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  • 99
    Millipore sp600125
    AdoR promotes transcription of TNF Egr in polarity-deficient epithelial cells. a sal > ent2 , AdoR expression enhances TNF egr transcript levels in the wing disc independently of <t>JNK</t> signalling. TNFR grnd expression, measured by RT-qPCR, is shown as a control. Fold changes are relative to rp49 , n = 6. b – d sal > ent2, AdoR expression upregulates expression of Egr-GFP from a genomic fosmid ( b , c ). This effect is not rescued by co-expression of JNK bskDN ( d ). The arrowhead in b indicates a suspected position effect of the attP site (VK00033) where the fosmid was integrated. e CADO treatment increases expression of TNF egr in explanted wild-type discs, but not in AdoR mutant discs. This effect is further enhanced by Ador overexpression. TNFR grnd expression, measured by RT-qPCR, is shown as a control. Fold changes are relative to rp49 , n ≥ 5. f en > scrib Ri discs upregulate TNF egr mRNA in an AdoR/Ent2-dependent and JNK-independent fashion. Fold changes are relative to rp49 , n ≥ 4. g Human HaCaT cells treated with adenosine upregulates TNF-α transcript levels (as assayed by qRT-PCR). Fold changes are relative to GAPDH , n = 5. Expression of PKG1 , a housekeeping gene and survivin , a JNK target gene, are also shown. h The effect shown in g was suppressed by three different adenosine receptor antagonists (CGS15943, SCH58261 or Caffeine) and by a PKA inhibitor cocktail (Merck # 20-114), but not by JNK inhibitor <t>SP600125.</t> Fold changes are relative to GAPDH , n ≥ 3 and expression of PKG1 is shown. i , j AdoR mutants live longer than wild-type flies when continuously fed with a low dose of Bleomycin ( i ) but live shorter than wild-type flies during acute desiccation (dry starvation, j ). **** P
    Sp600125, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7242 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Selleck Chemicals sp600125
    BMP14 induces tenogenic signaling in BMSCs via the JNK and Smad1 pathway in a Sirt1-dependent manner. (A) BMSC cells were infected with Sirt1 overexpressing or shSirt1 lentivirus. Following treatment with 50 ng/ml BMP14, the cell extracts were blotted with anti-P-p38, p38, P-JNK, JNK, P-smad2/3, smad2/3, P-smad1 and smad1 antibodies. Quantitative analysis of the blots was performed using ImageJ software and the relative ratios are indicated above the blots. (B) mRNA levels of Scx were detected using RT-qPCR. (C) BMSC cells were treated with 50 ng/ml BMP14 plus 1 µM LDN-193189 and <t>SP600125.</t> Following 48 h the cells were harvested and cell extracts were immunoprecipitated with PPARγ antibodies; the blots were immunoblotted with acetyl-lys and PPARγ antibodies. The total cell lysate was immunoblotted with anti-β-actin antibody. β-actin was used as the control. BMSC cells were treated with 50 ng/ml BMP14 with or without 1 µM LDN-193189 or SP600125. (D) The protein expression of Scx, collagen I, collagen III and Sirt1 in BMSC cells were detected by western blot analysis. β-actin was used as the loading control. The data are presented as the mean ± standard error of the mean. *P
    Sp600125, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 799 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Tocris sp600125
    FAF1-mediated JNK1 activation triggers mitochondrial dysfunction upon ischemic stress. a Faf1 +/+ MEFs were treated with oxygen glucose deprivation for the indicated times and then fractionated into cytoplasmic and mitochondrial fractions. The fractions were analyzed by immunoblotting with anti-P-JNK, JNK1, anti-COX IV (mitochondrial marker) and anti-β-actin (cytoplasmic marker) antibodies. b Faf1 +/+ and Faf1 gt/gt MEFs were treated with oxygen glucose deprivation for 2 h and then fractionated into cytoplasmic and mitochondrial fractions. The fractions were analyzed by immunoblotting with anti-P-JNK, JNK1, anti-COX IV and anti-β-actin antibodies. c Upper panel: Faf1 +/+ and Faf1 gt/gt MEFs were transfected with DsRed-Mito plasmid for 48 h. Subsequently, the cells were treated with oxygen glucose deprivation in the presence or absence of 20 μM <t>SP600125</t> for 2 h. Fluorescence images show the alterations in mitochondrial morphology . Scale bar = 20 μm. Bottom panel: the graph shows the quantification of mitochondrial fragmentation ( n = 3). d Faf1 +/+ and Faf1 gt/gt MEFs were untreated or treated with oxygen glucose deprivation in the presence or absence of 20 μM SP600125 for 5 h. Mitochondrial superoxide was stained using 10 μM MitoSOX reagent. The population of MitoSOX-positive cells was detected by flow cytometry. The graph shows the results of the quantitative analysis of MitoSOX-positive cells ( n = 3). e Faf1 +/+ and Faf1 gt/gt MEFs were untreated or treated with oxygen glucose deprivation in the presence or absence of 20 μM SP600125 for 5 h, and the mitochondrial potential was measured using a Muse analyzer. The graph was obtained from a quantitative analysis of depolarized cells ( n = 3). The data ( c - e ) are expressed as the mean ± S.E.M. of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Tukey’s HSD ( c - e ) post hoc analysis. *** P
    Sp600125, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 713 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc sp600125
    FSTL1 modulates myofibroblast differentiation by facilitating p38/JNK signaling. (a and b) Primary lung fibroblasts from Fstl1+/− and their WT littermates were treated with 5 ng/ml TGF-β1. (a) Immunofluorescence staining of α-SMA in lung fibroblasts. (b) Protein expression levels of α-SMA and type I collagen. (c-e) MLgs were pretreated with U0126, SB202190, and <t>SP600125.</t> Protein expression levels of α-SMA and type I collagen were detected by Western blotting. n = 4; Bars = 100 μm. * P
    Sp600125, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 638 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH sp600125
    Lack of JNK1 and JNK2 affects axonal elongation rate. A , Mean speed of growth cone advancement and retraction of wild-type and JNK-deficient DRG neurons. Note that jnk1 −/− and jnk2 −/− , but not jnk3 −/− , neurite outgrowth rates are significantly decreased compared with wild type. After <t>SP600125</t> application, the retraction rate of jnk2 −/− neurites is decreased compared with jnk3 −/− and wild-type neurites, and no retraction of jnk1 −/− neurites occurs. B , Advancement or retraction of growth cones measured 90 min before and 90 min after SP600125 application. Within a few minutes after JNK inhibition, neurites from jnk2 −/− and jnk3 −/− neurons retract, whereas jnk1 −/− neurites continue growing during the remaining recording period. Response time for growth cones to collapse after SP600125 application: jnk2 −/− and jnk3 −/− growth cones collapse rapidly, in contrast to jnk1 −/− growth cones. D–F , Frames of time-lapse recordings illustrating the outgrowth and typical response of neurites to JNK inhibition from jnk1 −/− , jnk2 −/− , and jnk3 −/− DRG neurons, 90 min before and after SP600125 addition. Arrowheads, Growth cones. Scale bars, 50 μm. *** p
    Sp600125, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 317 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Beyotime sp600125
    Blocking the MAPK signaling pathway diminished LPS-induced SLC15A3 upregulation. Mouse PMs (left panel) and BMDMs (right panel) were pretreated with or without 50 μM U0126, 10 μM SB203085 (SB203) and 10 μM <t>SP600125</t> (SP600) for 1 h, then the cells were treated with or without 100 ng/mL LPS for another 4 h. mRNA expression of Slc15a3 ( a , b ), Il-6 ( c , d ) and Tnf-α ( e , f ) were determined by real-time PCR. One-way ANOVA followed by Tukey’s test was used to evaluate the statistical differences, * P
    Sp600125, supplied by Beyotime, used in various techniques. Bioz Stars score: 93/100, based on 427 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Signal Pharmaceuticals sp600125
    Blocking the MAPK signaling pathway diminished LPS-induced SLC15A3 upregulation. Mouse PMs (left panel) and BMDMs (right panel) were pretreated with or without 50 μM U0126, 10 μM SB203085 (SB203) and 10 μM <t>SP600125</t> (SP600) for 1 h, then the cells were treated with or without 100 ng/mL LPS for another 4 h. mRNA expression of Slc15a3 ( a , b ), Il-6 ( c , d ) and Tnf-α ( e , f ) were determined by real-time PCR. One-way ANOVA followed by Tukey’s test was used to evaluate the statistical differences, * P
    Sp600125, supplied by Signal Pharmaceuticals, used in various techniques. Bioz Stars score: 92/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical sp600125
    JNK contributes to chronic morphine-induced activation of presynaptic NMDARs at primary afferent terminals. A , representative recordings and cumulative plots show a lack of effect of 50 μM AP5 on the frequency and amplitude of mEPSCs in a lamina II neuron (the spinal cord slice was pretreated with 40 μM <t>SP600125</t> for 1 h) in a morphine-treated rat. B , mean changes in the frequency and amplitude of mEPSCs in lamina II neurons show a lack of effect of AP5 on the frequency and amplitude of mEPSCs in spinal cord slices pretreated with SP600125 from morphine-treated rats (n = 10 neurons from 3 rats). C , representative current traces show a lack of effect of 50 μM AP5 on the amplitude of evoked monosynaptic EPSCs in a lamina II neuron from spinal cord slices pretreated with SP600125 in a morphine-treated rat. D , original recording traces show the lack of effect of AP5 on the PPR of evoked EPSCs in spinal cord slices pretreated with SP600125 in a morphine-treated rat. E , summary data show the lack of effect of 50 μM AP5 on the amplitude ( n = 10 neurons from 3 rats) and PPR ( n = 9 neurons from 3 rats) of monosynaptic EPSCs in lamina II neurons from spinal cord slices pretreated with SP600125 in morphine-treated rats. Data are presented as means ± SEM.
    Sp600125, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    LC Laboratories sp600125
    Effect of <t>SP600125</t> on proliferation, viability, polyploidy, expression of cyclin B1, cyclin D3, c-Myc, and survivin, and the translation-related proteins of Dami and CMK cells. Dami and CMK cells were seeded at 2×10 5 /ml in RPMI 1640 medium containing 10% FCS and treated with SP600125 at different concentrations for different periods of time as indicated. Dami and CMK cells treated with DMSO were used as a control. (A, B) The cell number and viability, presented as the mean±SEM, were determined from 4 separate experiments. (C) Representative DNA histograms of SP600125-induced Dami and CMK cells analyzed by flow cytometry. (D) Morphology was analyzed by Wright-Giemsa staining of cytocentrifuged preparations of Dami and CMK cells induced by SP600125 or nocodazole (Original magnification, 1000×). The Dami and CMK cells treated with DMSO or with SP600125 were lysed, and equal amounts of protein were loaded for western blots to evaluate the protein levels of cyclin B1, cyclin D3, c-Myc, and survivin (E), and the phosphorylation and protein levels of S6K1, eIF4E and 4E-BP1(F). β-actin was used as an internal control.
    Sp600125, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    AG Scientific sp600125
    Evaluation of inhibitory effect of <t>SP600125</t> on penetrating corneal wound induced corneal scarring in Wistar rats. A penetrating corneal wound model was created with Wistar rats and inhibition of JNK activation by subconjunctival injection of SP600125 daily post-wounding. (A) HE stained histological sections showed that corneal epithelial healing was almost complete at 3 d in both groups. Subconjunctival injection of SP600125 after injury daily markedly improved the architecture of cornea and reduced scarring and did not have a significant impact on wound stroma healing at 14 d (B), 21 d (C). n = 4 rat in each group, Bars: 40 µm.
    Sp600125, supplied by AG Scientific, used in various techniques. Bioz Stars score: 92/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem sp600125
    Whole cigarette smoke promoted human β-defensin-2 and -3 expression through the ERK1/2 MAP kinase and NFκB signaling pathways. Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor <t>(SP600125),</t> or 10 µM of NFκB inhibitor (IKK-2) for 45 min before exposure to cigarette smoke for 15 or 30 min. Six hours later, total RNA was extracted, and HBD gene expression was analyzed by qRT-PCR (n = 6). *, p
    Sp600125, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology sp600125
    TGF-β-associated gene expression profiles induced by treatment with mitogen-activated protein kinase inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured for 15-60 min in the presence or absence of 10 µ M U0126 (ERK i ) or 10 µ M <t>SP600125</t> (JNK i ) and subjected to western blot analysis for p-ERK1/2. Histone H3 was used as a loading control. Fibroblasts incubated with recombinant human FGF2 for 30 min were used as a positive control for p-ERK1/2 expression. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ) or 10 µ M SB202190 (p38 i ) until day 4. Expression levels of TGF-β-associated transcripts TGFB1 , TGFB2 , TGFBR1 , TGFBR2 and TGFBR3 were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Expression levels per transcript were compared using an one-way analysis of variance and post-hoc Holm-Sidak analysis. * P
    Sp600125, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA sp600125
    <t>SP600125</t> or PMA induce Prss14/epithin ectodomain shedding. A , diagram of Prss14/epithin domain structure and processed forms. Epi-S', Epi-S, aEpi-S are indicated. B , effects of MAP kinase inhibitors in Prss14/epithin shedding. 427.1.86 cells were treated with 10 μ m PD98059 ( PD ), 20 μ m SB203580 ( SB ), and 5 μ m SP600125 ( SP ) for 30 min and then with or without 0.5 μ m PMA for an additional 2 h. C , dose- and time-dependent profiles of Epi-S' and Epi-S. 427.1.86 cells were treated with the indicated concentration of SP600125 for 2 h ( left panel ) and 5 μ m SP600125 up to 2 h ( right panel ). D , 427.1.86 cells were pretreated with 10 μ m TAPI-0 ( TPI ) for 30 min, and then cells were treated with 5 μ m SP600125 or 0.5 μ m PMA for an additional 2 h. The TACE inhibitor abolished the appearance of Epi-S' while retaining Epi-S in the cell, regardless of shedding induction methods, PMA, and SP600125. E , removal of TACE with siRNA abolished shedding of Prss14/epithin. 427.1.86 cells were transfected with 200 n m TACE siRNA for 48 h, starved of serum for 4 h, and then treated with 5 μΜ SP600125 or 0.5 μΜ PMA for 2 h. The control samples were treated exactly the same way except for transfection with nontargeting control siRNA. F , SP600125 dose-dependently induced Epi-S' and aEpi-S in T47D cells. SP600125 was treated for 2 h. G , SP600125 time-dependently (with 5 μ m ) and dose-dependently (for 2 h) induced Epi-S' and aEpi-S in 4T1 cells. In all panels, Epi-S' collected from culture medium and other proteins, including Epi-S, from cell lysates were detected by Western blot analysis. Tubulin or β-actin was used for normalization. TM , transmembrane domain; SEA , sperm protein, enterokinase, and agrin domain; CUB1 , CUB2 , complement subcomponent C1r/C1s domain; 1, 2, 3, 4 LDLRA , low-density lipoprotein receptor class A repeats.
    Sp600125, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam sp600125
    <t>SP600125</t> or PMA induce Prss14/epithin ectodomain shedding. A , diagram of Prss14/epithin domain structure and processed forms. Epi-S', Epi-S, aEpi-S are indicated. B , effects of MAP kinase inhibitors in Prss14/epithin shedding. 427.1.86 cells were treated with 10 μ m PD98059 ( PD ), 20 μ m SB203580 ( SB ), and 5 μ m SP600125 ( SP ) for 30 min and then with or without 0.5 μ m PMA for an additional 2 h. C , dose- and time-dependent profiles of Epi-S' and Epi-S. 427.1.86 cells were treated with the indicated concentration of SP600125 for 2 h ( left panel ) and 5 μ m SP600125 up to 2 h ( right panel ). D , 427.1.86 cells were pretreated with 10 μ m TAPI-0 ( TPI ) for 30 min, and then cells were treated with 5 μ m SP600125 or 0.5 μ m PMA for an additional 2 h. The TACE inhibitor abolished the appearance of Epi-S' while retaining Epi-S in the cell, regardless of shedding induction methods, PMA, and SP600125. E , removal of TACE with siRNA abolished shedding of Prss14/epithin. 427.1.86 cells were transfected with 200 n m TACE siRNA for 48 h, starved of serum for 4 h, and then treated with 5 μΜ SP600125 or 0.5 μΜ PMA for 2 h. The control samples were treated exactly the same way except for transfection with nontargeting control siRNA. F , SP600125 dose-dependently induced Epi-S' and aEpi-S in T47D cells. SP600125 was treated for 2 h. G , SP600125 time-dependently (with 5 μ m ) and dose-dependently (for 2 h) induced Epi-S' and aEpi-S in 4T1 cells. In all panels, Epi-S' collected from culture medium and other proteins, including Epi-S, from cell lysates were detected by Western blot analysis. Tubulin or β-actin was used for normalization. TM , transmembrane domain; SEA , sperm protein, enterokinase, and agrin domain; CUB1 , CUB2 , complement subcomponent C1r/C1s domain; 1, 2, 3, 4 LDLRA , low-density lipoprotein receptor class A repeats.
    Sp600125, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Celgene sp600125
    The JNK cascade is the principal pathway inhibited by Reversine and <t>SP600125.</t> ( A–C ) Gene Ontology analysis of the common target genes (22) of Reversine and SP600125 was done by the Cytoscape associated-plugin ‘ClueGO’. ‘Positive regulation of JNK cascade’ was the most associated term with both Reversine and SP600125 ( A ). Network view of target genes of Reversine and/or SP600125 was performed by Cytoscape and GeneMANIA associated-plugin. Red colors represent the inhibited proteins through the Kinase panel test and JNK related nodes were highlighted in green zones after Reversine ( B ) and SP600125 ( C ) treatment. ( D ) Overexpression and activation of JNK was investigated in RKO colon cancer cell line comparatively to the normal human colon mucosal epithelial cell line NCM460. ( E ) Inactivation of JNK by SP600125 or Reversine in RKO cells was confirmed by Western blot. ( F ) Downstream targets of JNK pathway after treatment with Reversine or SP600125 were checked by western blot.
    Sp600125, supplied by Celgene, used in various techniques. Bioz Stars score: 92/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    InvivoGen sp600125
    The JNK cascade is the principal pathway inhibited by Reversine and <t>SP600125.</t> ( A–C ) Gene Ontology analysis of the common target genes (22) of Reversine and SP600125 was done by the Cytoscape associated-plugin ‘ClueGO’. ‘Positive regulation of JNK cascade’ was the most associated term with both Reversine and SP600125 ( A ). Network view of target genes of Reversine and/or SP600125 was performed by Cytoscape and GeneMANIA associated-plugin. Red colors represent the inhibited proteins through the Kinase panel test and JNK related nodes were highlighted in green zones after Reversine ( B ) and SP600125 ( C ) treatment. ( D ) Overexpression and activation of JNK was investigated in RKO colon cancer cell line comparatively to the normal human colon mucosal epithelial cell line NCM460. ( E ) Inactivation of JNK by SP600125 or Reversine in RKO cells was confirmed by Western blot. ( F ) Downstream targets of JNK pathway after treatment with Reversine or SP600125 were checked by western blot.
    Sp600125, supplied by InvivoGen, used in various techniques. Bioz Stars score: 95/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem jnk inhibitor sp600125
    Roles of MAPKs in FE-induced apoptosis. (A) MCF-7 cells were treated with 820 µg/mL FE for the indicated time periods. <t>JNK,</t> p38, and ERK1/2 MAPKs and their phosphorylated forms were determined by western blotting using specific antibodies. (B) Effects of MAPK inhibitors on apoptosis induced by FE. MCF-7 cells were pretreated with 10 µM <t>SP600125,</t> SB203580, or PD98059 for 1 h and then exposed to 820 µg/mL FE for 48 h (controls were not exposed to FE). After the treatment, annexin V-PI double staining was used to analyze apoptotic cell death using the IN Cell Analyzer 1000. All results were obtained from 3 independent experiments. Differences with p
    Jnk Inhibitor Sp600125, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck & Co sp600125
    Antiviral activity of type I IFN in combination with MAPK inhibitors. HCC (data for HepG2 cells are shown as representative data), PH5CH8, and PHH cells were treated with IFN-α/β (100 IU/ml) in combination with 25 μM <t>SP600125</t> (JNKi)
    Sp600125, supplied by Merck & Co, used in various techniques. Bioz Stars score: 93/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH jnk inhibitor sp600125
    ERK signaling regulates c-Fos expression and posttranslational modifications. (A) PDGF induction of c-Fos expression in NIH 3T3 cells. Cells were grown overnight in the absence of serum and then stimulated with PDGF (20 ng/ml) for the indicated times. Nuclear fractions were collected, and c-Fos was detected by Western blotting. (B) ERK dependency of c-Fos expression. Cells were serum starved overnight, incubated for 30 min in the absence (c) or presence of U0126 (U), <t>SP600125</t> (SP), or SB203580 (SB) (10 μM each), and stimulated with PDGF (20 ng/ml; 2 h), and c-Fos expression in nuclear fractions was determined as described for panel A. (C) ERK dependency of c- fos mRNA expression. Cells were serum starved overnight, incubated for 30 min in the absence or presence of U0126, and stimulated with PDGF (20 ng/ml) for the indicated times. c- fos transcript levels were detected by Northern blotting as described in Materials and Methods. (D) ERK dependency of c-Fos posttranslational modifications. Cells were transiently transfected with pCEFL-AU5-c-Fos (1 μg/well) and grown in serum-free medium overnight after transfection. Cells were stimulated with PDGF (20 ng/ml; 30 min), and total lysates were processed for Western blotting using anti-c-Fos antibodies (top panel). Pretreatment with MAPK inhibitors was performed as described above. Endogenous active (middle panel) and total (bottom panel) ERK expression from the same samples served as controls for PDGF and U0126 effects on ERK phosphorylation. (E) c-Fos phosphorylation induced by PDGF. NIH 3T3 cells were transfected with pCEFL-AU5-c-Fos (1 μg/well), cultured in serum-free medium overnight, and stimulated with PDGF for 10 or 30 min. Cell lysates were subjected to PP2A digestion followed by immunoblotting using anti-c-Fos antibodies (see Materials and Methods). (F) SP600125 (SP) and SB203580 (SB) inhibitory action on <t>JNK</t> and p38 activities. HEK-293T cells were transfected with HA-JNK1 or HA-p38α alone or in combination with their upstream activating kinases (MEKK and MEK3EE, respectively). In vitro kinase assays were performed as described in Materials and Methods in the absence or presence of the indicated final concentrations of the inhibitors. 32 P-labeled substrate (P-ATF2) is indicated. NT, nontransfected cells.
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    AdoR promotes transcription of TNF Egr in polarity-deficient epithelial cells. a sal > ent2 , AdoR expression enhances TNF egr transcript levels in the wing disc independently of JNK signalling. TNFR grnd expression, measured by RT-qPCR, is shown as a control. Fold changes are relative to rp49 , n = 6. b – d sal > ent2, AdoR expression upregulates expression of Egr-GFP from a genomic fosmid ( b , c ). This effect is not rescued by co-expression of JNK bskDN ( d ). The arrowhead in b indicates a suspected position effect of the attP site (VK00033) where the fosmid was integrated. e CADO treatment increases expression of TNF egr in explanted wild-type discs, but not in AdoR mutant discs. This effect is further enhanced by Ador overexpression. TNFR grnd expression, measured by RT-qPCR, is shown as a control. Fold changes are relative to rp49 , n ≥ 5. f en > scrib Ri discs upregulate TNF egr mRNA in an AdoR/Ent2-dependent and JNK-independent fashion. Fold changes are relative to rp49 , n ≥ 4. g Human HaCaT cells treated with adenosine upregulates TNF-α transcript levels (as assayed by qRT-PCR). Fold changes are relative to GAPDH , n = 5. Expression of PKG1 , a housekeeping gene and survivin , a JNK target gene, are also shown. h The effect shown in g was suppressed by three different adenosine receptor antagonists (CGS15943, SCH58261 or Caffeine) and by a PKA inhibitor cocktail (Merck # 20-114), but not by JNK inhibitor SP600125. Fold changes are relative to GAPDH , n ≥ 3 and expression of PKG1 is shown. i , j AdoR mutants live longer than wild-type flies when continuously fed with a low dose of Bleomycin ( i ) but live shorter than wild-type flies during acute desiccation (dry starvation, j ). **** P

