination. The experiments were repeated 3 times and the most representative results are shown in Fig. 3A, C, F and G. Note that S274A abolished ubiquitination, whereas S266A showed a lower level of ubiquitination compared to the WT.

    Article Snippet: Furthermore, 20 µM PD 98059 (Sigma-Aldrich, P215) or 10 µM N-acetyl-Leu-Leu-norleucinal (ALLN) (Sigma-Aldrich, A6185) was added to the cell culture 30 min or 2 h prior to harvesting to inhibit the ERK or proteasome pathway, respectively.

    Techniques: Western Blot, Mutagenesis

    Role ERK1/2 in PA-regulated MMP-2 and TIMP-2 expression in HeLa cells. ( A ) Cells were treated with various concentrations of PA (0 to 30 μM) for 24 h, then harvested and lysed for measurement target proteins by western blotting; ( B ) cells were treated with or without PA in the absence or presence of PD98059 (specific MEK1/2 inhibitor) for 24 h, followed by measurement of migration and invasion; ( C ) cells were treated as above for 24 h, followed by measurement of relative wound width; ( D , E ) Protein and mRNA expression of MMP-2 and TIMP-2 were measured by western blotting assay and RT-qPCR. Values are means and standard errors of 3 replicates. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Praeruptorin A Inhibits Human Cervical Cancer Cell Growth and Invasion by Suppressing MMP-2 Expression and ERK1/2 Signaling

    doi: 10.3390/ijms19010010

    Figure Lengend Snippet: Role ERK1/2 in PA-regulated MMP-2 and TIMP-2 expression in HeLa cells. ( A ) Cells were treated with various concentrations of PA (0 to 30 μM) for 24 h, then harvested and lysed for measurement target proteins by western blotting; ( B ) cells were treated with or without PA in the absence or presence of PD98059 (specific MEK1/2 inhibitor) for 24 h, followed by measurement of migration and invasion; ( C ) cells were treated as above for 24 h, followed by measurement of relative wound width; ( D , E ) Protein and mRNA expression of MMP-2 and TIMP-2 were measured by western blotting assay and RT-qPCR. Values are means and standard errors of 3 replicates. ** p

    Article Snippet: The MEK1/2 inhibitor PD98059 was purchased from Calbiochem (San Diego, CA, USA).

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

    STING and TLR9 pathways are essential for the cationic liposome-induced activation of pulmonary inflammation. WT , Sting -/- and Tlr9 -/- mice were treated with DOTAP liposomes for 48 h. (A) Representative H E staining of lung sections of mice treated with DOTAP liposomes (n=5); scale bar=50 μm. (B) Inflammatory monocyte (CD45 + CD11b + Ly6C + ) influx into the lungs of mice treated with DOTAP liposomes (n=5). (C) Inflammatory neutrophil (CD45 + CD11b + Ly6G + ) influx into the lungs of mice treated with DOTAP liposomes (n=5). (D) TNF-α expression in the inflammatory neutrophils (CD45 + CD11b + Ly6G + ) in the lungs of WT and Sting -/- mice (n=3). (E) IL-10 production in inflammatory monocytes (CD45 + CD11b + Ly6C + ) in the lungs of WT and Sting -/- mice (n=3). (F) Neutrophils were isolated from the bone marrow of WT and Sting -/- mice and were stimulated with necrotic lung cells or mtDNA (G) for 4 h in the presence of brefeldin A. TNF-α expression in inflammatory neutrophils (CD45 + CD11b + Ly6G + ) was determined by flow cytometry, (n=3). (H) Monocytes were isolated from the bone marrow of WT, Tlr9 -/- and Sting -/- mice and were cultured with mitochondrial DNA (5 μg/mL) at a concentration of 5×10 6 cells/mL; EIA assays were conducted for PGE 2 expression in supernatants (n=3). (I) Western blot analysis was performed. (J) The freshly isolated monocytes were cultured with mtDNA (5 μg/mL) at a concentration of 5×10 6 cells/mL. The inhibitors PD98059 (30 μM), SB203580 (10 μM), BAY117082 (10 μM) and indomethacin (10 μM) were added to the cells, and the cells were incubated for 2 h at 37 °C, then, Western blot analysis was performed. Data are representative of three independent experiments, and the results are expressed as the mean ± S.E.M. Statistical comparisons were performed using Student's t-test or Dunnet's t-test ( *P

    Journal: Theranostics

    Article Title: Negative regulation of cationic nanoparticle-induced inflammatory toxicity through the increased production of prostaglandin E2 via mitochondrial DNA-activated Ly6C+ monocytes

    doi: 10.7150/thno.21693

    Figure Lengend Snippet: STING and TLR9 pathways are essential for the cationic liposome-induced activation of pulmonary inflammation. WT , Sting -/- and Tlr9 -/- mice were treated with DOTAP liposomes for 48 h. (A) Representative H E staining of lung sections of mice treated with DOTAP liposomes (n=5); scale bar=50 μm. (B) Inflammatory monocyte (CD45 + CD11b + Ly6C + ) influx into the lungs of mice treated with DOTAP liposomes (n=5). (C) Inflammatory neutrophil (CD45 + CD11b + Ly6G + ) influx into the lungs of mice treated with DOTAP liposomes (n=5). (D) TNF-α expression in the inflammatory neutrophils (CD45 + CD11b + Ly6G + ) in the lungs of WT and Sting -/- mice (n=3). (E) IL-10 production in inflammatory monocytes (CD45 + CD11b + Ly6C + ) in the lungs of WT and Sting -/- mice (n=3). (F) Neutrophils were isolated from the bone marrow of WT and Sting -/- mice and were stimulated with necrotic lung cells or mtDNA (G) for 4 h in the presence of brefeldin A. TNF-α expression in inflammatory neutrophils (CD45 + CD11b + Ly6G + ) was determined by flow cytometry, (n=3). (H) Monocytes were isolated from the bone marrow of WT, Tlr9 -/- and Sting -/- mice and were cultured with mitochondrial DNA (5 μg/mL) at a concentration of 5×10 6 cells/mL; EIA assays were conducted for PGE 2 expression in supernatants (n=3). (I) Western blot analysis was performed. (J) The freshly isolated monocytes were cultured with mtDNA (5 μg/mL) at a concentration of 5×10 6 cells/mL. The inhibitors PD98059 (30 μM), SB203580 (10 μM), BAY117082 (10 μM) and indomethacin (10 μM) were added to the cells, and the cells were incubated for 2 h at 37 °C, then, Western blot analysis was performed. Data are representative of three independent experiments, and the results are expressed as the mean ± S.E.M. Statistical comparisons were performed using Student's t-test or Dunnet's t-test ( *P

    Article Snippet: The inhibitors PD98059 (EMD Chemicals), SB203580 (EMD Chemicals), BAY117082 (EMD Chemicals) and indomethacin (Sigma) were added to the cells, and the cells were incubated for 2 h at 37 °C.