    Journal: Nature Communications

    Article Title: Epithelial cells release adenosine to promote local TNF production in response to polarity disruption

    doi: 10.1038/s41467-018-07114-z

    Figure Lengend Snippet: AdoR promotes transcription of TNF Egr in polarity-deficient epithelial cells. a sal > ent2 , AdoR expression enhances TNF egr transcript levels in the wing disc independently of JNK signalling. TNFR grnd expression, measured by RT-qPCR, is shown as a control. Fold changes are relative to rp49 , n = 6. b – d sal > ent2, AdoR expression upregulates expression of Egr-GFP from a genomic fosmid ( b , c ). This effect is not rescued by co-expression of JNK bskDN ( d ). The arrowhead in b indicates a suspected position effect of the attP site (VK00033) where the fosmid was integrated. e CADO treatment increases expression of TNF egr in explanted wild-type discs, but not in AdoR mutant discs. This effect is further enhanced by Ador overexpression. TNFR grnd expression, measured by RT-qPCR, is shown as a control. Fold changes are relative to rp49 , n ≥ 5. f en > scrib Ri discs upregulate TNF egr mRNA in an AdoR/Ent2-dependent and JNK-independent fashion. Fold changes are relative to rp49 , n ≥ 4. g Human HaCaT cells treated with adenosine upregulates TNF-α transcript levels (as assayed by qRT-PCR). Fold changes are relative to GAPDH , n = 5. Expression of PKG1 , a housekeeping gene and survivin , a JNK target gene, are also shown. h The effect shown in g was suppressed by three different adenosine receptor antagonists (CGS15943, SCH58261 or Caffeine) and by a PKA inhibitor cocktail (Merck # 20-114), but not by JNK inhibitor SP600125. Fold changes are relative to GAPDH , n ≥ 3 and expression of PKG1 is shown. i , j AdoR mutants live longer than wild-type flies when continuously fed with a low dose of Bleomycin ( i ) but live shorter than wild-type flies during acute desiccation (dry starvation, j ). **** P

    Article Snippet: Adenosine, SCH58261, Caffeine and the JNK inhibitor SP600125 were obtained from Sigma.

    Techniques: Expressing, Quantitative RT-PCR, Mutagenesis, Over Expression

    TRBP is phosphorylated by ERK and JNK in response to arsenite-induced oxidative stress. ( A ) TRBP’s electrophoretic mobility shifts in response to sodium arsenite treatment. A western blot analysis of 50 μg protein per lane from HeLa-TRBP cells treated with 10 μM sodium arsenite at the indicated time points is shown. Western blot analysis was performed with anti-Flag and anti-β actin antibodies. The slower migrating TRBP band at 8 h, and 12 h is indicated by an asterisk. The line between lanes 1 and 2 as well as between lanes 3 and 4 represents where lanes from the same western blot were joined. ( B ) PKR phosphorylation and eIF2α phosphorylation kinetics in response to sodium arsenite treatment. HeLa cells were treated with 10 µM sodium arsenite and cell extracts were prepared at the indicated time points. PKR and eIF2α phosphorylation status at each time point was determined by a western blot analysis using anti-phospho-PKR and anti-phospho-eIF2α specific antibodies using 100 µg and 10 µg of total protein respectively. Each blot was subsequently stripped and re-probed with anti- eIF2α or anti-PKR antibody to ensure equal loading in all lanes. ( C ) TRBP is phosphorylated in response to oxidative stress. Extracts from untreated, and 8 h, or 12 h arsenite-treated TRBP-HeLa cells were prepared in the presence or absence of phosphatase inhibitor (PPi) as indicated above the lanes and subsequently treated with phosphatase (Ptase) or left untreated as indicated. Western blot to was performed with anti-Flag antibody followed by anti- β-actin antibody. ( D ) ERK is phosphorylated in response to oxidative stress and phosphorylates TRBP. TRBP overexpressing TRBP-HeLa cells were treated with 10 μM arsenite alone or in combination with the MEK inhibitor PD0325901 for 24 hours. Cell extracts were made at the indicated time points, and western blot analysis was performed using anti-Flag, anti-phospho-ERK, anti-total ERK, and anti-GAPDH antibodies. ( D ) JNK is phosphorylated in response to oxidative stress and phosphorylates TRBP. TRBP overexpressing TRBP-HeLa cells were treated with 10 μM arsenite alone or in combination with the MEK inhibitor SP600125 for 24 hours. Cell extracts were made at the indicated time points, and western blot analysis was performed using anti-Flag, anti-phospho-JNK, anti-total JNK, and anti-GAPDH antibodies.