    Techniques: Activation Assay, Mouse Assay, Staining, Expressing, Isolation, Flow Cytometry, Cytometry, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation

    O-GlcNAcylation regulated activations of Akt and Erk and expression of FOXO3 and MAN1A1. OGT was suppressed in parental (KKU-213 and KKU-214) and highly metastatic (KKU-213L5 and KKU-214L5) CCA cells using siOGT. a siOGT treatment inactivated the phosphorylation of Akt and Erk but not Ikk. b The modulations of Akt and Erk on FOXO3 expression were revealed using Akt and Erk inhibitors. CCA cells were treated with various concentrations of Akt inhibitor, MK-2206 and ( c ) Erk inhibitor, PD98059, for 24 h. The expression of pAkt/Akt, pErk/Erk, phospho-FOXO3, FOXO3 and MAN1A1 were determined using western blotting. d The MK-2206-treated L5 CCA cells were incubated with 20 µM of cycloheximide (CHX) for 1, 3, and 6 h. FOXO3 levels at each time point were determined using western blotting and compared with those of the untreated control cells. The data are represented as the mean ± SD from three independent experiments. Inhibition of Akt activation significantly reduced ( e ) migration and ( f ) invasion abilities of CCA cells and these effects could be rescued by kifunensine (Kif) treatment. The results are mean ± SEM of one representative from two independent experiments. * P

    Journal: Oncogene

    Article Title: O-GlcNAcylation mediates metastasis of cholangiocarcinoma through FOXO3 and MAN1A1

    doi: 10.1038/s41388-018-0366-1

    Figure Lengend Snippet: O-GlcNAcylation regulated activations of Akt and Erk and expression of FOXO3 and MAN1A1. OGT was suppressed in parental (KKU-213 and KKU-214) and highly metastatic (KKU-213L5 and KKU-214L5) CCA cells using siOGT. a siOGT treatment inactivated the phosphorylation of Akt and Erk but not Ikk. b The modulations of Akt and Erk on FOXO3 expression were revealed using Akt and Erk inhibitors. CCA cells were treated with various concentrations of Akt inhibitor, MK-2206 and ( c ) Erk inhibitor, PD98059, for 24 h. The expression of pAkt/Akt, pErk/Erk, phospho-FOXO3, FOXO3 and MAN1A1 were determined using western blotting. d The MK-2206-treated L5 CCA cells were incubated with 20 µM of cycloheximide (CHX) for 1, 3, and 6 h. FOXO3 levels at each time point were determined using western blotting and compared with those of the untreated control cells. The data are represented as the mean ± SD from three independent experiments. Inhibition of Akt activation significantly reduced ( e ) migration and ( f ) invasion abilities of CCA cells and these effects could be rescued by kifunensine (Kif) treatment. The results are mean ± SEM of one representative from two independent experiments. * P

    Article Snippet: Akt inhibitor, MK-2206 dihydrochloride (#sc-364537) was from Santa Cruz Biotechnology (Santa Cruz, CA) and Erk inhibitor, PD98059 (#9900) was obtained from Cell Signaling (Danvers, MA).

    Techniques: Expressing, Western Blot, Incubation, Inhibition, Activation Assay, Migration

    Gelatinolytic activities in bronchoalveolar lavage fluid (A, D) and the supernatants of alveolar macrophages in culture (B, E). MMP-9 expression in bronchoalveolar lavage fluid (C, F). The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125(IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO); LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Alveolar macrophages (10 6 /m1 of RPMI medium) were incubated for 24 hours. BAL fluid and culture supernatants were analyzed by sensitive zymography, followed by scanning densitometry. 92 kD and 66 kD gelatinolytic bands correspond to MMP-9 and MMP-2, respectively. Densitometry of 92 kD bands is expressed in arbitrary densitometric units. Western blots of BAL fluid with anti-MMP-9 antibodywere employed to monitor MMP-9. Values are represented as means ± SEM of results from 5 rats in each group. * Significant differences between saline, p

    Journal: Respiratory Research

    Article Title: Inhibition of c-Jun NH2-terminal kinase or extracellular signal-regulated kinase improves lung injury

    doi: 10.1186/1465-9921-5-23

    Figure Lengend Snippet: Gelatinolytic activities in bronchoalveolar lavage fluid (A, D) and the supernatants of alveolar macrophages in culture (B, E). MMP-9 expression in bronchoalveolar lavage fluid (C, F). The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125(IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO); LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Alveolar macrophages (10 6 /m1 of RPMI medium) were incubated for 24 hours. BAL fluid and culture supernatants were analyzed by sensitive zymography, followed by scanning densitometry. 92 kD and 66 kD gelatinolytic bands correspond to MMP-9 and MMP-2, respectively. Densitometry of 92 kD bands is expressed in arbitrary densitometric units. Western blots of BAL fluid with anti-MMP-9 antibodywere employed to monitor MMP-9. Values are represented as means ± SEM of results from 5 rats in each group. * Significant differences between saline, p

    Article Snippet: PD98059, however, caused no significant changes in the LPS-induced phosphorylation and degradation of IκB-α (Figure and lane 3 ).

    Techniques: Expressing, Incubation, Zymography, Western Blot

    Phosphorylation (A, B) and degradation (C, D) of IκB-α in lung tissue. The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125 (IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO); LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Western blots with anti-serine phospho-IκBα (Ser32)/IκBα antibody were employed to monitor phosphorylated IκB-α and IκB-α. Densitometry of phospho-IκB-α/IκB-α bands is expressed in arbitrary densitometric units. Values represent means ± SEM of results from 5 rats in each group. * Significant differences compared with saline, p

    Journal: Respiratory Research

    Article Title: Inhibition of c-Jun NH2-terminal kinase or extracellular signal-regulated kinase improves lung injury

    doi: 10.1186/1465-9921-5-23

    Figure Lengend Snippet: Phosphorylation (A, B) and degradation (C, D) of IκB-α in lung tissue. The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125 (IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO); LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Western blots with anti-serine phospho-IκBα (Ser32)/IκBα antibody were employed to monitor phosphorylated IκB-α and IκB-α. Densitometry of phospho-IκB-α/IκB-α bands is expressed in arbitrary densitometric units. Values represent means ± SEM of results from 5 rats in each group. * Significant differences compared with saline, p

    Article Snippet: PD98059, however, caused no significant changes in the LPS-induced phosphorylation and degradation of IκB-α (Figure and lane 3 ).

    Techniques: Western Blot

    EMSA illustrating DNA-binding activity of NF-κB to the NF-κB motif in lung tissue (A, C), and alveolar macrophages (B, D). The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125 (IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO); LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Nuclear extracts were prepared in lung tissue and alveolar macrophages (5 × 10 6 alveolar macrophages). Addition of 100 ng of unlabeled cold competitor to the LPS samples successfully competed for NF-κB binding, and eliminated the specific band. Densitometry of NF-κB bands on EMSA is expressed in arbitrary densitometric units. Values are represented as means ± SEM of results from 5 rats in each group. * Significant differences compared with saline, p

    Journal: Respiratory Research

    Article Title: Inhibition of c-Jun NH2-terminal kinase or extracellular signal-regulated kinase improves lung injury

    doi: 10.1186/1465-9921-5-23

    Figure Lengend Snippet: EMSA illustrating DNA-binding activity of NF-κB to the NF-κB motif in lung tissue (A, C), and alveolar macrophages (B, D). The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125 (IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO); LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Nuclear extracts were prepared in lung tissue and alveolar macrophages (5 × 10 6 alveolar macrophages). Addition of 100 ng of unlabeled cold competitor to the LPS samples successfully competed for NF-κB binding, and eliminated the specific band. Densitometry of NF-κB bands on EMSA is expressed in arbitrary densitometric units. Values are represented as means ± SEM of results from 5 rats in each group. * Significant differences compared with saline, p

    Article Snippet: PD98059, however, caused no significant changes in the LPS-induced phosphorylation and degradation of IκB-α (Figure and lane 3 ).

    Techniques: Binding Assay, Activity Assay

    Phosphorylation of JNK (A, B), ERK (C, D)and p38 MAP kinase (E, F) in lung tissue 4 hours after saline or LPS treatment. The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125 (IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO);LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Western blots with anti-phospho-JNK/JNK antibody, phospho-ERK/ERK antibody or phospho-p38 MAP kinase/p38 MAP kinase were employed in order to monitor JNK, ERK or p38 MAP kinase phosphorylation. Relative values for levels of phosphorylated JNK1/2, ERK1/2 or p38 MAP kinse normalized to JNK1/2, ERK1/2 or p38 MAP kinase are indicated below the gel. Results are representative results from 5 rats in each group.

    Journal: Respiratory Research

    Article Title: Inhibition of c-Jun NH2-terminal kinase or extracellular signal-regulated kinase improves lung injury

    doi: 10.1186/1465-9921-5-23

    Figure Lengend Snippet: Phosphorylation of JNK (A, B), ERK (C, D)and p38 MAP kinase (E, F) in lung tissue 4 hours after saline or LPS treatment. The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125 (IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO);LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Western blots with anti-phospho-JNK/JNK antibody, phospho-ERK/ERK antibody or phospho-p38 MAP kinase/p38 MAP kinase were employed in order to monitor JNK, ERK or p38 MAP kinase phosphorylation. Relative values for levels of phosphorylated JNK1/2, ERK1/2 or p38 MAP kinse normalized to JNK1/2, ERK1/2 or p38 MAP kinase are indicated below the gel. Results are representative results from 5 rats in each group.