    Journal: Scientific Reports

    Article Title: Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival

    doi: 10.1038/s41598-018-19360-8

    Figure Lengend Snippet: TRBP is phosphorylated by ERK and JNK in response to arsenite-induced oxidative stress. ( A ) TRBP’s electrophoretic mobility shifts in response to sodium arsenite treatment. A western blot analysis of 50 μg protein per lane from HeLa-TRBP cells treated with 10 μM sodium arsenite at the indicated time points is shown. Western blot analysis was performed with anti-Flag and anti-β actin antibodies. The slower migrating TRBP band at 8 h, and 12 h is indicated by an asterisk. The line between lanes 1 and 2 as well as between lanes 3 and 4 represents where lanes from the same western blot were joined. ( B ) PKR phosphorylation and eIF2α phosphorylation kinetics in response to sodium arsenite treatment. HeLa cells were treated with 10 µM sodium arsenite and cell extracts were prepared at the indicated time points. PKR and eIF2α phosphorylation status at each time point was determined by a western blot analysis using anti-phospho-PKR and anti-phospho-eIF2α specific antibodies using 100 µg and 10 µg of total protein respectively. Each blot was subsequently stripped and re-probed with anti- eIF2α or anti-PKR antibody to ensure equal loading in all lanes. ( C ) TRBP is phosphorylated in response to oxidative stress. Extracts from untreated, and 8 h, or 12 h arsenite-treated TRBP-HeLa cells were prepared in the presence or absence of phosphatase inhibitor (PPi) as indicated above the lanes and subsequently treated with phosphatase (Ptase) or left untreated as indicated. Western blot to was performed with anti-Flag antibody followed by anti- β-actin antibody. ( D ) ERK is phosphorylated in response to oxidative stress and phosphorylates TRBP. TRBP overexpressing TRBP-HeLa cells were treated with 10 μM arsenite alone or in combination with the MEK inhibitor PD0325901 for 24 hours. Cell extracts were made at the indicated time points, and western blot analysis was performed using anti-Flag, anti-phospho-ERK, anti-total ERK, and anti-GAPDH antibodies. ( D ) JNK is phosphorylated in response to oxidative stress and phosphorylates TRBP. TRBP overexpressing TRBP-HeLa cells were treated with 10 μM arsenite alone or in combination with the MEK inhibitor SP600125 for 24 hours. Cell extracts were made at the indicated time points, and western blot analysis was performed using anti-Flag, anti-phospho-JNK, anti-total JNK, and anti-GAPDH antibodies.

    Article Snippet: Sodium arsenite, phosphatase inhibitor cocktail (Phosphatase Inhibitor Cocktail 2 – P5726), and the JNK inhibitor (SP600125, Catalog number S5567) were purchased from Sigma Aldrich.

    Techniques: Western Blot

    Inhibition of JNK signaling prevents rotenone-induced apoptosis. (A) The SH-SY5Y cells were pretreated with 25 μM SP600125 (SP) for 1 h, and then treated with rotenone for 36 h, cleaved PARP, p-JNK and JNK1 were detected by western blot, JNK1 was used as loading control, β-Actin was used as an internal control. Results are representative of at least three experiments. The levels of p-JNK (B) , cleaved caspase-9 (C) , cleaved caspase-3 (D) , cleaved PARP (E) were quantified by densitometry. ** p

    Journal: Frontiers in Neuroscience

    Article Title: Neuroprotective Effects of Proanthocyanidins, Natural Flavonoids Derived From Plants, on Rotenone-Induced Oxidative Stress and Apoptotic Cell Death in Human Neuroblastoma SH-SY5Y Cells

    doi: 10.3389/fnins.2018.00369

    Figure Lengend Snippet: Inhibition of JNK signaling prevents rotenone-induced apoptosis. (A) The SH-SY5Y cells were pretreated with 25 μM SP600125 (SP) for 1 h, and then treated with rotenone for 36 h, cleaved PARP, p-JNK and JNK1 were detected by western blot, JNK1 was used as loading control, β-Actin was used as an internal control. Results are representative of at least three experiments. The levels of p-JNK (B) , cleaved caspase-9 (C) , cleaved caspase-3 (D) , cleaved PARP (E) were quantified by densitometry. ** p

    Article Snippet: Inhibitors SB203580 (p38 MAPK) and SP600125 (JNK), were from Sigma Aldrich.

    Techniques: Inhibition, Western Blot

    BMP14 induces tenogenic signaling in BMSCs via the JNK and Smad1 pathway in a Sirt1-dependent manner. (A) BMSC cells were infected with Sirt1 overexpressing or shSirt1 lentivirus. Following treatment with 50 ng/ml BMP14, the cell extracts were blotted with anti-P-p38, p38, P-JNK, JNK, P-smad2/3, smad2/3, P-smad1 and smad1 antibodies. Quantitative analysis of the blots was performed using ImageJ software and the relative ratios are indicated above the blots. (B) mRNA levels of Scx were detected using RT-qPCR. (C) BMSC cells were treated with 50 ng/ml BMP14 plus 1 µM LDN-193189 and SP600125. Following 48 h the cells were harvested and cell extracts were immunoprecipitated with PPARγ antibodies; the blots were immunoblotted with acetyl-lys and PPARγ antibodies. The total cell lysate was immunoblotted with anti-β-actin antibody. β-actin was used as the control. BMSC cells were treated with 50 ng/ml BMP14 with or without 1 µM LDN-193189 or SP600125. (D) The protein expression of Scx, collagen I, collagen III and Sirt1 in BMSC cells were detected by western blot analysis. β-actin was used as the loading control. The data are presented as the mean ± standard error of the mean. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: BMP14 induces tenogenic differentiation of bone marrow mesenchymal stem cells in vitro

    doi: 10.3892/etm.2018.6293

    Figure Lengend Snippet: BMP14 induces tenogenic signaling in BMSCs via the JNK and Smad1 pathway in a Sirt1-dependent manner. (A) BMSC cells were infected with Sirt1 overexpressing or shSirt1 lentivirus. Following treatment with 50 ng/ml BMP14, the cell extracts were blotted with anti-P-p38, p38, P-JNK, JNK, P-smad2/3, smad2/3, P-smad1 and smad1 antibodies. Quantitative analysis of the blots was performed using ImageJ software and the relative ratios are indicated above the blots. (B) mRNA levels of Scx were detected using RT-qPCR. (C) BMSC cells were treated with 50 ng/ml BMP14 plus 1 µM LDN-193189 and SP600125. Following 48 h the cells were harvested and cell extracts were immunoprecipitated with PPARγ antibodies; the blots were immunoblotted with acetyl-lys and PPARγ antibodies. The total cell lysate was immunoblotted with anti-β-actin antibody. β-actin was used as the control. BMSC cells were treated with 50 ng/ml BMP14 with or without 1 µM LDN-193189 or SP600125. (D) The protein expression of Scx, collagen I, collagen III and Sirt1 in BMSC cells were detected by western blot analysis. β-actin was used as the loading control. The data are presented as the mean ± standard error of the mean. *P

    Article Snippet: LDN-193189 (S2618) and SP600125 (S1460) were obtained from Selleck (Shanghai, China).

    Techniques: Infection, Software, Quantitative RT-PCR, Immunoprecipitation, Expressing, Western Blot

    Signaling pathways involved in BMP4-activated autophagy to promote HCC proliferation: a Western blot was applied to detect the expression of JNK1, p-JNK, Bcl-2 and p-Bcl-2 in HepG2 and HCCLM3 cells. HCC cells were appointed to the indicated treatment respectively. b The effect of JNK inhibitor SP600125 was determined by Western blot in HepG2 and HCCLM3 cells. c Western blot was performed to detect the expression of LC3-II, p62 and BECN1 in HCC cells. JNK pathway inhibition significantly attenuated the BMP4-activated autophagy in HepG2 and HCCLM3 cells. d Quantification of LC3-II expression level by densitometric analysis and was normalized to the control (blank) groups. GAPDH was used as the internal control. n = 3, one-way ANOVA with post-hoc Tukey’s test. e Effects of JNK inhibitor SP600125 on long term colony formation promoted by BMP4 in HCC cells. JNK pathway inhibition significantly attenuated the promotion effects on the number of colonies both in HepG2 and HCCLM3. n = 3, one-way ANOVA with post-hoc Tukey’s test. f Effects of JNK inhibitor SP600125 on BMP4-promoted HCC cells growth. JNK pathway inhibition significantly attenuated the promotion effects on the cell viability both in HepG2 and HCCLM3

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: BMP4 promotes hepatocellular carcinoma proliferation by autophagy activation through JNK1-mediated Bcl-2 phosphorylation

    doi: 10.1186/s13046-018-0828-x

    Figure Lengend Snippet: Signaling pathways involved in BMP4-activated autophagy to promote HCC proliferation: a Western blot was applied to detect the expression of JNK1, p-JNK, Bcl-2 and p-Bcl-2 in HepG2 and HCCLM3 cells. HCC cells were appointed to the indicated treatment respectively. b The effect of JNK inhibitor SP600125 was determined by Western blot in HepG2 and HCCLM3 cells. c Western blot was performed to detect the expression of LC3-II, p62 and BECN1 in HCC cells. JNK pathway inhibition significantly attenuated the BMP4-activated autophagy in HepG2 and HCCLM3 cells. d Quantification of LC3-II expression level by densitometric analysis and was normalized to the control (blank) groups. GAPDH was used as the internal control. n = 3, one-way ANOVA with post-hoc Tukey’s test. e Effects of JNK inhibitor SP600125 on long term colony formation promoted by BMP4 in HCC cells. JNK pathway inhibition significantly attenuated the promotion effects on the number of colonies both in HepG2 and HCCLM3. n = 3, one-way ANOVA with post-hoc Tukey’s test. f Effects of JNK inhibitor SP600125 on BMP4-promoted HCC cells growth. JNK pathway inhibition significantly attenuated the promotion effects on the cell viability both in HepG2 and HCCLM3

    Article Snippet: 3-Methyladenine (3-MA) (S2767) and JNK inhibitor SP600125 (S1460) were purchased from Selleckchem (Houston, TX, USA).

    Techniques: Western Blot, Expressing, Inhibition

    FAF1-mediated JNK1 activation triggers mitochondrial dysfunction upon ischemic stress. a Faf1 +/+ MEFs were treated with oxygen glucose deprivation for the indicated times and then fractionated into cytoplasmic and mitochondrial fractions. The fractions were analyzed by immunoblotting with anti-P-JNK, JNK1, anti-COX IV (mitochondrial marker) and anti-β-actin (cytoplasmic marker) antibodies. b Faf1 +/+ and Faf1 gt/gt MEFs were treated with oxygen glucose deprivation for 2 h and then fractionated into cytoplasmic and mitochondrial fractions. The fractions were analyzed by immunoblotting with anti-P-JNK, JNK1, anti-COX IV and anti-β-actin antibodies. c Upper panel: Faf1 +/+ and Faf1 gt/gt MEFs were transfected with DsRed-Mito plasmid for 48 h. Subsequently, the cells were treated with oxygen glucose deprivation in the presence or absence of 20 μM SP600125 for 2 h. Fluorescence images show the alterations in mitochondrial morphology . Scale bar = 20 μm. Bottom panel: the graph shows the quantification of mitochondrial fragmentation ( n = 3). d Faf1 +/+ and Faf1 gt/gt MEFs were untreated or treated with oxygen glucose deprivation in the presence or absence of 20 μM SP600125 for 5 h. Mitochondrial superoxide was stained using 10 μM MitoSOX reagent. The population of MitoSOX-positive cells was detected by flow cytometry. The graph shows the results of the quantitative analysis of MitoSOX-positive cells ( n = 3). e Faf1 +/+ and Faf1 gt/gt MEFs were untreated or treated with oxygen glucose deprivation in the presence or absence of 20 μM SP600125 for 5 h, and the mitochondrial potential was measured using a Muse analyzer. The graph was obtained from a quantitative analysis of depolarized cells ( n = 3). The data ( c - e ) are expressed as the mean ± S.E.M. of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Tukey’s HSD ( c - e ) post hoc analysis. *** P

    Journal: Cell Communication and Signaling : CCS

    Article Title: FAF1 mediates necrosis through JNK1-mediated mitochondrial dysfunction leading to retinal degeneration in the ganglion cell layer upon ischemic insult

    doi: 10.1186/s12964-018-0265-7

    Figure Lengend Snippet: FAF1-mediated JNK1 activation triggers mitochondrial dysfunction upon ischemic stress. a Faf1 +/+ MEFs were treated with oxygen glucose deprivation for the indicated times and then fractionated into cytoplasmic and mitochondrial fractions. The fractions were analyzed by immunoblotting with anti-P-JNK, JNK1, anti-COX IV (mitochondrial marker) and anti-β-actin (cytoplasmic marker) antibodies. b Faf1 +/+ and Faf1 gt/gt MEFs were treated with oxygen glucose deprivation for 2 h and then fractionated into cytoplasmic and mitochondrial fractions. The fractions were analyzed by immunoblotting with anti-P-JNK, JNK1, anti-COX IV and anti-β-actin antibodies. c Upper panel: Faf1 +/+ and Faf1 gt/gt MEFs were transfected with DsRed-Mito plasmid for 48 h. Subsequently, the cells were treated with oxygen glucose deprivation in the presence or absence of 20 μM SP600125 for 2 h. Fluorescence images show the alterations in mitochondrial morphology . Scale bar = 20 μm. Bottom panel: the graph shows the quantification of mitochondrial fragmentation ( n = 3). d Faf1 +/+ and Faf1 gt/gt MEFs were untreated or treated with oxygen glucose deprivation in the presence or absence of 20 μM SP600125 for 5 h. Mitochondrial superoxide was stained using 10 μM MitoSOX reagent. The population of MitoSOX-positive cells was detected by flow cytometry. The graph shows the results of the quantitative analysis of MitoSOX-positive cells ( n = 3). e Faf1 +/+ and Faf1 gt/gt MEFs were untreated or treated with oxygen glucose deprivation in the presence or absence of 20 μM SP600125 for 5 h, and the mitochondrial potential was measured using a Muse analyzer. The graph was obtained from a quantitative analysis of depolarized cells ( n = 3). The data ( c - e ) are expressed as the mean ± S.E.M. of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Tukey’s HSD ( c - e ) post hoc analysis. *** P

    Article Snippet: The weakening of the protective potential provided by SP600125 in Fig. might be due to possible off-target effects of SP600125 on other survival kinases, such as AMP-activated protein k inase and phosphatidylinositol 3-kinase [ – ].

    Techniques: Activation Assay, Marker, Transfection, Plasmid Preparation, Fluorescence, Staining, Flow Cytometry, Cytometry

    JNK1 contributes to necrosis upon ischemic stress induced by oxygen-glucose deprivation in MEFs. a MEFs were treated with oxygen glucose deprivation for the indicated times and/or 1 μM staurosporine (STS) for 12 h. The type of cell death was determined by flow cytometry using double staining with annexin V and PI. b The graph shows the quantitative results of the flow cytometry analysis. c and d MEFs were treated with oxygen glucose deprivation or 1 μM STS for the indicated times ( n = 3). Caspase-3 activity was measured using fluorometric assay ( n = 3) ( c ) and western blot analysis ( d ). e MEFs were untreated or treated with oxygen glucose deprivation for 8 h in the presence or absence of zVAD-fmk (50 μM), and cell death was determined by measuring PI uptake using a flow cytometer ( n = 3). f MEFs were pretreated with Nec-1 (50 μM), DPQ (30 μM), or SP600125 (20 μM) for 30 min and then with oxygen glucose deprivation for 8 h in the presence of the individual compounds. Cell death was detected by flow cytometry ( n = 3). g Left panel: Jnk1+/+ and Jnk1−/− MEFs were untreated or treated with oxygen glucose deprivation for 8 h. Cell death was detected using flow cytometry ( n = 3). Right panel: representative immunoblots show the JNK1, FAF1 and β-actin expression levels. The data ( b , c , e - g ) are expressed as the mean ± S.E.M. of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Dunnett’s T3 ( b, c, and e ) and Tukey’s HSD ( f and g ) post hoc analysis. *** p

    Journal: Cell Communication and Signaling : CCS

    Article Title: FAF1 mediates necrosis through JNK1-mediated mitochondrial dysfunction leading to retinal degeneration in the ganglion cell layer upon ischemic insult

    doi: 10.1186/s12964-018-0265-7

    Figure Lengend Snippet: JNK1 contributes to necrosis upon ischemic stress induced by oxygen-glucose deprivation in MEFs. a MEFs were treated with oxygen glucose deprivation for the indicated times and/or 1 μM staurosporine (STS) for 12 h. The type of cell death was determined by flow cytometry using double staining with annexin V and PI. b The graph shows the quantitative results of the flow cytometry analysis. c and d MEFs were treated with oxygen glucose deprivation or 1 μM STS for the indicated times ( n = 3). Caspase-3 activity was measured using fluorometric assay ( n = 3) ( c ) and western blot analysis ( d ). e MEFs were untreated or treated with oxygen glucose deprivation for 8 h in the presence or absence of zVAD-fmk (50 μM), and cell death was determined by measuring PI uptake using a flow cytometer ( n = 3). f MEFs were pretreated with Nec-1 (50 μM), DPQ (30 μM), or SP600125 (20 μM) for 30 min and then with oxygen glucose deprivation for 8 h in the presence of the individual compounds. Cell death was detected by flow cytometry ( n = 3). g Left panel: Jnk1+/+ and Jnk1−/− MEFs were untreated or treated with oxygen glucose deprivation for 8 h. Cell death was detected using flow cytometry ( n = 3). Right panel: representative immunoblots show the JNK1, FAF1 and β-actin expression levels. The data ( b , c , e - g ) are expressed as the mean ± S.E.M. of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Dunnett’s T3 ( b, c, and e ) and Tukey’s HSD ( f and g ) post hoc analysis. *** p

    Article Snippet: The weakening of the protective potential provided by SP600125 in Fig. might be due to possible off-target effects of SP600125 on other survival kinases, such as AMP-activated protein k inase and phosphatidylinositol 3-kinase [ – ].

    Techniques: Flow Cytometry, Cytometry, Double Staining, Activity Assay, Western Blot, Expressing

    FSTL1 modulates myofibroblast differentiation by facilitating p38/JNK signaling. (a and b) Primary lung fibroblasts from Fstl1+/− and their WT littermates were treated with 5 ng/ml TGF-β1. (a) Immunofluorescence staining of α-SMA in lung fibroblasts. (b) Protein expression levels of α-SMA and type I collagen. (c-e) MLgs were pretreated with U0126, SB202190, and SP600125. Protein expression levels of α-SMA and type I collagen were detected by Western blotting. n = 4; Bars = 100 μm. * P

    Journal: Chinese Medical Journal

    Article Title: Follistatin-Like 1 Promotes Bleomycin-Induced Pulmonary Fibrosis through the Transforming Growth Factor Beta 1/Mitogen-Activated Protein Kinase Signaling Pathway

    doi: 10.4103/0366-6999.238151

    Figure Lengend Snippet: FSTL1 modulates myofibroblast differentiation by facilitating p38/JNK signaling. (a and b) Primary lung fibroblasts from Fstl1+/− and their WT littermates were treated with 5 ng/ml TGF-β1. (a) Immunofluorescence staining of α-SMA in lung fibroblasts. (b) Protein expression levels of α-SMA and type I collagen. (c-e) MLgs were pretreated with U0126, SB202190, and SP600125. Protein expression levels of α-SMA and type I collagen were detected by Western blotting. n = 4; Bars = 100 μm. * P

    Article Snippet: MLgs were cultured in 96-well plates for 24 h, and serum starved for 24 h. The cells were preincubated for 1 h with U0126 (ERK inhibitor, Cell Signaling Technology, MA, USA), SB202190 (p38 inhibitor, Cell Signaling Technology, MA, USA), SP600125 (JNK inhibitor, Cell Signaling Technology, MA, USA), and SB525334 (Smad2/3 inhibitor, R and D Systems, MN, USA) before treatment with 5 ng/ml TGF-β1 (R and D Systems, MN, USA) in the presence or absence of 100 ng/ml recombinant human FSTL1 protein (R and D Systems, MN, USA) for 24 h and finally incubated with MTT (final concentration of 0.5 mg/ml) for 4 h. The supernatant was removed, and 150 ml/well of dimethylsulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) was added to dissolve the blue formazan crystals by shaking the plates for 15 min on an orbital shaker at 25°C.

    Techniques: Immunofluorescence, Staining, Expressing, Western Blot

    Lack of JNK1 and JNK2 affects axonal elongation rate. A , Mean speed of growth cone advancement and retraction of wild-type and JNK-deficient DRG neurons. Note that jnk1 −/− and jnk2 −/− , but not jnk3 −/− , neurite outgrowth rates are significantly decreased compared with wild type. After SP600125 application, the retraction rate of jnk2 −/− neurites is decreased compared with jnk3 −/− and wild-type neurites, and no retraction of jnk1 −/− neurites occurs. B , Advancement or retraction of growth cones measured 90 min before and 90 min after SP600125 application. Within a few minutes after JNK inhibition, neurites from jnk2 −/− and jnk3 −/− neurons retract, whereas jnk1 −/− neurites continue growing during the remaining recording period. Response time for growth cones to collapse after SP600125 application: jnk2 −/− and jnk3 −/− growth cones collapse rapidly, in contrast to jnk1 −/− growth cones. D–F , Frames of time-lapse recordings illustrating the outgrowth and typical response of neurites to JNK inhibition from jnk1 −/− , jnk2 −/− , and jnk3 −/− DRG neurons, 90 min before and after SP600125 addition. Arrowheads, Growth cones. Scale bars, 50 μm. *** p

    Journal: The Journal of Neuroscience

    Article Title: Distinct Roles of c-Jun N-Terminal Kinase Isoforms in Neurite Initiation and Elongation during Axonal Regeneration

    doi: 10.1523/JNEUROSCI.0372-10.2010

    Figure Lengend Snippet: Lack of JNK1 and JNK2 affects axonal elongation rate. A , Mean speed of growth cone advancement and retraction of wild-type and JNK-deficient DRG neurons. Note that jnk1 −/− and jnk2 −/− , but not jnk3 −/− , neurite outgrowth rates are significantly decreased compared with wild type. After SP600125 application, the retraction rate of jnk2 −/− neurites is decreased compared with jnk3 −/− and wild-type neurites, and no retraction of jnk1 −/− neurites occurs. B , Advancement or retraction of growth cones measured 90 min before and 90 min after SP600125 application. Within a few minutes after JNK inhibition, neurites from jnk2 −/− and jnk3 −/− neurons retract, whereas jnk1 −/− neurites continue growing during the remaining recording period. Response time for growth cones to collapse after SP600125 application: jnk2 −/− and jnk3 −/− growth cones collapse rapidly, in contrast to jnk1 −/− growth cones. D–F , Frames of time-lapse recordings illustrating the outgrowth and typical response of neurites to JNK inhibition from jnk1 −/− , jnk2 −/− , and jnk3 −/− DRG neurons, 90 min before and after SP600125 addition. Arrowheads, Growth cones. Scale bars, 50 μm. *** p

    Article Snippet: To analyze the effect of JNK inhibition on neurite initiation, SP600125 (20 μ m ; BIOMOL International) was added to culture medium 4 h after plating (i.e., before any neurites emerged), and cell cultures were maintained for 24 and 48 h before fixation.

    Techniques: Inhibition

    Scaffold protein JIP1 is required for neurite regeneration. A , B , and merge in C , JIP1 immunostaining is enriched in growth cones of regenerating DRG neurites, shafts of which are visualized by total MAP1B immunostaining. D , Western blot analysis of P-JNK in protein extracts from wild-type or jip1 −/− DRG neurons cultured 48 h before lysis; the expression of GAPDH was monitored in the lower part of the same membrane as loading control. Lack of JIP1 results in a reduction of the 46 kDa form of P-JNK. E–G , Tubulin immunostaining of DRG neurons cultured for 48 h derived from wild-type ( E ), jip1 −/− ( F ), and jip1 −/− neurons with additional SP600125 treatment ( G ). Note the abnormal “curly” morphology of neurites from jip1 −/− DRG exhibiting more terminal branching; additional SP600125 treatment has no effect on jip1 −/− neurites. H , Quantification of total neuritic length of wild-type versus jip1 −/− neurons reveals a difference of 70.4%; in contrast, additional SP600125 treatment has only a slight effect on neurite length of jip1 −/− DRG neurons ( n ≥ 55). I , Diagram showing the distribution of total neuritic lengths of wild-type versus jip1 −/− DRG neurons, the latter cultured with or without additional SP600125 treatment. J , Mean speed of growth cone advancement from wild-type and JIP1-deficient DRG neurons showing that the outgrowth rate of jip1 −/− neurite is drastically decreased. K , Growth cone advancement or retraction measured 90 min before and 90 min after SP600125 application, showing that no neurite retraction occurs from jip1 −/− neurons during the remaining recording time, as illustrated in L , showing corresponding frames of time-lapse recordings. Arrowheads, Growth cones. Scale bars: A , C , 15 μm; E–G , 100 μm; L , 50 μm. *** p

    Journal: The Journal of Neuroscience

    Article Title: Distinct Roles of c-Jun N-Terminal Kinase Isoforms in Neurite Initiation and Elongation during Axonal Regeneration

    doi: 10.1523/JNEUROSCI.0372-10.2010

    Figure Lengend Snippet: Scaffold protein JIP1 is required for neurite regeneration. A , B , and merge in C , JIP1 immunostaining is enriched in growth cones of regenerating DRG neurites, shafts of which are visualized by total MAP1B immunostaining. D , Western blot analysis of P-JNK in protein extracts from wild-type or jip1 −/− DRG neurons cultured 48 h before lysis; the expression of GAPDH was monitored in the lower part of the same membrane as loading control. Lack of JIP1 results in a reduction of the 46 kDa form of P-JNK. E–G , Tubulin immunostaining of DRG neurons cultured for 48 h derived from wild-type ( E ), jip1 −/− ( F ), and jip1 −/− neurons with additional SP600125 treatment ( G ). Note the abnormal “curly” morphology of neurites from jip1 −/− DRG exhibiting more terminal branching; additional SP600125 treatment has no effect on jip1 −/− neurites. H , Quantification of total neuritic length of wild-type versus jip1 −/− neurons reveals a difference of 70.4%; in contrast, additional SP600125 treatment has only a slight effect on neurite length of jip1 −/− DRG neurons ( n ≥ 55). I , Diagram showing the distribution of total neuritic lengths of wild-type versus jip1 −/− DRG neurons, the latter cultured with or without additional SP600125 treatment. J , Mean speed of growth cone advancement from wild-type and JIP1-deficient DRG neurons showing that the outgrowth rate of jip1 −/− neurite is drastically decreased. K , Growth cone advancement or retraction measured 90 min before and 90 min after SP600125 application, showing that no neurite retraction occurs from jip1 −/− neurons during the remaining recording time, as illustrated in L , showing corresponding frames of time-lapse recordings. Arrowheads, Growth cones. Scale bars: A , C , 15 μm; E–G , 100 μm; L , 50 μm. *** p

    Article Snippet: To analyze the effect of JNK inhibition on neurite initiation, SP600125 (20 μ m ; BIOMOL International) was added to culture medium 4 h after plating (i.e., before any neurites emerged), and cell cultures were maintained for 24 and 48 h before fixation.