    Article Snippet: PD98059, however, caused no significant changes in the LPS-induced phosphorylation and degradation of IκB-α (Figure and lane 3 ).

    Techniques: Western Blot

    Levels of total protein (A), activity of LDH (B) and neutrophil numbers (C) in bronchoalveolar lavage fluid. The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125 (IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO);LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Values represent means ± SEM of results from 5 rats in each group. * Significant differences between saline, p

    Journal: Respiratory Research

    Article Title: Inhibition of c-Jun NH2-terminal kinase or extracellular signal-regulated kinase improves lung injury

    doi: 10.1186/1465-9921-5-23

    Figure Lengend Snippet: Levels of total protein (A), activity of LDH (B) and neutrophil numbers (C) in bronchoalveolar lavage fluid. The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125 (IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO);LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Values represent means ± SEM of results from 5 rats in each group. * Significant differences between saline, p

    Article Snippet: PD98059, however, caused no significant changes in the LPS-induced phosphorylation and degradation of IκB-α (Figure and lane 3 ).

    Techniques: Activity Assay

    CINC expression in lung tissue (A, C) and bronchoalveolar lavage fluid (B, D). The groups represent rats treated as follows: The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125(IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125(IO); LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Western blots with anti-CINC antibodies were performed on the samples of lung tissue and BAL fluid. Densitometry of CINC bands is expressed in arbitrary densitometric units. Values are represented as means ± SEM of results from 5 rats in each group. * Significant differences between saline, p

    Journal: Respiratory Research

    Article Title: Inhibition of c-Jun NH2-terminal kinase or extracellular signal-regulated kinase improves lung injury

    doi: 10.1186/1465-9921-5-23

    Figure Lengend Snippet: CINC expression in lung tissue (A, C) and bronchoalveolar lavage fluid (B, D). The groups represent rats treated as follows: The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125(IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125(IO); LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Western blots with anti-CINC antibodies were performed on the samples of lung tissue and BAL fluid. Densitometry of CINC bands is expressed in arbitrary densitometric units. Values are represented as means ± SEM of results from 5 rats in each group. * Significant differences between saline, p

    Article Snippet: PD98059, however, caused no significant changes in the LPS-induced phosphorylation and degradation of IκB-α (Figure and lane 3 ).

    Techniques: Expressing, Western Blot

    NO production in BAL fluid (A) and alveolar macrophages in culture (B). The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125 (IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO); LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Alveolar macrophages (10 6 /m1 of RPMI medium) were incubated for 24 hours. BAL fluid and culture supernatants were analyzed using nitrite assays. Values are represented as means ± SEM of results from 5 rats in each group. * Significant differences compared with saline, p

    Journal: Respiratory Research

    Article Title: Inhibition of c-Jun NH2-terminal kinase or extracellular signal-regulated kinase improves lung injury

    doi: 10.1186/1465-9921-5-23

    Figure Lengend Snippet: NO production in BAL fluid (A) and alveolar macrophages in culture (B). The groups represent rats treated as follows: Saline, saline (IT); LPS, LPS (IT); LPS-SP600125, LPS (IT) and a pretreatment with SP600125 (IO), Saline-SP600125, saline (IT) and a pretreatment with SP600125 (IO); LPS-PD98059, LPS (IT) and a pretreatment with PD98059 (IO), Saline-PD98059, saline (IT) and a pretreatment with PD98059 (IO). Animals were sacrificed 4 hours after LPS treatment. Alveolar macrophages (10 6 /m1 of RPMI medium) were incubated for 24 hours. BAL fluid and culture supernatants were analyzed using nitrite assays. Values are represented as means ± SEM of results from 5 rats in each group. * Significant differences compared with saline, p

    Article Snippet: PD98059, however, caused no significant changes in the LPS-induced phosphorylation and degradation of IκB-α (Figure and lane 3 ).

    Techniques: Incubation

    Dabrafenib reduced the expression of phosphorylated JNK and c-jun in both SH-SY5Y cells and mice ( A ) LDH/cell viability assay under the condition of ERK inhibitor. Neuroprotective effects of dabrafenib (5 μM) were not dependent on the downregulation of activated ERK expression by PD98059 (30 μM) or U0126 (3 μM). Neuroprotective effects were assessed by the LDH and cell viability assay in SH-SY5Y cells at 24 h after MPP + (3 mM) exposure. ** , P -value

    Journal: Human Molecular Genetics

    Article Title: In silico drug screening by using genome-wide association study data repurposed dabrafenib, an anti-melanoma drug, for Parkinson’s disease

    doi: 10.1093/hmg/ddy279

    Figure Lengend Snippet: Dabrafenib reduced the expression of phosphorylated JNK and c-jun in both SH-SY5Y cells and mice ( A ) LDH/cell viability assay under the condition of ERK inhibitor. Neuroprotective effects of dabrafenib (5 μM) were not dependent on the downregulation of activated ERK expression by PD98059 (30 μM) or U0126 (3 μM). Neuroprotective effects were assessed by the LDH and cell viability assay in SH-SY5Y cells at 24 h after MPP + (3 mM) exposure. ** , P -value

    Article Snippet: PD98059 and U0126 were from Cayman Chemical Company.

    Techniques: Expressing, Mouse Assay, Viability Assay

    While PD98059 or compound C had little effect on influencing the effect of β-elemene in phosphorylation of AMPKα or ERK1/2, metformin reversed the effect of β-elemene on phosphorylation of Akt. A549 (A) and PC9 (B) cells were treated with PD98059 (20 μM) or compound C (20 μM) for 2 hrs before exposure of the cells to β-elemene (20 μg/ml) for an additional 24 hrs. Afterwards, the expression of p-ERK1/2 and p-AMPKα protein and their total forms was detected by Western blot. (C) A549 and PC9 cells were treated with metformin (5 mM) and β-elemene (20 μg/ml) for up to 2 hrs. Afterwards, the expression of p-Akt and p-AMPKα protein and total ones were detected by Western blot. The bar graphs represent the mean ± SD of p-Akt/GAPDH of three independent experiments. * indicates significant difference from untreated control cells. ** indicates significant difference from β-elemene treated alone ( P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: β-elemene inhibited expression of DNA methyltransferase 1 through activation of ERK1/2 and AMPKα signalling pathways in human lung cancer cells: the role of Sp1

    doi: 10.1111/jcmm.12476

    Figure Lengend Snippet: While PD98059 or compound C had little effect on influencing the effect of β-elemene in phosphorylation of AMPKα or ERK1/2, metformin reversed the effect of β-elemene on phosphorylation of Akt. A549 (A) and PC9 (B) cells were treated with PD98059 (20 μM) or compound C (20 μM) for 2 hrs before exposure of the cells to β-elemene (20 μg/ml) for an additional 24 hrs. Afterwards, the expression of p-ERK1/2 and p-AMPKα protein and their total forms was detected by Western blot. (C) A549 and PC9 cells were treated with metformin (5 mM) and β-elemene (20 μg/ml) for up to 2 hrs. Afterwards, the expression of p-Akt and p-AMPKα protein and total ones were detected by Western blot. The bar graphs represent the mean ± SD of p-Akt/GAPDH of three independent experiments. * indicates significant difference from untreated control cells. ** indicates significant difference from β-elemene treated alone ( P

    Article Snippet: PD98059 (ERK inhibitor), compound C (a special inhibitor of AMPK) and metformin (an activator of AMPK) were purchased from Merck Millipore (Darmstadt, Germany), MTT powder was purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Expressing, Western Blot

    β-elemene inhibited protein expression of DNMT1 in the dose-dependent manner, overexpression of DNMT1 reversed the effect of β-elemene on cell growth. A549 (A) and PC9 (B) cells were exposed to increased concentrations of β-elemene for 24 hrs. Afterwards, the expression of DNMT1 protein were detected by Western blot. A549 and PC9 cells were treated with PD98059 (20 μM; C) or compound C (20 μM; D) for 2 hrs before exposure of the cells to β-elemene (20 μg/ml) for an additional up to 24 hrs. Afterwards, the expression of DNMT1 protein was detected by Western blot. (E) A549 and PC9 cells were treated with metformin (20 μM) and β-elemene (20 μg/ml) for up to 24 hrs. Afterwards, the expression of DNMT1 protein was detected by Western blot. The bar graphs represent the mean ± SD of DNMT1/GAPDH of three independent experiments. (F) Cells were transfected with control or DNMT1 expression vector for 24 hrs before exposing the cells to β-elemene for an additional 24 hrs. Afterwards, the expression of DNMT1 protein and cell growth were detected by Western blot and MTT assays, respectively. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. * indicates significant difference as compared to the untreated control group ( P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: β-elemene inhibited expression of DNA methyltransferase 1 through activation of ERK1/2 and AMPKα signalling pathways in human lung cancer cells: the role of Sp1

    doi: 10.1111/jcmm.12476

    Figure Lengend Snippet: β-elemene inhibited protein expression of DNMT1 in the dose-dependent manner, overexpression of DNMT1 reversed the effect of β-elemene on cell growth. A549 (A) and PC9 (B) cells were exposed to increased concentrations of β-elemene for 24 hrs. Afterwards, the expression of DNMT1 protein were detected by Western blot. A549 and PC9 cells were treated with PD98059 (20 μM; C) or compound C (20 μM; D) for 2 hrs before exposure of the cells to β-elemene (20 μg/ml) for an additional up to 24 hrs. Afterwards, the expression of DNMT1 protein was detected by Western blot. (E) A549 and PC9 cells were treated with metformin (20 μM) and β-elemene (20 μg/ml) for up to 24 hrs. Afterwards, the expression of DNMT1 protein was detected by Western blot. The bar graphs represent the mean ± SD of DNMT1/GAPDH of three independent experiments. (F) Cells were transfected with control or DNMT1 expression vector for 24 hrs before exposing the cells to β-elemene for an additional 24 hrs. Afterwards, the expression of DNMT1 protein and cell growth were detected by Western blot and MTT assays, respectively. Values in bar graphs were given as the mean ± SD from three independent experiments performed in triplicate. * indicates significant difference as compared to the untreated control group ( P

    Article Snippet: PD98059 (ERK inhibitor), compound C (a special inhibitor of AMPK) and metformin (an activator of AMPK) were purchased from Merck Millipore (Darmstadt, Germany), MTT powder was purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Expressing, Over Expression, Western Blot, Transfection, Plasmid Preparation, MTT Assay

    β-elemene inhibits transcription factor Sp1 protein expression, which was abrogated by the either ERK or AMPK inhibitor. A549 (A) and PC9 (B) cells were exposed to increased concentration of β-elemene for 24 hrs, followed by measuring the protein expression of Sp1 by Western blot. The bar graphs represent the mean ± SD of Sp1/GAPDH of three independent experiments. A549 cells were treated with PD98059 (20 μM; C) or compound C (20 μM; D) for 2 hrs before exposure of the cells to β-elemene (20 μg/ml) for an additional 24 hrs. Afterwards, the expression of Sp1 protein was detected by Western blot.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: β-elemene inhibited expression of DNA methyltransferase 1 through activation of ERK1/2 and AMPKα signalling pathways in human lung cancer cells: the role of Sp1

    doi: 10.1111/jcmm.12476

    Figure Lengend Snippet: β-elemene inhibits transcription factor Sp1 protein expression, which was abrogated by the either ERK or AMPK inhibitor. A549 (A) and PC9 (B) cells were exposed to increased concentration of β-elemene for 24 hrs, followed by measuring the protein expression of Sp1 by Western blot. The bar graphs represent the mean ± SD of Sp1/GAPDH of three independent experiments. A549 cells were treated with PD98059 (20 μM; C) or compound C (20 μM; D) for 2 hrs before exposure of the cells to β-elemene (20 μg/ml) for an additional 24 hrs. Afterwards, the expression of Sp1 protein was detected by Western blot.

    Article Snippet: PD98059 (ERK inhibitor), compound C (a special inhibitor of AMPK) and metformin (an activator of AMPK) were purchased from Merck Millipore (Darmstadt, Germany), MTT powder was purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Expressing, Concentration Assay, Western Blot

    Phosphor-serine activation of m-calpain dominants KCC2 regulation in 0 Mg 2+ seizure model. (A,B) Immunoblotting detection of both plasma membrane (A) and cytoplasm (B) KCC2 expression level change in hippocampal slice 0 Mg 2+ model treated with either μ-calpain or m-calpain selective inhibitor CI I and CI IV, respectively. (C) Expression of either μ- or m-calpain from seizure slices induced by 0 Mg 2+ . (D) Serine phosphorylation level by IP method. Naïve or 0 Mg 2+ treated slices were used as inputs to form immune complex with μ- or m-calpain antibodies and detected by phosphor-serine antibody. Phosphor-serine levels were normalized to corresponded calpain levels. (E) Phosphor-serine level of m-calpain from 0 Mg 2+ , 0 Mg 2+ + PD98059 or K252a treated slices. (F) Immunoblotting showed KCC2 level in 0 Mg 2+ + MDL-28170 or PD98059 treated slices, compared with none treated or 0 Mg 2+ treated alone slices. (G) KCC2 level of in vivo rats pre-treated with either MDL-28170 or SL-327 before PTZ kindling, in compared with vehicle PTZ treated rats. CI I, Calpain Inhibitor I; CI IV, Calpain Inhibitor IV; Calp, calpain; p-Ser, Phosphor-serine. * p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: M-Calpain Activation Facilitates Seizure Induced KCC2 Down Regulation

    doi: 10.3389/fnmol.2018.00287

    Figure Lengend Snippet: Phosphor-serine activation of m-calpain dominants KCC2 regulation in 0 Mg 2+ seizure model. (A,B) Immunoblotting detection of both plasma membrane (A) and cytoplasm (B) KCC2 expression level change in hippocampal slice 0 Mg 2+ model treated with either μ-calpain or m-calpain selective inhibitor CI I and CI IV, respectively. (C) Expression of either μ- or m-calpain from seizure slices induced by 0 Mg 2+ . (D) Serine phosphorylation level by IP method. Naïve or 0 Mg 2+ treated slices were used as inputs to form immune complex with μ- or m-calpain antibodies and detected by phosphor-serine antibody. Phosphor-serine levels were normalized to corresponded calpain levels. (E) Phosphor-serine level of m-calpain from 0 Mg 2+ , 0 Mg 2+ + PD98059 or K252a treated slices. (F) Immunoblotting showed KCC2 level in 0 Mg 2+ + MDL-28170 or PD98059 treated slices, compared with none treated or 0 Mg 2+ treated alone slices. (G) KCC2 level of in vivo rats pre-treated with either MDL-28170 or SL-327 before PTZ kindling, in compared with vehicle PTZ treated rats. CI I, Calpain Inhibitor I; CI IV, Calpain Inhibitor IV; Calp, calpain; p-Ser, Phosphor-serine. * p

    Article Snippet: In corresponding vehicle control or intervention groups, slices were also treated at same time with one of the following agents: MDL-28170: #ab145601, abcam, 50 μM; PD98059: #ab120234, abcam, 25 μM ; K252a: #420298, Calbiochem, 200 nM; Tautomycetin: #2305, Tocris, 20 nM; BAPTA-AM: # A1076, Sigma, 10 μM; Calpain Inhibitor I: # A6185-5MG, Sigma, 100 μM; Calpain Inhibitor IV: # 208724, Calbiochem, 200 μM.