    Techniques: Immunostaining, Western Blot, Cell Culture, Lysis, Expressing, Derivative Assay

    Differential involvement of JNK isoforms in neurite initiation and elongation. A , Western blots of protein extracts from wild-type and JNK-deficient mice DRG neurons, cultured 48 h, reacted with antibodies specific for individual JNK proteins. In regenerating wild-type neurons, all isoforms are expressed. B , Quantification of neurons bearing neurites after 24 and 48 h of culture: At 24 h, jnk2 −/− and jnk3 −/− neurons exhibit a strong decrease in the percentage of neurons with neurites compared with wild-type neurons. At 48 h, only jnk2 −/− neurons still display a slight but significant difference. Neurite initiation is not affected in regenerating jnk1 −/− neurons ( n ≥ 297). C , D , Quantification of total neurite length and length of the longest neurite for wild-type versus JNK-deficient neurons without (gray bars) or with SP600125 treatment (black bars). Total neurite lengths for jnk1 −/− and jnk2 −/− , but not jnk3 −/− neurons, are decreased compared with wild-type neurons; the average length of the longest neurite is significantly affected only by lack of JNK2. SP600125 treatment induces an additional reduction in total neurite length for neurons derived from all three jnk -ko mice, but induces a specific decrease in length of the longest neurite only in jnk3 −/− neurons ( n ≥ 50). E , F , The effect on neurite length of JNK1 and JNK2, but not JNK3 deficiency, is reflected in a leftward shift in the distribution diagrams for total neurite length; values for the length of the longest neurite per neuron display a leftward shift only for jnk2 −/− neurons; those for jnk1 −/− and jnk3 −/− neurons are similar to wild type. *** p

    Journal: The Journal of Neuroscience

    Article Title: Distinct Roles of c-Jun N-Terminal Kinase Isoforms in Neurite Initiation and Elongation during Axonal Regeneration

    doi: 10.1523/JNEUROSCI.0372-10.2010

    Figure Lengend Snippet: Differential involvement of JNK isoforms in neurite initiation and elongation. A , Western blots of protein extracts from wild-type and JNK-deficient mice DRG neurons, cultured 48 h, reacted with antibodies specific for individual JNK proteins. In regenerating wild-type neurons, all isoforms are expressed. B , Quantification of neurons bearing neurites after 24 and 48 h of culture: At 24 h, jnk2 −/− and jnk3 −/− neurons exhibit a strong decrease in the percentage of neurons with neurites compared with wild-type neurons. At 48 h, only jnk2 −/− neurons still display a slight but significant difference. Neurite initiation is not affected in regenerating jnk1 −/− neurons ( n ≥ 297). C , D , Quantification of total neurite length and length of the longest neurite for wild-type versus JNK-deficient neurons without (gray bars) or with SP600125 treatment (black bars). Total neurite lengths for jnk1 −/− and jnk2 −/− , but not jnk3 −/− neurons, are decreased compared with wild-type neurons; the average length of the longest neurite is significantly affected only by lack of JNK2. SP600125 treatment induces an additional reduction in total neurite length for neurons derived from all three jnk -ko mice, but induces a specific decrease in length of the longest neurite only in jnk3 −/− neurons ( n ≥ 50). E , F , The effect on neurite length of JNK1 and JNK2, but not JNK3 deficiency, is reflected in a leftward shift in the distribution diagrams for total neurite length; values for the length of the longest neurite per neuron display a leftward shift only for jnk2 −/− neurons; those for jnk1 −/− and jnk3 −/− neurons are similar to wild type. *** p

    Article Snippet: To analyze the effect of JNK inhibition on neurite initiation, SP600125 (20 μ m ; BIOMOL International) was added to culture medium 4 h after plating (i.e., before any neurites emerged), and cell cultures were maintained for 24 and 48 h before fixation.

    Techniques: Western Blot, Mouse Assay, Cell Culture, Derivative Assay

    JNK activation is required for both neuritogenesis and sustained extension of regenerating neurites. A–D , P-JNK and tubulin immunolabeling of DRG neurons cultured for 48 h. P-JNK is found in soma (including the nucleus), axon shafts, and growth cones. E , Quantification of neurite-bearing neurons cultured for 24 and 48 h, without (gray bars) or with chronic SP600125 treatment (black bars), reveals that neurite formation is blocked by JNK inhibition ( n ≥ 446). F , G , Neurons treated with or without SP600125 and visualized by tubulin immunostaining. JNK inhibition induces loss of growth cones and leads to retraction bulb formation and trailing remnants along neurites (arrowheads); insets show higher magnifications of a typical growth cone under control conditions and a drug treatment-induced retraction bulb. H , I , Quantitative analysis reveals that JNK inhibition decreases total neuritic and longest neurite lengths of regenerating neurons ( n ≥ 55). J , K , Distribution diagrams showing the percentage of neurons exhibiting a given total neuritic length and length of the longest neurite. Whereas the majority of neurons cultured under control conditions displays a total neurite length > 3500 μm and a longest neurite > 400 μm, JNK inhibition induces a shift of both values toward neurons with shorter neurites. L , M , Frames of time-lapse recordings illustrating the typical response of neurites to JNK inhibition: Before SP600125 addition ( t = 60 min), growth cones (arrowheads) are highly dynamic and neurites elongate steadily. Shortly after drug addition, growth cones collapse, and neurites stop extending and start to retract. Scale bars: A , C , F , G , L , M , 50 μm; B , D , 15 μm. *** p

    Journal: The Journal of Neuroscience

    Article Title: Distinct Roles of c-Jun N-Terminal Kinase Isoforms in Neurite Initiation and Elongation during Axonal Regeneration

    doi: 10.1523/JNEUROSCI.0372-10.2010

    Figure Lengend Snippet: JNK activation is required for both neuritogenesis and sustained extension of regenerating neurites. A–D , P-JNK and tubulin immunolabeling of DRG neurons cultured for 48 h. P-JNK is found in soma (including the nucleus), axon shafts, and growth cones. E , Quantification of neurite-bearing neurons cultured for 24 and 48 h, without (gray bars) or with chronic SP600125 treatment (black bars), reveals that neurite formation is blocked by JNK inhibition ( n ≥ 446). F , G , Neurons treated with or without SP600125 and visualized by tubulin immunostaining. JNK inhibition induces loss of growth cones and leads to retraction bulb formation and trailing remnants along neurites (arrowheads); insets show higher magnifications of a typical growth cone under control conditions and a drug treatment-induced retraction bulb. H , I , Quantitative analysis reveals that JNK inhibition decreases total neuritic and longest neurite lengths of regenerating neurons ( n ≥ 55). J , K , Distribution diagrams showing the percentage of neurons exhibiting a given total neuritic length and length of the longest neurite. Whereas the majority of neurons cultured under control conditions displays a total neurite length > 3500 μm and a longest neurite > 400 μm, JNK inhibition induces a shift of both values toward neurons with shorter neurites. L , M , Frames of time-lapse recordings illustrating the typical response of neurites to JNK inhibition: Before SP600125 addition ( t = 60 min), growth cones (arrowheads) are highly dynamic and neurites elongate steadily. Shortly after drug addition, growth cones collapse, and neurites stop extending and start to retract. Scale bars: A , C , F , G , L , M , 50 μm; B , D , 15 μm. *** p

    Article Snippet: To analyze the effect of JNK inhibition on neurite initiation, SP600125 (20 μ m ; BIOMOL International) was added to culture medium 4 h after plating (i.e., before any neurites emerged), and cell cultures were maintained for 24 and 48 h before fixation.

    Techniques: Activation Assay, Immunolabeling, Cell Culture, Inhibition, Immunostaining

    JNK inhibition affects MAP1B phosphorylation in regenerating neurons. A–C , In control DRG neurons, distal parts of neurites are enriched in MAP1B-P and characterized by low levels of detyrosinated tubulin (representing stable microtubules). D–F , Higher magnification of a growth cone showing the presence of MAP1B-P at the leading edge, where no colocalization with detyrosinated tubulin can be found. G–I , Double immunostaining reveals that total MAP1B is distributed in all parts of the neuron (green), whereas MAP1B-P (red) is mainly localized in neurites and exhibits a proximo-distal gradient. J , K , Application of SP600125, although not altering total MAP1B levels, dramatically decreases the neurite content in MAP1B-P, concomitantly with growth cone retraction (see also insets). L , M , Immunostaining for MAP1B-P, compared with total MAP1B, on regenerating jip1 −/− neurons reveals that lack of JIP strongly affects the level of MAP1B phosphorylation. Scale bars: A–C , 50 μm; D–F , 10 μm; G–M , 100 μm.

    Journal: The Journal of Neuroscience

    Article Title: Distinct Roles of c-Jun N-Terminal Kinase Isoforms in Neurite Initiation and Elongation during Axonal Regeneration

    doi: 10.1523/JNEUROSCI.0372-10.2010

    Figure Lengend Snippet: JNK inhibition affects MAP1B phosphorylation in regenerating neurons. A–C , In control DRG neurons, distal parts of neurites are enriched in MAP1B-P and characterized by low levels of detyrosinated tubulin (representing stable microtubules). D–F , Higher magnification of a growth cone showing the presence of MAP1B-P at the leading edge, where no colocalization with detyrosinated tubulin can be found. G–I , Double immunostaining reveals that total MAP1B is distributed in all parts of the neuron (green), whereas MAP1B-P (red) is mainly localized in neurites and exhibits a proximo-distal gradient. J , K , Application of SP600125, although not altering total MAP1B levels, dramatically decreases the neurite content in MAP1B-P, concomitantly with growth cone retraction (see also insets). L , M , Immunostaining for MAP1B-P, compared with total MAP1B, on regenerating jip1 −/− neurons reveals that lack of JIP strongly affects the level of MAP1B phosphorylation. Scale bars: A–C , 50 μm; D–F , 10 μm; G–M , 100 μm.

    Article Snippet: To analyze the effect of JNK inhibition on neurite initiation, SP600125 (20 μ m ; BIOMOL International) was added to culture medium 4 h after plating (i.e., before any neurites emerged), and cell cultures were maintained for 24 and 48 h before fixation.

    Techniques: Inhibition, Double Immunostaining, Immunostaining

    Regulation of stathmin and MAP1B phosphorylation by JNKs: Western blot analysis with GADPH used as internal loading control. A , Relative amounts of total stathmin and stathmin phosphorylated on serine 25 (P-Ser25) in lysates of wild-type and JNK-deficient DRG neurons cultured for 48 h; note the decrease in stathmin phosphorylation in regenerating jnk1 −/− neurons. B , MAP1B staining of protein extracts from wild-type and JNK-deficient DRG neurons cultured for 48 h, showing a shift in the protein band representing MAP1B from higher to lower apparent molecular weight for jnk1 −/− and jnk2 −/− , but not jnk3 −/− neurons. This shift reflects a reduced level of MAP1B phosphorylation as demonstrated in C , MAP1B in lysates of N2A neuroblastoma cells cultured for 48 h is phosphorylated under control conditions (lane 1); experimental dephosphorylation by AP treatment of lysates (lanes 3, 4) results in the same shift to a lower molecular weight as JNK inhibition by SP600125 (SP) addition to the N2A cultures (lane 2).

    Journal: The Journal of Neuroscience

    Article Title: Distinct Roles of c-Jun N-Terminal Kinase Isoforms in Neurite Initiation and Elongation during Axonal Regeneration

    doi: 10.1523/JNEUROSCI.0372-10.2010

    Figure Lengend Snippet: Regulation of stathmin and MAP1B phosphorylation by JNKs: Western blot analysis with GADPH used as internal loading control. A , Relative amounts of total stathmin and stathmin phosphorylated on serine 25 (P-Ser25) in lysates of wild-type and JNK-deficient DRG neurons cultured for 48 h; note the decrease in stathmin phosphorylation in regenerating jnk1 −/− neurons. B , MAP1B staining of protein extracts from wild-type and JNK-deficient DRG neurons cultured for 48 h, showing a shift in the protein band representing MAP1B from higher to lower apparent molecular weight for jnk1 −/− and jnk2 −/− , but not jnk3 −/− neurons. This shift reflects a reduced level of MAP1B phosphorylation as demonstrated in C , MAP1B in lysates of N2A neuroblastoma cells cultured for 48 h is phosphorylated under control conditions (lane 1); experimental dephosphorylation by AP treatment of lysates (lanes 3, 4) results in the same shift to a lower molecular weight as JNK inhibition by SP600125 (SP) addition to the N2A cultures (lane 2).

    Article Snippet: To analyze the effect of JNK inhibition on neurite initiation, SP600125 (20 μ m ; BIOMOL International) was added to culture medium 4 h after plating (i.e., before any neurites emerged), and cell cultures were maintained for 24 and 48 h before fixation.

    Techniques: Western Blot, Cell Culture, Staining, Molecular Weight, De-Phosphorylation Assay, Inhibition

    Blocking the MAPK signaling pathway diminished LPS-induced SLC15A3 upregulation. Mouse PMs (left panel) and BMDMs (right panel) were pretreated with or without 50 μM U0126, 10 μM SB203085 (SB203) and 10 μM SP600125 (SP600) for 1 h, then the cells were treated with or without 100 ng/mL LPS for another 4 h. mRNA expression of Slc15a3 ( a , b ), Il-6 ( c , d ) and Tnf-α ( e , f ) were determined by real-time PCR. One-way ANOVA followed by Tukey’s test was used to evaluate the statistical differences, * P

    Journal: Cell Death & Disease

    Article Title: Regulation and biological role of the peptide/histidine transporter SLC15A3 in Toll-like receptor-mediated inflammatory responses in macrophage

    doi: 10.1038/s41419-018-0809-1

    Figure Lengend Snippet: Blocking the MAPK signaling pathway diminished LPS-induced SLC15A3 upregulation. Mouse PMs (left panel) and BMDMs (right panel) were pretreated with or without 50 μM U0126, 10 μM SB203085 (SB203) and 10 μM SP600125 (SP600) for 1 h, then the cells were treated with or without 100 ng/mL LPS for another 4 h. mRNA expression of Slc15a3 ( a , b ), Il-6 ( c , d ) and Tnf-α ( e , f ) were determined by real-time PCR. One-way ANOVA followed by Tukey’s test was used to evaluate the statistical differences, * P

    Article Snippet: For inhibition studies, the cells were pretreated with 10 μM BAY 11–7082, 50 μM U0126, 10 μM SB203580, 10 μM SP600125 (Beyotime Biotechnology, Shanghai, China) or 2 μM MRT67307 (Monmouth Junction, NJ) for 1 h and then incubated with TLR ligands for another 4 h. Cells were analyzed by real-time PCR and western blot.

    Techniques: Blocking Assay, Expressing, Real-time Polymerase Chain Reaction

    JNK contributes to chronic morphine-induced activation of presynaptic NMDARs at primary afferent terminals. A , representative recordings and cumulative plots show a lack of effect of 50 μM AP5 on the frequency and amplitude of mEPSCs in a lamina II neuron (the spinal cord slice was pretreated with 40 μM SP600125 for 1 h) in a morphine-treated rat. B , mean changes in the frequency and amplitude of mEPSCs in lamina II neurons show a lack of effect of AP5 on the frequency and amplitude of mEPSCs in spinal cord slices pretreated with SP600125 from morphine-treated rats (n = 10 neurons from 3 rats). C , representative current traces show a lack of effect of 50 μM AP5 on the amplitude of evoked monosynaptic EPSCs in a lamina II neuron from spinal cord slices pretreated with SP600125 in a morphine-treated rat. D , original recording traces show the lack of effect of AP5 on the PPR of evoked EPSCs in spinal cord slices pretreated with SP600125 in a morphine-treated rat. E , summary data show the lack of effect of 50 μM AP5 on the amplitude ( n = 10 neurons from 3 rats) and PPR ( n = 9 neurons from 3 rats) of monosynaptic EPSCs in lamina II neurons from spinal cord slices pretreated with SP600125 in morphine-treated rats. Data are presented as means ± SEM.