    Techniques: Activation Assay, Expressing, In Vivo

    The regulation of KCC2 by m-calpain is independent on [Ca 2+ ] i but is related to KCC2 endocytosis. (A) BAPTA is able to reverse 0 Mg 2+ induced mKCC2 down-regulation, but failed to reverse BDNF induced mKCC2 down-regulation. (B) BAPTA partially reverses the 0 Mg 2+ induced, but not BDNF induced Changes in cytoplasmic KCC2 (cKCC2) down-regulation. (C) Phosphor-serine level of m-calpain from 0 Mg 2+ , 0 Mg 2+ + BAPTA, BDNF and BDNF + BAPTA treated slices. BAPTA intervention partially reverses the 0 Mg 2+ induced, but not BDNF induced the increase of phosphor-serine level of m-calpain. (D–E) Tautomycetin (TMC) can reverse KCC2 down-regulation in plasm membrane (left), but the expression of KCC2 in cytoplasm remained significantly lower than DMSO group (right). In TMC + PD98059 co-treated group the expression of KCC2 is rescued to control level. * p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: M-Calpain Activation Facilitates Seizure Induced KCC2 Down Regulation

    doi: 10.3389/fnmol.2018.00287

    Figure Lengend Snippet: The regulation of KCC2 by m-calpain is independent on [Ca 2+ ] i but is related to KCC2 endocytosis. (A) BAPTA is able to reverse 0 Mg 2+ induced mKCC2 down-regulation, but failed to reverse BDNF induced mKCC2 down-regulation. (B) BAPTA partially reverses the 0 Mg 2+ induced, but not BDNF induced Changes in cytoplasmic KCC2 (cKCC2) down-regulation. (C) Phosphor-serine level of m-calpain from 0 Mg 2+ , 0 Mg 2+ + BAPTA, BDNF and BDNF + BAPTA treated slices. BAPTA intervention partially reverses the 0 Mg 2+ induced, but not BDNF induced the increase of phosphor-serine level of m-calpain. (D–E) Tautomycetin (TMC) can reverse KCC2 down-regulation in plasm membrane (left), but the expression of KCC2 in cytoplasm remained significantly lower than DMSO group (right). In TMC + PD98059 co-treated group the expression of KCC2 is rescued to control level. * p

    Article Snippet: In corresponding vehicle control or intervention groups, slices were also treated at same time with one of the following agents: MDL-28170: #ab145601, abcam, 50 μM; PD98059: #ab120234, abcam, 25 μM ; K252a: #420298, Calbiochem, 200 nM; Tautomycetin: #2305, Tocris, 20 nM; BAPTA-AM: # A1076, Sigma, 10 μM; Calpain Inhibitor I: # A6185-5MG, Sigma, 100 μM; Calpain Inhibitor IV: # 208724, Calbiochem, 200 μM.

    Techniques: Expressing

    EBV lytic reactivation by C7 requires the activation of ERK and hypoxia signaling pathways. ( a ) Gene sets enriched with a false discovery rate (FDR) smaller than 0.05 were subject to leading edge analysis to obtain a leading edge subset of genes. Clustered map of leading edge subsets upregulated in C7_24 h treatment compared to solvent control. Degree of upregulation is shown with color intensity (darker color indicates greater fold change). ( b ) AGS-BX1 cells were incubated with 10 μM C7 for 1, 2, 4, 8, 16, 24 and 48 h and the expression of Zta, Rta, and HIF-1α was examined by Western blotting. ( c ) SNU-719 cells were incubated with 20 μM C7 for 8, 16, 24, 48 and 72 h and the expression ofZta, Rta, and HIF-1α was examined by Western blotting. ( d ) AGS-BX1 cells were pretreated with 50 μM PD98059 (mitogen-activated protein kinase kinase (MEK) inhibitor) for 1 h, and then incubated with 10 μM M C7 or Dp44mT for 48 h. The expression of Zta, Rta, p-ERK1/2, and HIF-1α was analyzed by Western blotting. ( e ) AGS-BX1 cells were treated with either iron-precomplexed C7 or Dp44mT for 48 h. The expression of Zta, Rta, p-ERK1/2 and HIF-1α was analyzed by Western blotting. ( f ) AGS-BDneo cells were either untreated or treated with 1000 μM deferoxamine, 20 μM C7, or 150 μM CoCl 2 for 48 h. The expression of Zta and HIF-1α was analyzed by Western blotting. ( g ) HA cells were untreated, or treated with either 1000 μM deferoxamine, 20 μM C7, or 150 μM CoCl 2 for 48 h. The expression of Zta and HIF-1α was analyzed by Western blotting. ( h ) HA cells were either untreated or treated with either 20 μM C7, 100, 150 or 500 μM CoCl 2 for 48 h. The expression of Zta and HIF-1α was analyzed by Western blotting. ( i ) Wild-type and HIF-1α knockout HA cells were treated with 20 μM C7 for 24 h. The expression of Zta, EA-D, and HIF-1α was analyzed by Western blotting. Cellular α-tubulin was detected as a loading control.

    Journal: Cancers

    Article Title: Intracellular Iron Chelation by a Novel Compound, C7, Reactivates Epstein–Barr Virus (EBV) Lytic Cycle via the ERK-Autophagy Axis in EBV-Positive Epithelial Cancers

    doi: 10.3390/cancers10120505

    Figure Lengend Snippet: EBV lytic reactivation by C7 requires the activation of ERK and hypoxia signaling pathways. ( a ) Gene sets enriched with a false discovery rate (FDR) smaller than 0.05 were subject to leading edge analysis to obtain a leading edge subset of genes. Clustered map of leading edge subsets upregulated in C7_24 h treatment compared to solvent control. Degree of upregulation is shown with color intensity (darker color indicates greater fold change). ( b ) AGS-BX1 cells were incubated with 10 μM C7 for 1, 2, 4, 8, 16, 24 and 48 h and the expression of Zta, Rta, and HIF-1α was examined by Western blotting. ( c ) SNU-719 cells were incubated with 20 μM C7 for 8, 16, 24, 48 and 72 h and the expression ofZta, Rta, and HIF-1α was examined by Western blotting. ( d ) AGS-BX1 cells were pretreated with 50 μM PD98059 (mitogen-activated protein kinase kinase (MEK) inhibitor) for 1 h, and then incubated with 10 μM M C7 or Dp44mT for 48 h. The expression of Zta, Rta, p-ERK1/2, and HIF-1α was analyzed by Western blotting. ( e ) AGS-BX1 cells were treated with either iron-precomplexed C7 or Dp44mT for 48 h. The expression of Zta, Rta, p-ERK1/2 and HIF-1α was analyzed by Western blotting. ( f ) AGS-BDneo cells were either untreated or treated with 1000 μM deferoxamine, 20 μM C7, or 150 μM CoCl 2 for 48 h. The expression of Zta and HIF-1α was analyzed by Western blotting. ( g ) HA cells were untreated, or treated with either 1000 μM deferoxamine, 20 μM C7, or 150 μM CoCl 2 for 48 h. The expression of Zta and HIF-1α was analyzed by Western blotting. ( h ) HA cells were either untreated or treated with either 20 μM C7, 100, 150 or 500 μM CoCl 2 for 48 h. The expression of Zta and HIF-1α was analyzed by Western blotting. ( i ) Wild-type and HIF-1α knockout HA cells were treated with 20 μM C7 for 24 h. The expression of Zta, EA-D, and HIF-1α was analyzed by Western blotting. Cellular α-tubulin was detected as a loading control.

    Article Snippet: Chemical compounds used: deferoxamine (Novartis NDC 0078-0467-91, Basel, Switzerland), deferiprone (ApoPharma, Toronto, ON, Canada), deferasirox (Novartis), Dp44mT (gift from Prof. Des Richardson, University of Sydney, Australia), C7, C7-1, C7-2, C7-3, C7-4, C7-5, C7-6 (ChemBridge, ID#5632947, ID#5636413, ID#6120380, ID#5631431, ID#5335854, ID#5630707, and ID#5636784, respectively), PD98059 (Merck 513000, Kenilworth, NJ, USA), romidepsin (Selleck S3020, Houston, TX, USA), 3-MA (Sigma Aldrich M9281, St. Louis, MO, USA), and chloroquine (Sigma Aldrich C6628).

    Techniques: Activation Assay, Incubation, Expressing, Western Blot, Knock-Out

    C7 and iron chelators reactivate EBV lytic cycle via intracellular iron chelation and activation of the ERK-autophagy axis. ( a ) HA cells were incubated with either 20 μM C7 or 20 μM iron-precomplexed C7 for 48 h. Expression of Zta (red signals) and LC3B (green signals) was analyzed by immunofluorescence staining. DAPI (blue signals) stained cell nuclei. Scale Bar: 250 μm. ( b ) AGS-BDneo cells were incubated with either 20 μM C7, 20 μM iron-precomplexed C7, 1000 μM deferoxamine, or 1000 μM iron-precomplexed deferoxamine for 48 h. Expression of Zta (red signals) and LC3B (green signals) was analyzed by immunofluorescent staining. DAPI (blue signals) stained cell nuclei. Scale Bar: 250 μm. ( c ) AGS-BDneo cells were treated with either 20 μM C7, 20 μM iron-precomplexed C7, 20 μM Dp44mT, or 20 μM iron-precomplexed Dp44mT for 48 h. The expression of Zta and LC3B was analyzed by Western blotting. ( d ) HA cells were incubated with either 20 μM C7 or 20 μM C7 in combination with 50 μM PD98059 (MEK inhibitor) for 48 h. Expression of Zta (red signals) and LC3B (green signals) was analyzed by immunofluorescent staining. DAPI (blue signals) stained cell nuclei. Scale Bar: 250 μm. ( e ) AGS-BDneo cells were treated with either 20 μM C7, 20 μM C7 in combination with 10 μM chloroquine, 20 μM C7 in combination with 50 μM PD98059 (MEK inhibitor), 20 μM C7 in combination with 50 μM PD98059 (MEK inhibitor), or 10 μM chloroquine for 48 h. The expression of Zta and LC3B was analyzed by Western blotting. ( f ) HA cells were treated with either 20 μM C7, 20 μM C7 in combination with 5mM 3-MA, or 20 μM Dp44mT or 20 μM Dp44mT in combination with 5 mM 3-MA for 48 h. The expression of Zta and LC3B was analyzed by Western blotting. ( g ) Wild-type, atg5 knockdown and scramble control knockdown HA cells were treated with 20 μM C7 for 48 h. The expression of phosphorylated-c-Jun, phosphorylated-ERK1/2, ATG5, and Zta was analyzed by Western blotting. ( h ) SNU-719 cells were treated with either 20 μM C7, 20 μM C7 in combination with 50 μM PD98059, 50 μM PD98059 alone, 20 μM iron-precomplexed C7, 20 μM C7 in combination with 3-MA, or 3-MA alone for 48 h. The expression of HIF-1α, p-ERK1/2, Zta, and LC3B was analyzed by Western blotting. ( i ) Schematic illustration of EBV lytic reactivation via the proposed intracellular iron chelation-ERK-autophagy axis.

    Journal: Cancers

    Article Title: Intracellular Iron Chelation by a Novel Compound, C7, Reactivates Epstein–Barr Virus (EBV) Lytic Cycle via the ERK-Autophagy Axis in EBV-Positive Epithelial Cancers

    doi: 10.3390/cancers10120505

    Figure Lengend Snippet: C7 and iron chelators reactivate EBV lytic cycle via intracellular iron chelation and activation of the ERK-autophagy axis. ( a ) HA cells were incubated with either 20 μM C7 or 20 μM iron-precomplexed C7 for 48 h. Expression of Zta (red signals) and LC3B (green signals) was analyzed by immunofluorescence staining. DAPI (blue signals) stained cell nuclei. Scale Bar: 250 μm. ( b ) AGS-BDneo cells were incubated with either 20 μM C7, 20 μM iron-precomplexed C7, 1000 μM deferoxamine, or 1000 μM iron-precomplexed deferoxamine for 48 h. Expression of Zta (red signals) and LC3B (green signals) was analyzed by immunofluorescent staining. DAPI (blue signals) stained cell nuclei. Scale Bar: 250 μm. ( c ) AGS-BDneo cells were treated with either 20 μM C7, 20 μM iron-precomplexed C7, 20 μM Dp44mT, or 20 μM iron-precomplexed Dp44mT for 48 h. The expression of Zta and LC3B was analyzed by Western blotting. ( d ) HA cells were incubated with either 20 μM C7 or 20 μM C7 in combination with 50 μM PD98059 (MEK inhibitor) for 48 h. Expression of Zta (red signals) and LC3B (green signals) was analyzed by immunofluorescent staining. DAPI (blue signals) stained cell nuclei. Scale Bar: 250 μm. ( e ) AGS-BDneo cells were treated with either 20 μM C7, 20 μM C7 in combination with 10 μM chloroquine, 20 μM C7 in combination with 50 μM PD98059 (MEK inhibitor), 20 μM C7 in combination with 50 μM PD98059 (MEK inhibitor), or 10 μM chloroquine for 48 h. The expression of Zta and LC3B was analyzed by Western blotting. ( f ) HA cells were treated with either 20 μM C7, 20 μM C7 in combination with 5mM 3-MA, or 20 μM Dp44mT or 20 μM Dp44mT in combination with 5 mM 3-MA for 48 h. The expression of Zta and LC3B was analyzed by Western blotting. ( g ) Wild-type, atg5 knockdown and scramble control knockdown HA cells were treated with 20 μM C7 for 48 h. The expression of phosphorylated-c-Jun, phosphorylated-ERK1/2, ATG5, and Zta was analyzed by Western blotting. ( h ) SNU-719 cells were treated with either 20 μM C7, 20 μM C7 in combination with 50 μM PD98059, 50 μM PD98059 alone, 20 μM iron-precomplexed C7, 20 μM C7 in combination with 3-MA, or 3-MA alone for 48 h. The expression of HIF-1α, p-ERK1/2, Zta, and LC3B was analyzed by Western blotting. ( i ) Schematic illustration of EBV lytic reactivation via the proposed intracellular iron chelation-ERK-autophagy axis.

    Article Snippet: Chemical compounds used: deferoxamine (Novartis NDC 0078-0467-91, Basel, Switzerland), deferiprone (ApoPharma, Toronto, ON, Canada), deferasirox (Novartis), Dp44mT (gift from Prof. Des Richardson, University of Sydney, Australia), C7, C7-1, C7-2, C7-3, C7-4, C7-5, C7-6 (ChemBridge, ID#5632947, ID#5636413, ID#6120380, ID#5631431, ID#5335854, ID#5630707, and ID#5636784, respectively), PD98059 (Merck 513000, Kenilworth, NJ, USA), romidepsin (Selleck S3020, Houston, TX, USA), 3-MA (Sigma Aldrich M9281, St. Louis, MO, USA), and chloroquine (Sigma Aldrich C6628).

    Techniques: Activation Assay, Incubation, Expressing, Immunofluorescence, Staining, Western Blot

    Cell cycle distribution of nucleus pulposus cells . Serum-deprived nucleus pulposus cells were cultured with no supplements for 24 h (a). The cells were treated with 5 ng/mL transforming growth factor β1 (TGFβ1) for 24 h (b). At 2 h before the addition of TGFβ1, the cells were treated with 16 μM 10058-F4 (c), or with 30 μM PD98059 (d). The cells were harvested 24 h after the addition of TGFβ1 and the nuclei were stained with propidium iodide. DNA histograms were generated using flow cytometry. Each plot represents the analysis of 10,000 events. The histograms present typical results and the percentage of cells in G 0 /G 1 , S and G 2 /M phases are shown as the average of duplicated measurements.