    Journal: Journal of neurochemistry

    Article Title: Mitogen-Activated Protein Kinase Signaling Mediates Opioid-induced Presynaptic NMDA Receptor Activation and Analgesic Tolerance

    doi: 10.1111/jnc.14628

    Figure Lengend Snippet: JNK contributes to chronic morphine-induced activation of presynaptic NMDARs at primary afferent terminals. A , representative recordings and cumulative plots show a lack of effect of 50 μM AP5 on the frequency and amplitude of mEPSCs in a lamina II neuron (the spinal cord slice was pretreated with 40 μM SP600125 for 1 h) in a morphine-treated rat. B , mean changes in the frequency and amplitude of mEPSCs in lamina II neurons show a lack of effect of AP5 on the frequency and amplitude of mEPSCs in spinal cord slices pretreated with SP600125 from morphine-treated rats (n = 10 neurons from 3 rats). C , representative current traces show a lack of effect of 50 μM AP5 on the amplitude of evoked monosynaptic EPSCs in a lamina II neuron from spinal cord slices pretreated with SP600125 in a morphine-treated rat. D , original recording traces show the lack of effect of AP5 on the PPR of evoked EPSCs in spinal cord slices pretreated with SP600125 in a morphine-treated rat. E , summary data show the lack of effect of 50 μM AP5 on the amplitude ( n = 10 neurons from 3 rats) and PPR ( n = 9 neurons from 3 rats) of monosynaptic EPSCs in lamina II neurons from spinal cord slices pretreated with SP600125 in morphine-treated rats. Data are presented as means ± SEM.

    Article Snippet: U0126 (5 μg; EMD-Calbiochem, La Jolla, CA), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580, 5 μg; EMD-Calbiochem), and SP600125 (30 μg; Cayman Chemical, Ann Arbor, MI) were dissolved in DMSO and intrathecally injected 20 min before morphine administration on each testing day (days 1–8).

    Techniques: Activation Assay

    Inhibition of ERK1/2, p38, or JNK activity at the spinal cord level attenuates both opioid-induced hyperalgesia and analgesic tolerance caused by chronic morphine treatment. A , time course of changes in the baseline nociceptive withdrawal threshold in response to a noxious pressure stimulus of the hindpaw in rats given systemic morphine plus intrathecal injections of a vehicle (n = 9 rats), 5 μg U0126 (n = 7 rats), 5 μg SB203580 (n = 7 rats), 30 μg SP600125 (n = 8 rats), or combination of 5 μg U0126, 5 μg SB203580, and 30 μg SP600125 (n = 8 rats). B , time course of changes in the baseline withdrawal latency measured with a noxious heat stimulus in rats given systemic morphine plus intrathecal injections of a vehicle (n = 9 rats), 5 μg U0126 (n = 7 rats), 5 μg SB203580 (n = 7 rats), 30 μg SP600125 (n = 8 rats), or combination of 5 μg U0126, 5 μg SB203580, and 30 μg SP600125 (n = 8 rats). C , time course of the analgesic effect of morphine on the mechanical nociceptive withdrawal threshold in rats given intrathecal injections of a vehicle (n = 9 rats), 5 μg U0126 (n = 7 rats), 5 μg SB203580 (n = 7 rats), 30 μg SP600125 (n = 8 rats), or combination of 5 μg U0126, 5 μg SB203580, and 30 μg SP600125 (n = 8 rats). D , time course of the analgesic effect of morphine on the heat withdrawal latency in rats given intrathecal injections of a vehicle (n = 9 rats), 5 μg U0126 (n = 7 rats), 5 μg SB203580 (n = 7 rats), 30 μg SP600125 (n = 8 rats), or combination of 5 μg U0126, 5 μg SB203580, and 30 μg SP600125 (n = 8 rats). The baseline withdrawal threshold was measured before morphine injection each day, and the analgesic effect of morphine was tested 30 min after each morphine injection (5 mg/kg, intraperitoneal). Data are presented as means ± SEM. *p

    Journal: Journal of neurochemistry

    Article Title: Mitogen-Activated Protein Kinase Signaling Mediates Opioid-induced Presynaptic NMDA Receptor Activation and Analgesic Tolerance

    doi: 10.1111/jnc.14628

    Figure Lengend Snippet: Inhibition of ERK1/2, p38, or JNK activity at the spinal cord level attenuates both opioid-induced hyperalgesia and analgesic tolerance caused by chronic morphine treatment. A , time course of changes in the baseline nociceptive withdrawal threshold in response to a noxious pressure stimulus of the hindpaw in rats given systemic morphine plus intrathecal injections of a vehicle (n = 9 rats), 5 μg U0126 (n = 7 rats), 5 μg SB203580 (n = 7 rats), 30 μg SP600125 (n = 8 rats), or combination of 5 μg U0126, 5 μg SB203580, and 30 μg SP600125 (n = 8 rats). B , time course of changes in the baseline withdrawal latency measured with a noxious heat stimulus in rats given systemic morphine plus intrathecal injections of a vehicle (n = 9 rats), 5 μg U0126 (n = 7 rats), 5 μg SB203580 (n = 7 rats), 30 μg SP600125 (n = 8 rats), or combination of 5 μg U0126, 5 μg SB203580, and 30 μg SP600125 (n = 8 rats). C , time course of the analgesic effect of morphine on the mechanical nociceptive withdrawal threshold in rats given intrathecal injections of a vehicle (n = 9 rats), 5 μg U0126 (n = 7 rats), 5 μg SB203580 (n = 7 rats), 30 μg SP600125 (n = 8 rats), or combination of 5 μg U0126, 5 μg SB203580, and 30 μg SP600125 (n = 8 rats). D , time course of the analgesic effect of morphine on the heat withdrawal latency in rats given intrathecal injections of a vehicle (n = 9 rats), 5 μg U0126 (n = 7 rats), 5 μg SB203580 (n = 7 rats), 30 μg SP600125 (n = 8 rats), or combination of 5 μg U0126, 5 μg SB203580, and 30 μg SP600125 (n = 8 rats). The baseline withdrawal threshold was measured before morphine injection each day, and the analgesic effect of morphine was tested 30 min after each morphine injection (5 mg/kg, intraperitoneal). Data are presented as means ± SEM. *p

    Article Snippet: U0126 (5 μg; EMD-Calbiochem, La Jolla, CA), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580, 5 μg; EMD-Calbiochem), and SP600125 (30 μg; Cayman Chemical, Ann Arbor, MI) were dissolved in DMSO and intrathecally injected 20 min before morphine administration on each testing day (days 1–8).

    Techniques: Inhibition, Activity Assay, Injection

    Effect of SP600125 on proliferation, viability, polyploidy, expression of cyclin B1, cyclin D3, c-Myc, and survivin, and the translation-related proteins of Dami and CMK cells. Dami and CMK cells were seeded at 2×10 5 /ml in RPMI 1640 medium containing 10% FCS and treated with SP600125 at different concentrations for different periods of time as indicated. Dami and CMK cells treated with DMSO were used as a control. (A, B) The cell number and viability, presented as the mean±SEM, were determined from 4 separate experiments. (C) Representative DNA histograms of SP600125-induced Dami and CMK cells analyzed by flow cytometry. (D) Morphology was analyzed by Wright-Giemsa staining of cytocentrifuged preparations of Dami and CMK cells induced by SP600125 or nocodazole (Original magnification, 1000×). The Dami and CMK cells treated with DMSO or with SP600125 were lysed, and equal amounts of protein were loaded for western blots to evaluate the protein levels of cyclin B1, cyclin D3, c-Myc, and survivin (E), and the phosphorylation and protein levels of S6K1, eIF4E and 4E-BP1(F). β-actin was used as an internal control.

    Journal: PLoS ONE

    Article Title: Phosphorylation of Ribosomal Protein S6 Kinase 1 at Thr421/Ser424 and Dephosphorylation at Thr389 Regulates SP600125-Induced Polyploidization of Megakaryocytic Cell Lines

    doi: 10.1371/journal.pone.0114389

    Figure Lengend Snippet: Effect of SP600125 on proliferation, viability, polyploidy, expression of cyclin B1, cyclin D3, c-Myc, and survivin, and the translation-related proteins of Dami and CMK cells. Dami and CMK cells were seeded at 2×10 5 /ml in RPMI 1640 medium containing 10% FCS and treated with SP600125 at different concentrations for different periods of time as indicated. Dami and CMK cells treated with DMSO were used as a control. (A, B) The cell number and viability, presented as the mean±SEM, were determined from 4 separate experiments. (C) Representative DNA histograms of SP600125-induced Dami and CMK cells analyzed by flow cytometry. (D) Morphology was analyzed by Wright-Giemsa staining of cytocentrifuged preparations of Dami and CMK cells induced by SP600125 or nocodazole (Original magnification, 1000×). The Dami and CMK cells treated with DMSO or with SP600125 were lysed, and equal amounts of protein were loaded for western blots to evaluate the protein levels of cyclin B1, cyclin D3, c-Myc, and survivin (E), and the phosphorylation and protein levels of S6K1, eIF4E and 4E-BP1(F). β-actin was used as an internal control.

    Article Snippet: SP600125 and H-89 were purchased from LC Laboratories (Woburn, MA).

    Techniques: Expressing, Flow Cytometry, Cytometry, Staining, Western Blot

    Effects of S6K1 mutant plasmids on SP600125-induced Dami cells. Dami cells were transfected with or without S6K1-WT, S6K1-T389E, S6K1-T389A and S6K1-D3E and cultured overnight. The cells were then induced with SP600125 for 72 hours after pretreatment with or without LY294002 for 1 hour. (A) Representative DNA histograms of Dami cells in each condition. (B) The data are presented as the mean±SEM of the level of polyploidy and were obtained from 4 separate experiments; *p

    Journal: PLoS ONE

    Article Title: Phosphorylation of Ribosomal Protein S6 Kinase 1 at Thr421/Ser424 and Dephosphorylation at Thr389 Regulates SP600125-Induced Polyploidization of Megakaryocytic Cell Lines

    doi: 10.1371/journal.pone.0114389

    Figure Lengend Snippet: Effects of S6K1 mutant plasmids on SP600125-induced Dami cells. Dami cells were transfected with or without S6K1-WT, S6K1-T389E, S6K1-T389A and S6K1-D3E and cultured overnight. The cells were then induced with SP600125 for 72 hours after pretreatment with or without LY294002 for 1 hour. (A) Representative DNA histograms of Dami cells in each condition. (B) The data are presented as the mean±SEM of the level of polyploidy and were obtained from 4 separate experiments; *p

    Article Snippet: SP600125 and H-89 were purchased from LC Laboratories (Woburn, MA).

    Techniques: Mutagenesis, Transfection, Cell Culture

    H-89 blocked polyploidization of the SP600125 treated-Dami and CMK cells. Dami and CMK cells were treated with SP600125 at 32 µM for 72 hours after pretreatment without or with H-89 at 10 µM or 20 µM for 1 hour. Dami and CMK cells treated with DMSO were used as the vehicle-treated control, and Dami or CMK cells treated with H-89 alone were used as the pretreatment control. After incubation, the cells were fixed, stained with PI and analyzed with a flow cytometer for DNA ploidy (A). The data are presented as the mean±SEM polyploidy and were obtained from 4 separate experiments (B). All bar graphs depict the means ± SEM; *p

    Journal: PLoS ONE

    Article Title: Phosphorylation of Ribosomal Protein S6 Kinase 1 at Thr421/Ser424 and Dephosphorylation at Thr389 Regulates SP600125-Induced Polyploidization of Megakaryocytic Cell Lines

    doi: 10.1371/journal.pone.0114389

    Figure Lengend Snippet: H-89 blocked polyploidization of the SP600125 treated-Dami and CMK cells. Dami and CMK cells were treated with SP600125 at 32 µM for 72 hours after pretreatment without or with H-89 at 10 µM or 20 µM for 1 hour. Dami and CMK cells treated with DMSO were used as the vehicle-treated control, and Dami or CMK cells treated with H-89 alone were used as the pretreatment control. After incubation, the cells were fixed, stained with PI and analyzed with a flow cytometer for DNA ploidy (A). The data are presented as the mean±SEM polyploidy and were obtained from 4 separate experiments (B). All bar graphs depict the means ± SEM; *p

    Article Snippet: SP600125 and H-89 were purchased from LC Laboratories (Woburn, MA).

    Techniques: Incubation, Staining, Flow Cytometry, Cytometry

    H-89 blocked the polyploidization of Dami and CMK cells independent of PKA. Dami and CMK cells were treated with SP600125 at 32 µM for 72 hours after pretreatment with or without H-89 at increasing concentrations as indicated for 1 hour. Dami or CMK cells treated with DMSO were used as the vehicle-treated control, and Dami or CMK cells treated with H-89 alone were used as the pretreatment control. (A) The cells were lysed, and equal amounts of protein were analyzed by western blot for Phospho-PKA Substrate (RRXS*/T*), Phospho-(Ser/Thr) PKA Substrate, S6K1, phospho-S6K1 (Thr421/Ser424), and phospho-S6K1 (Thr389). (B) The cells were fixed, stained with PI and analyzed with a flow cytometer for DNA ploidy.

    Journal: PLoS ONE

    Article Title: Phosphorylation of Ribosomal Protein S6 Kinase 1 at Thr421/Ser424 and Dephosphorylation at Thr389 Regulates SP600125-Induced Polyploidization of Megakaryocytic Cell Lines

    doi: 10.1371/journal.pone.0114389

    Figure Lengend Snippet: H-89 blocked the polyploidization of Dami and CMK cells independent of PKA. Dami and CMK cells were treated with SP600125 at 32 µM for 72 hours after pretreatment with or without H-89 at increasing concentrations as indicated for 1 hour. Dami or CMK cells treated with DMSO were used as the vehicle-treated control, and Dami or CMK cells treated with H-89 alone were used as the pretreatment control. (A) The cells were lysed, and equal amounts of protein were analyzed by western blot for Phospho-PKA Substrate (RRXS*/T*), Phospho-(Ser/Thr) PKA Substrate, S6K1, phospho-S6K1 (Thr421/Ser424), and phospho-S6K1 (Thr389). (B) The cells were fixed, stained with PI and analyzed with a flow cytometer for DNA ploidy.

    Article Snippet: SP600125 and H-89 were purchased from LC Laboratories (Woburn, MA).

    Techniques: Western Blot, Staining, Flow Cytometry, Cytometry

    Partial inhibition of phosphorylation of S6K1 at Thr421/Ser424 is not sufficient to block polyploidization. Dami and CMK cells were incubated with SP600125 at 32 µM for 72 hours after pretreatment with or without PD184352 (2 µM), U0126 (10 µM), or LY294002 (30 µM) for 1 hour. Dami and CMK cells treated with DMSO were used as the control. After incubation, the cells were fixed, stained with PI and analyzed with a flow cytometer for DNA ploidy. (A) A typical representative DNA histogram. (B) The data are presented as the mean±SEM level of polyploidy and were obtained from 4 separate experiments; *p

    Journal: PLoS ONE

    Article Title: Phosphorylation of Ribosomal Protein S6 Kinase 1 at Thr421/Ser424 and Dephosphorylation at Thr389 Regulates SP600125-Induced Polyploidization of Megakaryocytic Cell Lines

    doi: 10.1371/journal.pone.0114389

    Figure Lengend Snippet: Partial inhibition of phosphorylation of S6K1 at Thr421/Ser424 is not sufficient to block polyploidization. Dami and CMK cells were incubated with SP600125 at 32 µM for 72 hours after pretreatment with or without PD184352 (2 µM), U0126 (10 µM), or LY294002 (30 µM) for 1 hour. Dami and CMK cells treated with DMSO were used as the control. After incubation, the cells were fixed, stained with PI and analyzed with a flow cytometer for DNA ploidy. (A) A typical representative DNA histogram. (B) The data are presented as the mean±SEM level of polyploidy and were obtained from 4 separate experiments; *p

    Article Snippet: SP600125 and H-89 were purchased from LC Laboratories (Woburn, MA).

    Techniques: Inhibition, Blocking Assay, Incubation, Staining, Flow Cytometry, Cytometry

    Evaluation of inhibitory effect of SP600125 on penetrating corneal wound induced corneal scarring in Wistar rats. A penetrating corneal wound model was created with Wistar rats and inhibition of JNK activation by subconjunctival injection of SP600125 daily post-wounding. (A) HE stained histological sections showed that corneal epithelial healing was almost complete at 3 d in both groups. Subconjunctival injection of SP600125 after injury daily markedly improved the architecture of cornea and reduced scarring and did not have a significant impact on wound stroma healing at 14 d (B), 21 d (C). n = 4 rat in each group, Bars: 40 µm.

    Journal: PLoS ONE

    Article Title: Activation of JNK Signaling Mediates Connective Tissue Growth Factor Expression and Scar Formation in Corneal Wound Healing

    doi: 10.1371/journal.pone.0032128

    Figure Lengend Snippet: Evaluation of inhibitory effect of SP600125 on penetrating corneal wound induced corneal scarring in Wistar rats. A penetrating corneal wound model was created with Wistar rats and inhibition of JNK activation by subconjunctival injection of SP600125 daily post-wounding. (A) HE stained histological sections showed that corneal epithelial healing was almost complete at 3 d in both groups. Subconjunctival injection of SP600125 after injury daily markedly improved the architecture of cornea and reduced scarring and did not have a significant impact on wound stroma healing at 14 d (B), 21 d (C). n = 4 rat in each group, Bars: 40 µm.

    Article Snippet: PD98059 and SB203580 were purchased from Calbiochem (San Diego, CA), SP600125 was obtained from A. G. Scientific, Inc (San Diego, CA).

    Techniques: Inhibition, Activation Assay, Injection, Staining

    Effect of MAPK-specific inhibitors on expression of fibronectin and collagen I induced by TGF -β1 in THSF cells. THSF cells were pretreated with ERK inhibitor (PD98059, 30 µM), p38MAPK inhibitor (SB203580, 10 µM) or JNK inhibitor (SP600125, 30 µM) for 1 hour, respectively. Subsequently they were treated with TGF-β1 (3 ng/ml) for 24 hour. Expression of fibronectin and collagen I protein was determined by Western blot analysis. (A) SB203580 or SP600125 significant inhibited TGF-β1 induced fibronectin expression. (B) SP600125 significant suppressed TGF-β1 induced collagen I expression. Data are representative of three independent experiments. *, P

    Journal: PLoS ONE

    Article Title: Activation of JNK Signaling Mediates Connective Tissue Growth Factor Expression and Scar Formation in Corneal Wound Healing

    doi: 10.1371/journal.pone.0032128

    Figure Lengend Snippet: Effect of MAPK-specific inhibitors on expression of fibronectin and collagen I induced by TGF -β1 in THSF cells. THSF cells were pretreated with ERK inhibitor (PD98059, 30 µM), p38MAPK inhibitor (SB203580, 10 µM) or JNK inhibitor (SP600125, 30 µM) for 1 hour, respectively. Subsequently they were treated with TGF-β1 (3 ng/ml) for 24 hour. Expression of fibronectin and collagen I protein was determined by Western blot analysis. (A) SB203580 or SP600125 significant inhibited TGF-β1 induced fibronectin expression. (B) SP600125 significant suppressed TGF-β1 induced collagen I expression. Data are representative of three independent experiments. *, P

    Article Snippet: PD98059 and SB203580 were purchased from Calbiochem (San Diego, CA), SP600125 was obtained from A. G. Scientific, Inc (San Diego, CA).

    Techniques: Expressing, Western Blot

    Evaluation of inhibitory effect of SP600125 on penetrating corneal wound induced JNK phosphorylation in Wistar rats. (A) p-JNK was examined by immunofluorescence analysis. Five micrometer corneal sections were stained with antibodies to p-JNK (green) as well as with nuclear staining dye (blue). There was little expression of P-JNK in the normal mice cornea of normal rats. (B) Penetrating injury was made in the central cornea of Wistar rats, control group received daily subconjunctival injection of physiological saline. Positive p-JNK staining was markedly increased in the corneal stroma at 1 d after injury. (C) Subconjunctival injection of SP600125 notably inhibited JNK activation in the corneal stroma at 1 d after injury. n = 4 rat in each group, Bars: 40 µm.

    Journal: PLoS ONE

    Article Title: Activation of JNK Signaling Mediates Connective Tissue Growth Factor Expression and Scar Formation in Corneal Wound Healing

    doi: 10.1371/journal.pone.0032128

    Figure Lengend Snippet: Evaluation of inhibitory effect of SP600125 on penetrating corneal wound induced JNK phosphorylation in Wistar rats. (A) p-JNK was examined by immunofluorescence analysis. Five micrometer corneal sections were stained with antibodies to p-JNK (green) as well as with nuclear staining dye (blue). There was little expression of P-JNK in the normal mice cornea of normal rats. (B) Penetrating injury was made in the central cornea of Wistar rats, control group received daily subconjunctival injection of physiological saline. Positive p-JNK staining was markedly increased in the corneal stroma at 1 d after injury. (C) Subconjunctival injection of SP600125 notably inhibited JNK activation in the corneal stroma at 1 d after injury. n = 4 rat in each group, Bars: 40 µm.

    Article Snippet: PD98059 and SB203580 were purchased from Calbiochem (San Diego, CA), SP600125 was obtained from A. G. Scientific, Inc (San Diego, CA).

    Techniques: Immunofluorescence, Staining, Expressing, Mouse Assay, Injection, Activation Assay

    SP600125 inhibited TGF-β1 induced CTGF expression and secretion in THSF cells. THSF cells were pretreated with ERK inhibitor (PD98059, 30 µM), p38MAPK inhibitor (SB203580, 10 µM) or JNK inhibitor (SP600125, 30 µM) for 1 hour, respectively. Subsequently they were treated with TGF-β1 (3 ng/ml) for 24 hour. (A) CTGF mRNA expression levels were detected by real time PCR. (B) CTGF protein was measured in conditioned medium samples using ELISA and results were normalized for total protein concentration. Data are representative of tree independent experiments. *, P

    Journal: PLoS ONE

    Article Title: Activation of JNK Signaling Mediates Connective Tissue Growth Factor Expression and Scar Formation in Corneal Wound Healing

    doi: 10.1371/journal.pone.0032128

    Figure Lengend Snippet: SP600125 inhibited TGF-β1 induced CTGF expression and secretion in THSF cells. THSF cells were pretreated with ERK inhibitor (PD98059, 30 µM), p38MAPK inhibitor (SB203580, 10 µM) or JNK inhibitor (SP600125, 30 µM) for 1 hour, respectively. Subsequently they were treated with TGF-β1 (3 ng/ml) for 24 hour. (A) CTGF mRNA expression levels were detected by real time PCR. (B) CTGF protein was measured in conditioned medium samples using ELISA and results were normalized for total protein concentration. Data are representative of tree independent experiments. *, P

    Article Snippet: PD98059 and SB203580 were purchased from Calbiochem (San Diego, CA), SP600125 was obtained from A. G. Scientific, Inc (San Diego, CA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Protein Concentration

    Evaluation of inhibitory effect of SP600125 on penetrating corneal wound induced CTGF expression in Wistar rats. (A) Real time PCR was used to measure CTGF mRNA expression. The expression of CTGF mRNA was upregulated significantly in wounded corneas and reached a peak at 3 d after injury, subconjunctival injection of SP600125 significantly inhibited injury-induced CTGF mRNA expression. (B) Real time PCR was used to measure TGF-β1 mRNA expression. Subconjunctival injection of SP600125 did not influence the expression of TGF-β1. Data are representative of three independent experiments. *, P

    Journal: PLoS ONE

    Article Title: Activation of JNK Signaling Mediates Connective Tissue Growth Factor Expression and Scar Formation in Corneal Wound Healing

    doi: 10.1371/journal.pone.0032128

    Figure Lengend Snippet: Evaluation of inhibitory effect of SP600125 on penetrating corneal wound induced CTGF expression in Wistar rats. (A) Real time PCR was used to measure CTGF mRNA expression. The expression of CTGF mRNA was upregulated significantly in wounded corneas and reached a peak at 3 d after injury, subconjunctival injection of SP600125 significantly inhibited injury-induced CTGF mRNA expression. (B) Real time PCR was used to measure TGF-β1 mRNA expression. Subconjunctival injection of SP600125 did not influence the expression of TGF-β1. Data are representative of three independent experiments. *, P

    Article Snippet: PD98059 and SB203580 were purchased from Calbiochem (San Diego, CA), SP600125 was obtained from A. G. Scientific, Inc (San Diego, CA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Injection

    Inhibitory effect of PD98059, SB203580 or SP600125 on TGF-β1 induced MAPK pathways phosphorylation in THSF cells. THSF cells were pretreated with ERK inhibitor (PD98059, 30 µM), p38 inhibitor (SB203580, 10 µM) or JNK inhibitor (SP600125, 30 µM) for 1 h, respectively. Then the cells were subsequently treated with TGF-β1 (3 ng/ml) for 15 min (A) or 30 min (B, C), followed by protein extraction and Western blot analysis for total and phosphorylated ERK1/2 (A), p38 (B), and JNK (C). Data are representative of three independent experiments. *, P

    Journal: PLoS ONE

    Article Title: Activation of JNK Signaling Mediates Connective Tissue Growth Factor Expression and Scar Formation in Corneal Wound Healing

    doi: 10.1371/journal.pone.0032128

    Figure Lengend Snippet: Inhibitory effect of PD98059, SB203580 or SP600125 on TGF-β1 induced MAPK pathways phosphorylation in THSF cells. THSF cells were pretreated with ERK inhibitor (PD98059, 30 µM), p38 inhibitor (SB203580, 10 µM) or JNK inhibitor (SP600125, 30 µM) for 1 h, respectively. Then the cells were subsequently treated with TGF-β1 (3 ng/ml) for 15 min (A) or 30 min (B, C), followed by protein extraction and Western blot analysis for total and phosphorylated ERK1/2 (A), p38 (B), and JNK (C). Data are representative of three independent experiments. *, P

    Article Snippet: PD98059 and SB203580 were purchased from Calbiochem (San Diego, CA), SP600125 was obtained from A. G. Scientific, Inc (San Diego, CA).

    Techniques: Protein Extraction, Western Blot

    Whole cigarette smoke promoted human β-defensin-2 and -3 expression through the ERK1/2 MAP kinase and NFκB signaling pathways. Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NFκB inhibitor (IKK-2) for 45 min before exposure to cigarette smoke for 15 or 30 min. Six hours later, total RNA was extracted, and HBD gene expression was analyzed by qRT-PCR (n = 6). *, p

    Journal: PLoS ONE

    Article Title: Whole Cigarette Smoke Increased the Expression of TLRs, HBDs, and Proinflammory Cytokines by Human Gingival Epithelial Cells through Different Signaling Pathways

    doi: 10.1371/journal.pone.0052614

    Figure Lengend Snippet: Whole cigarette smoke promoted human β-defensin-2 and -3 expression through the ERK1/2 MAP kinase and NFκB signaling pathways. Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NFκB inhibitor (IKK-2) for 45 min before exposure to cigarette smoke for 15 or 30 min. Six hours later, total RNA was extracted, and HBD gene expression was analyzed by qRT-PCR (n = 6). *, p

    Article Snippet: PD98059, an inhibitor of ERK1/2 kinase, and SB202190, a specific inhibitor for p38 kinase, were obtained from EMD Millipore (Billerica, MA, USA), while SP600125, a JNK inhibitor, and IKK-2 inhibitor to block NFκB were obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA).

    Techniques: Expressing, Incubation, Quantitative RT-PCR

    Whole cigarette smoke promoted human IL-1β and IL-6 secretions through the ERK1/2 MAP kinase and NFκB signaling pathways. Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NFκB inhibitor (IKK-2) for 45 min before exposure or not to cigarette smoke for 15 min. supernatants were collected 24 h later and used to measure the secretion of IL-1β and IL-6 by ELISA kits. Data are expressed the means+SD, (n = 4).

    Journal: PLoS ONE

    Article Title: Whole Cigarette Smoke Increased the Expression of TLRs, HBDs, and Proinflammory Cytokines by Human Gingival Epithelial Cells through Different Signaling Pathways

    doi: 10.1371/journal.pone.0052614

    Figure Lengend Snippet: Whole cigarette smoke promoted human IL-1β and IL-6 secretions through the ERK1/2 MAP kinase and NFκB signaling pathways. Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NFκB inhibitor (IKK-2) for 45 min before exposure or not to cigarette smoke for 15 min. supernatants were collected 24 h later and used to measure the secretion of IL-1β and IL-6 by ELISA kits. Data are expressed the means+SD, (n = 4).

    Article Snippet: PD98059, an inhibitor of ERK1/2 kinase, and SB202190, a specific inhibitor for p38 kinase, were obtained from EMD Millipore (Billerica, MA, USA), while SP600125, a JNK inhibitor, and IKK-2 inhibitor to block NFκB were obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA).

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay

    Whole cigarette smoke promoted human β-defensin-2 and -3 secretions through the ERK1/2 MAP kinase and NFκB signaling pathways. Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NF-κB inhibitor (IKK-2) for 45 min before exposure or not to cigarette smoke for 15 mn. 24 hours later, supernatants were collected and used to measure the β-defensin secretion levels by ELISA kits. Data are expressed the means+SD, (n = 4).