    Journal: Arthritis Research & Therapy

    Article Title: Synergistic role of c-Myc and ERK1/2 in the mitogenic response to TGF?-1 in cultured rat nucleus pulposus cells

    doi: 10.1186/ar2567

    Figure Lengend Snippet: Cell cycle distribution of nucleus pulposus cells . Serum-deprived nucleus pulposus cells were cultured with no supplements for 24 h (a). The cells were treated with 5 ng/mL transforming growth factor β1 (TGFβ1) for 24 h (b). At 2 h before the addition of TGFβ1, the cells were treated with 16 μM 10058-F4 (c), or with 30 μM PD98059 (d). The cells were harvested 24 h after the addition of TGFβ1 and the nuclei were stained with propidium iodide. DNA histograms were generated using flow cytometry. Each plot represents the analysis of 10,000 events. The histograms present typical results and the percentage of cells in G 0 /G 1 , S and G 2 /M phases are shown as the average of duplicated measurements.

    Article Snippet: For experiments using pathway specific inhibitors, appropriate concentrations of 10058-F4 or PD98059 were added to the medium as concentrated stock solutions dissolved in dimethyl sulfoxide (DMSO, Wako).

    Techniques: Cell Culture, Staining, Generated, Flow Cytometry, Cytometry

    Time course study of c-Myc and phospho-extracellular signal regulated kinase (ERK)1/2 expression by western blot analysis . Serum-deprived nucleus pulposus cells were treated with or without 16 μM 10058-F4 or 30 μM PD98059 before the addition of 5 ng/mL transforming growth factor β1 (TGFβ1). The cells were harvested at the times indicated and lysed. Aliquots of the lysates were electrophoresed on 5% to 20% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE). The protein bands were blotted to a polyvinylidene diflouride (PVDF) membrane and probed with antibodies against c-Myc, total ERK1/2 and phospho-ERK1/2. β-Actin was used as a quantity loading control. (a) TGFβ1 treatment induced immediate phosphorylation of ERK1/2 with robust c-Myc expression for 2 h. The expression of c-Myc, phospho-ERK1/2, and total ERK1/2 were detected throughout the experimental period. The right lane indicates the result of 24 h treatment with 10% FBS; c-Myc and phospho-ERK1/2 appear distinctly. (b) Pretreatment with ERK1/2 inhibitor 30 μM PD98059 diminished the expression of c-Myc and interrupted the phosphorylation of ERK1/2. Note that a single isoform corresponding to phospho-ERK2 was detected at all times. (c) Pretreatment with c-Myc inhibitor 16 μM 10058-F4 diminished c-Myc expression and limited ERK1/2 phosphorylation for a short time under TGFβ1 stimulation. Graphs show relative intensities in expression of c-Myc normalized to β-actin levels and in expression of phospho-ERK1/2 normalized to total ERK1/2 levels, respectively.

    Journal: Arthritis Research & Therapy

    Article Title: Synergistic role of c-Myc and ERK1/2 in the mitogenic response to TGF?-1 in cultured rat nucleus pulposus cells

    doi: 10.1186/ar2567

    Figure Lengend Snippet: Time course study of c-Myc and phospho-extracellular signal regulated kinase (ERK)1/2 expression by western blot analysis . Serum-deprived nucleus pulposus cells were treated with or without 16 μM 10058-F4 or 30 μM PD98059 before the addition of 5 ng/mL transforming growth factor β1 (TGFβ1). The cells were harvested at the times indicated and lysed. Aliquots of the lysates were electrophoresed on 5% to 20% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE). The protein bands were blotted to a polyvinylidene diflouride (PVDF) membrane and probed with antibodies against c-Myc, total ERK1/2 and phospho-ERK1/2. β-Actin was used as a quantity loading control. (a) TGFβ1 treatment induced immediate phosphorylation of ERK1/2 with robust c-Myc expression for 2 h. The expression of c-Myc, phospho-ERK1/2, and total ERK1/2 were detected throughout the experimental period. The right lane indicates the result of 24 h treatment with 10% FBS; c-Myc and phospho-ERK1/2 appear distinctly. (b) Pretreatment with ERK1/2 inhibitor 30 μM PD98059 diminished the expression of c-Myc and interrupted the phosphorylation of ERK1/2. Note that a single isoform corresponding to phospho-ERK2 was detected at all times. (c) Pretreatment with c-Myc inhibitor 16 μM 10058-F4 diminished c-Myc expression and limited ERK1/2 phosphorylation for a short time under TGFβ1 stimulation. Graphs show relative intensities in expression of c-Myc normalized to β-actin levels and in expression of phospho-ERK1/2 normalized to total ERK1/2 levels, respectively.

    Article Snippet: For experiments using pathway specific inhibitors, appropriate concentrations of 10058-F4 or PD98059 were added to the medium as concentrated stock solutions dissolved in dimethyl sulfoxide (DMSO, Wako).

    Techniques: Expressing, Western Blot, Polyacrylamide Gel Electrophoresis, SDS Page

    Western blot analysis of cell cycle regulators . After 24 h incubation in a medium containing 2% fetal bovine serum (FBS), this medium was replaced with medium containing 0.5% FBS. Nucleus pulposus cells were cultured with no supplements for an additional 24 h (a). The cells were treated with 5 ng/mL transforming growth factor β1 (TGFβ1) for 24 h (b). At 2 h before the addition of TGFβ1, the cells were treated with 16 μM 10058-F4 (c), or with 30 μM PD98059 (d). The cells were harvested 24 h after the TGFβ1 treatment and lysed. Aliquots of the lysates were electrophoresed on 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein bands were blotted to a polyvinylidene diflouride (PVDF) membrane and probed with antibodies against c-Myc, p15, p21, and p27. β-Actin was used as a quantity loading control. Treatment with TGFβ1 without inhibitors (b) did not abolish c-Myc expression but decreased the level of cyclin-dependent kinase inhibitors (CKIs) (p21, p27) compared to the control, while treatments with inhibitors (c, d) diminished c-Myc and upregulated p21 and p27. In contrast, p15 levels were unchanged by any of these treatments.

    Journal: Arthritis Research & Therapy

    Article Title: Synergistic role of c-Myc and ERK1/2 in the mitogenic response to TGF?-1 in cultured rat nucleus pulposus cells

    doi: 10.1186/ar2567

    Figure Lengend Snippet: Western blot analysis of cell cycle regulators . After 24 h incubation in a medium containing 2% fetal bovine serum (FBS), this medium was replaced with medium containing 0.5% FBS. Nucleus pulposus cells were cultured with no supplements for an additional 24 h (a). The cells were treated with 5 ng/mL transforming growth factor β1 (TGFβ1) for 24 h (b). At 2 h before the addition of TGFβ1, the cells were treated with 16 μM 10058-F4 (c), or with 30 μM PD98059 (d). The cells were harvested 24 h after the TGFβ1 treatment and lysed. Aliquots of the lysates were electrophoresed on 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein bands were blotted to a polyvinylidene diflouride (PVDF) membrane and probed with antibodies against c-Myc, p15, p21, and p27. β-Actin was used as a quantity loading control. Treatment with TGFβ1 without inhibitors (b) did not abolish c-Myc expression but decreased the level of cyclin-dependent kinase inhibitors (CKIs) (p21, p27) compared to the control, while treatments with inhibitors (c, d) diminished c-Myc and upregulated p21 and p27. In contrast, p15 levels were unchanged by any of these treatments.

    Article Snippet: For experiments using pathway specific inhibitors, appropriate concentrations of 10058-F4 or PD98059 were added to the medium as concentrated stock solutions dissolved in dimethyl sulfoxide (DMSO, Wako).

    Techniques: Western Blot, Incubation, Cell Culture, Polyacrylamide Gel Electrophoresis, SDS Page, Expressing

    The inhibition of extracellular signal regulated kinase (ERK)1/2 phosphorylation prevents transforming growth factor β1 (TGFβ1)-stimulated cell proliferation . Serum-deprived cells in 96-well plates were treated with 5 or 20 ng/mL TGFβ1 (abbreviated to T) with or without 10, 20, 30 μM PD98059. Cell proliferation was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on day 3 after treatment. Five replicates per experimental condition were made. Data are normalized to values obtained for untreated cells cultured in 0.5% serum containing medium and represented as mean ± standard error of the mean (SEM) (*p

    Journal: Arthritis Research & Therapy

    Article Title: Synergistic role of c-Myc and ERK1/2 in the mitogenic response to TGF?-1 in cultured rat nucleus pulposus cells

    doi: 10.1186/ar2567

    Figure Lengend Snippet: The inhibition of extracellular signal regulated kinase (ERK)1/2 phosphorylation prevents transforming growth factor β1 (TGFβ1)-stimulated cell proliferation . Serum-deprived cells in 96-well plates were treated with 5 or 20 ng/mL TGFβ1 (abbreviated to T) with or without 10, 20, 30 μM PD98059. Cell proliferation was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on day 3 after treatment. Five replicates per experimental condition were made. Data are normalized to values obtained for untreated cells cultured in 0.5% serum containing medium and represented as mean ± standard error of the mean (SEM) (*p

    Article Snippet: For experiments using pathway specific inhibitors, appropriate concentrations of 10058-F4 or PD98059 were added to the medium as concentrated stock solutions dissolved in dimethyl sulfoxide (DMSO, Wako).

    Techniques: Inhibition, MTT Assay, Cell Culture

    Corylin reduces VCAM-1 expression in TNF-α-treated VSMCs through ROS/MAP kinase inhibition. VSMCs were treated with 20 µM corylin, 2 mM NAC or 10 µM DPI for 1 h and then incubated with or without 10 ng/mL TNF-α for 15 min. ( A ) Phosphorylated P38, ERK, and JNK levels were examined by Immunoblot analysis. Total ERK (T-ERK), total P38 (T-P38), and total JNK (T-JNK) were used as an internal control for sample loading. ( B ) VSMCs were pretreated (1 h) with corylin (20 µM), a P38 inhibitor (SB203580; SB; 10, 20, or 30 µM), PD98059 (PD; 10, 20, or 30 µM), or a JNK inhibitor (SP600125; SP; 10, 20, or 30 nM) and then treated with 10 ng/mL TNF-α for 24 h. Western blot analysis for VCAM-1 and quantification of VCAM-1 to GAPDH in VSMCs. ( C – E ) After P38, ERK and JNK silencing, cells were treated with TNF-α and corylin. VCAM-1 expression was determined by Western blot analysis. Total P38 (T-P38), total ERK (T-ERK), and total JNK (T-JNK) proteins were used to examine the siRNA effects. GAPDH was used as an internal control for sample loading. ( F , G ) Confluent VSMCs were pretreated (1 h) with 20 µM corylin (C20), 30 µM SB203580 (SB), 30 µM PD98059 (PD), 30 nM SP600125 (SP), 10 µM parthenolide (PAR), and 1 µg/mL anti-VCAM-1 antibody or IgG and then treated with 10 ng/mL TNF-α (T) for 24 h. BCECF/AM-labeled U937 cells were added and cocultured for another 1 h. The adherent U937 cells were counted to evaluate the overall VCAM-1 expression level. The data are shown as the mean ± SD. * p

    Journal: Antioxidants

    Article Title: Corylin Inhibits Vascular Cell Inflammation, Proliferation and Migration and Reduces Atherosclerosis in ApoE-Deficient Mice

    doi: 10.3390/antiox9040275

    Figure Lengend Snippet: Corylin reduces VCAM-1 expression in TNF-α-treated VSMCs through ROS/MAP kinase inhibition. VSMCs were treated with 20 µM corylin, 2 mM NAC or 10 µM DPI for 1 h and then incubated with or without 10 ng/mL TNF-α for 15 min. ( A ) Phosphorylated P38, ERK, and JNK levels were examined by Immunoblot analysis. Total ERK (T-ERK), total P38 (T-P38), and total JNK (T-JNK) were used as an internal control for sample loading. ( B ) VSMCs were pretreated (1 h) with corylin (20 µM), a P38 inhibitor (SB203580; SB; 10, 20, or 30 µM), PD98059 (PD; 10, 20, or 30 µM), or a JNK inhibitor (SP600125; SP; 10, 20, or 30 nM) and then treated with 10 ng/mL TNF-α for 24 h. Western blot analysis for VCAM-1 and quantification of VCAM-1 to GAPDH in VSMCs. ( C – E ) After P38, ERK and JNK silencing, cells were treated with TNF-α and corylin. VCAM-1 expression was determined by Western blot analysis. Total P38 (T-P38), total ERK (T-ERK), and total JNK (T-JNK) proteins were used to examine the siRNA effects. GAPDH was used as an internal control for sample loading. ( F , G ) Confluent VSMCs were pretreated (1 h) with 20 µM corylin (C20), 30 µM SB203580 (SB), 30 µM PD98059 (PD), 30 nM SP600125 (SP), 10 µM parthenolide (PAR), and 1 µg/mL anti-VCAM-1 antibody or IgG and then treated with 10 ng/mL TNF-α (T) for 24 h. BCECF/AM-labeled U937 cells were added and cocultured for another 1 h. The adherent U937 cells were counted to evaluate the overall VCAM-1 expression level. The data are shown as the mean ± SD. * p

    Article Snippet: PD98059 was purchased from LC Labs (Woburn, MA, USA).

    Techniques: Expressing, Inhibition, Incubation, Western Blot, Labeling

    Corylin reduces the activation of NF-κB p65 in TNF-α-treated HUVECs and VSMCs. ( A ) HUVECs were pretreated (1 h) with 20 µM corylin (C20) or 30 nM SP600125 (SP30) and then incubated with 10 ng/mL TNF-α (T) for 15 min. VSMCs were pretreated (1 h) with 20 µM corylin, 30 µM SB203580 (SB30), 30 µM PD98059 (PD30), or 30 nM SP600125 (SP30) and then incubated with 10 ng/mL TNF-α (T) for 15 min. Immunofluorescence staining was performed to examine the nuclear localization and expression level of phosphorylated NF-κB p65 (P-p65). Nuclei were labeled with DAPI (blue). Scale bar = 25 μm. ( B , C ) HUVECs and VSMCs were transfected with NF-κB p65 luciferase reporter constructs, pretreated (1 h) with 20 µM corylin (C20), 30 µM SB203580 (SB30), 30 µM PD98059 (PD30), or 30 nM SP600125 (SP30) and then incubated with 10 ng/mL TNF-α for 6 h. Total cell lysates were collected, and luciferase activity was detected. The data are shown as the mean ± SD. * p

    Journal: Antioxidants

    Article Title: Corylin Inhibits Vascular Cell Inflammation, Proliferation and Migration and Reduces Atherosclerosis in ApoE-Deficient Mice

    doi: 10.3390/antiox9040275

    Figure Lengend Snippet: Corylin reduces the activation of NF-κB p65 in TNF-α-treated HUVECs and VSMCs. ( A ) HUVECs were pretreated (1 h) with 20 µM corylin (C20) or 30 nM SP600125 (SP30) and then incubated with 10 ng/mL TNF-α (T) for 15 min. VSMCs were pretreated (1 h) with 20 µM corylin, 30 µM SB203580 (SB30), 30 µM PD98059 (PD30), or 30 nM SP600125 (SP30) and then incubated with 10 ng/mL TNF-α (T) for 15 min. Immunofluorescence staining was performed to examine the nuclear localization and expression level of phosphorylated NF-κB p65 (P-p65). Nuclei were labeled with DAPI (blue). Scale bar = 25 μm. ( B , C ) HUVECs and VSMCs were transfected with NF-κB p65 luciferase reporter constructs, pretreated (1 h) with 20 µM corylin (C20), 30 µM SB203580 (SB30), 30 µM PD98059 (PD30), or 30 nM SP600125 (SP30) and then incubated with 10 ng/mL TNF-α for 6 h. Total cell lysates were collected, and luciferase activity was detected. The data are shown as the mean ± SD. * p

    Article Snippet: PD98059 was purchased from LC Labs (Woburn, MA, USA).

    Techniques: Activation Assay, Incubation, Immunofluorescence, Staining, Expressing, Labeling, Transfection, Luciferase, Construct, Activity Assay