    Journal: PLoS ONE

    Article Title: Whole Cigarette Smoke Increased the Expression of TLRs, HBDs, and Proinflammory Cytokines by Human Gingival Epithelial Cells through Different Signaling Pathways

    doi: 10.1371/journal.pone.0052614

    Figure Lengend Snippet: Whole cigarette smoke promoted human β-defensin-2 and -3 secretions through the ERK1/2 MAP kinase and NFκB signaling pathways. Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NF-κB inhibitor (IKK-2) for 45 min before exposure or not to cigarette smoke for 15 mn. 24 hours later, supernatants were collected and used to measure the β-defensin secretion levels by ELISA kits. Data are expressed the means+SD, (n = 4).

    Article Snippet: PD98059, an inhibitor of ERK1/2 kinase, and SB202190, a specific inhibitor for p38 kinase, were obtained from EMD Millipore (Billerica, MA, USA), while SP600125, a JNK inhibitor, and IKK-2 inhibitor to block NFκB were obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA).

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay

    TLR modulation by whole cigarette smoke leads to ERK1/2, p38, JNK, MAP kinase and NFκB activations. Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NFκB inhibitor (IKK-2) for 45 min before exposure to cigarette smoke for 15 or 30 min. The cells were then incubated 6 h, after which the total RNA was extracted, and TLR expression was analyzed by qRT-PCR (n = 6). *, p

    Journal: PLoS ONE

    Article Title: Whole Cigarette Smoke Increased the Expression of TLRs, HBDs, and Proinflammory Cytokines by Human Gingival Epithelial Cells through Different Signaling Pathways

    doi: 10.1371/journal.pone.0052614

    Figure Lengend Snippet: TLR modulation by whole cigarette smoke leads to ERK1/2, p38, JNK, MAP kinase and NFκB activations. Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NFκB inhibitor (IKK-2) for 45 min before exposure to cigarette smoke for 15 or 30 min. The cells were then incubated 6 h, after which the total RNA was extracted, and TLR expression was analyzed by qRT-PCR (n = 6). *, p

    Article Snippet: PD98059, an inhibitor of ERK1/2 kinase, and SB202190, a specific inhibitor for p38 kinase, were obtained from EMD Millipore (Billerica, MA, USA), while SP600125, a JNK inhibitor, and IKK-2 inhibitor to block NFκB were obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA).

    Techniques: Incubation, Expressing, Quantitative RT-PCR

    Whole cigarette smoke promoted IL-1β and IL-6 expression through the ERK1/2 and NFκB signaling pathways. Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NFκB inhibitor (IKK-2) for 45 min before exposure to cigarette smoke for 15 or 30 min. Six hours later, total RNA was extracted, and cytokine gene expression was analyzed by qRT-PCR (n = 6). *, p

    Journal: PLoS ONE

    Article Title: Whole Cigarette Smoke Increased the Expression of TLRs, HBDs, and Proinflammory Cytokines by Human Gingival Epithelial Cells through Different Signaling Pathways

    doi: 10.1371/journal.pone.0052614

    Figure Lengend Snippet: Whole cigarette smoke promoted IL-1β and IL-6 expression through the ERK1/2 and NFκB signaling pathways. Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NFκB inhibitor (IKK-2) for 45 min before exposure to cigarette smoke for 15 or 30 min. Six hours later, total RNA was extracted, and cytokine gene expression was analyzed by qRT-PCR (n = 6). *, p

    Article Snippet: PD98059, an inhibitor of ERK1/2 kinase, and SB202190, a specific inhibitor for p38 kinase, were obtained from EMD Millipore (Billerica, MA, USA), while SP600125, a JNK inhibitor, and IKK-2 inhibitor to block NFκB were obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA).

    Techniques: Expressing, Incubation, Quantitative RT-PCR

    TGF-β-associated gene expression profiles induced by treatment with mitogen-activated protein kinase inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured for 15-60 min in the presence or absence of 10 µ M U0126 (ERK i ) or 10 µ M SP600125 (JNK i ) and subjected to western blot analysis for p-ERK1/2. Histone H3 was used as a loading control. Fibroblasts incubated with recombinant human FGF2 for 30 min were used as a positive control for p-ERK1/2 expression. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ) or 10 µ M SB202190 (p38 i ) until day 4. Expression levels of TGF-β-associated transcripts TGFB1 , TGFB2 , TGFBR1 , TGFBR2 and TGFBR3 were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Expression levels per transcript were compared using an one-way analysis of variance and post-hoc Holm-Sidak analysis. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts

    doi: 10.3892/ijmm.2018.3949

    Figure Lengend Snippet: TGF-β-associated gene expression profiles induced by treatment with mitogen-activated protein kinase inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured for 15-60 min in the presence or absence of 10 µ M U0126 (ERK i ) or 10 µ M SP600125 (JNK i ) and subjected to western blot analysis for p-ERK1/2. Histone H3 was used as a loading control. Fibroblasts incubated with recombinant human FGF2 for 30 min were used as a positive control for p-ERK1/2 expression. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ) or 10 µ M SB202190 (p38 i ) until day 4. Expression levels of TGF-β-associated transcripts TGFB1 , TGFB2 , TGFBR1 , TGFBR2 and TGFBR3 were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Expression levels per transcript were compared using an one-way analysis of variance and post-hoc Holm-Sidak analysis. * P

    Article Snippet: The MAPK inhibitors used were U0126, SP600125 and SB202190 (all from Santa Cruz Biotechnology, Inc.).

    Techniques: Expressing, Cell Culture, Western Blot, Incubation, Recombinant, Positive Control, Real-time Polymerase Chain Reaction

    Effects of MAPK inhibitors on fibroblast activation. (A–D) CRL-2097 human dermal fibroblasts were cultured under control conditions, or in the presence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), 10 µ M SB202190 (p38 i ) or 10 ng/ml exogenous recombinant human TGF-β1, and harvested at day 4 in culture. (A) Expression of myofibroblast marker transcripts ACTA2 , CNN1 and TAGLN were determined by reverse transcription-quantitative polymerase chain reaction, normalized to the expression of GAPDH , and expressed as fold change relative to the expression of control samples. There were 4 biological replicates/condition. Error bars represent standard deviation. Statistics were performed by one-way ANOVA with multiple comparisons using the Holm-Sidak post-hoc test. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts

    doi: 10.3892/ijmm.2018.3949

    Figure Lengend Snippet: Effects of MAPK inhibitors on fibroblast activation. (A–D) CRL-2097 human dermal fibroblasts were cultured under control conditions, or in the presence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), 10 µ M SB202190 (p38 i ) or 10 ng/ml exogenous recombinant human TGF-β1, and harvested at day 4 in culture. (A) Expression of myofibroblast marker transcripts ACTA2 , CNN1 and TAGLN were determined by reverse transcription-quantitative polymerase chain reaction, normalized to the expression of GAPDH , and expressed as fold change relative to the expression of control samples. There were 4 biological replicates/condition. Error bars represent standard deviation. Statistics were performed by one-way ANOVA with multiple comparisons using the Holm-Sidak post-hoc test. * P

    Article Snippet: The MAPK inhibitors used were U0126, SP600125 and SB202190 (all from Santa Cruz Biotechnology, Inc.).

    Techniques: Activation Assay, Cell Culture, Recombinant, Expressing, Marker, Real-time Polymerase Chain Reaction, Standard Deviation

    Effects of mitogen-activated protein kinase inhibitors on the ECM. CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ) or 10 µ M SB202190 (p38 i ), and harvested at day 4. Expression levels of ECM-associated transcripts (A) Col1A1 , Col1A2 , Col3A1 , MMP1 and LOX , and (B) MYOCD , CCN2 and ED-A Fn were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Expression levels per transcript were compared using a one-way analysis of variance and post-hoc Holm-Sidak analysis. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts

    doi: 10.3892/ijmm.2018.3949

    Figure Lengend Snippet: Effects of mitogen-activated protein kinase inhibitors on the ECM. CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ) or 10 µ M SB202190 (p38 i ), and harvested at day 4. Expression levels of ECM-associated transcripts (A) Col1A1 , Col1A2 , Col3A1 , MMP1 and LOX , and (B) MYOCD , CCN2 and ED-A Fn were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Expression levels per transcript were compared using a one-way analysis of variance and post-hoc Holm-Sidak analysis. * P

    Article Snippet: The MAPK inhibitors used were U0126, SP600125 and SB202190 (all from Santa Cruz Biotechnology, Inc.).

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

    Cooperative effects of mitogen-activated protein kinase inhibition and exogenous TGF-β on fibroblast activation. CRL-2097 human dermal fibroblasts were cultured under control conditions or in the presence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), and/or 10 ng/ml exogenous recombinant human TGF-β1, and analyzed at day 4 in culture. (A) Protein lysates were examined for expression of myofibroblast-associated protein markers α-SMA, calponin, and SM22α by western blot analysis. Histone H3 was used as a loading control. (B) Fibroblasts were fixed and stained for the myofibroblast marker protein α-SMA and nuclei were counterstained with Hoechst. Scale bar, 100 µ m. TGF-β, transforming growth factor-β; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; α-SMA, smooth muscle α actin; i , inhibitor.

    Journal: International Journal of Molecular Medicine

    Article Title: Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts

    doi: 10.3892/ijmm.2018.3949

    Figure Lengend Snippet: Cooperative effects of mitogen-activated protein kinase inhibition and exogenous TGF-β on fibroblast activation. CRL-2097 human dermal fibroblasts were cultured under control conditions or in the presence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), and/or 10 ng/ml exogenous recombinant human TGF-β1, and analyzed at day 4 in culture. (A) Protein lysates were examined for expression of myofibroblast-associated protein markers α-SMA, calponin, and SM22α by western blot analysis. Histone H3 was used as a loading control. (B) Fibroblasts were fixed and stained for the myofibroblast marker protein α-SMA and nuclei were counterstained with Hoechst. Scale bar, 100 µ m. TGF-β, transforming growth factor-β; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; α-SMA, smooth muscle α actin; i , inhibitor.

    Article Snippet: The MAPK inhibitors used were U0126, SP600125 and SB202190 (all from Santa Cruz Biotechnology, Inc.).

    Techniques: Inhibition, Activation Assay, Cell Culture, Recombinant, Expressing, Western Blot, Staining, Marker

    Antagonism of p38 inhibitor towards ERK and JNK inhibitor-mediated fibroblast activation. CRL-2097 human dermal fibroblasts were cultured under control conditions or in the presence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), and/or 10 µ M SB202190 (p38 i ), and analyzed at day 4 in culture. (A) Expression levels of myofibroblast-associated transcripts ACTA2 , CNN1 and TAGLN were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Student’s t-tests were performed in order to compare the expression between each culture condition and its p38 i -treated counterpart. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts

    doi: 10.3892/ijmm.2018.3949

    Figure Lengend Snippet: Antagonism of p38 inhibitor towards ERK and JNK inhibitor-mediated fibroblast activation. CRL-2097 human dermal fibroblasts were cultured under control conditions or in the presence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), and/or 10 µ M SB202190 (p38 i ), and analyzed at day 4 in culture. (A) Expression levels of myofibroblast-associated transcripts ACTA2 , CNN1 and TAGLN were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Student’s t-tests were performed in order to compare the expression between each culture condition and its p38 i -treated counterpart. * P

    Article Snippet: The MAPK inhibitors used were U0126, SP600125 and SB202190 (all from Santa Cruz Biotechnology, Inc.).

    Techniques: Activation Assay, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

    Effects of TGF-βR1 inhibition on fibroblast activation induced by ERK or JNK inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of the indicated concentration of the TGF-βR1 inhibitor RepSox until day 4 and then subjected to Western blot analysis for myofibroblast markers α-SMA and calponin. Histone H3 was used as a loading control. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), 10 ng/ml TGF-β1 and 100 nM RepSox until day 4 and then subjected to western blot analysis for myofibroblast marker α-SMA. Histone H3 was used as a loading control. (C) Western blots were analyzed using densitometry, and relative density of α-SMA/Histone H3 was compared for each mitogen-activated protein kinase inhibitor-treated or TGF-β1-treated sample compared with its corresponding TGF-βR1 inhibitor-treated sample. Statistical significance was determined using a two-tailed Student’s t-test. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts

    doi: 10.3892/ijmm.2018.3949

    Figure Lengend Snippet: Effects of TGF-βR1 inhibition on fibroblast activation induced by ERK or JNK inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of the indicated concentration of the TGF-βR1 inhibitor RepSox until day 4 and then subjected to Western blot analysis for myofibroblast markers α-SMA and calponin. Histone H3 was used as a loading control. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), 10 ng/ml TGF-β1 and 100 nM RepSox until day 4 and then subjected to western blot analysis for myofibroblast marker α-SMA. Histone H3 was used as a loading control. (C) Western blots were analyzed using densitometry, and relative density of α-SMA/Histone H3 was compared for each mitogen-activated protein kinase inhibitor-treated or TGF-β1-treated sample compared with its corresponding TGF-βR1 inhibitor-treated sample. Statistical significance was determined using a two-tailed Student’s t-test. * P

    Article Snippet: The MAPK inhibitors used were U0126, SP600125 and SB202190 (all from Santa Cruz Biotechnology, Inc.).

    Techniques: Inhibition, Activation Assay, Cell Culture, Concentration Assay, Western Blot, Marker, Two Tailed Test

    SP600125 or PMA induce Prss14/epithin ectodomain shedding. A , diagram of Prss14/epithin domain structure and processed forms. Epi-S', Epi-S, aEpi-S are indicated. B , effects of MAP kinase inhibitors in Prss14/epithin shedding. 427.1.86 cells were treated with 10 μ m PD98059 ( PD ), 20 μ m SB203580 ( SB ), and 5 μ m SP600125 ( SP ) for 30 min and then with or without 0.5 μ m PMA for an additional 2 h. C , dose- and time-dependent profiles of Epi-S' and Epi-S. 427.1.86 cells were treated with the indicated concentration of SP600125 for 2 h ( left panel ) and 5 μ m SP600125 up to 2 h ( right panel ). D , 427.1.86 cells were pretreated with 10 μ m TAPI-0 ( TPI ) for 30 min, and then cells were treated with 5 μ m SP600125 or 0.5 μ m PMA for an additional 2 h. The TACE inhibitor abolished the appearance of Epi-S' while retaining Epi-S in the cell, regardless of shedding induction methods, PMA, and SP600125. E , removal of TACE with siRNA abolished shedding of Prss14/epithin. 427.1.86 cells were transfected with 200 n m TACE siRNA for 48 h, starved of serum for 4 h, and then treated with 5 μΜ SP600125 or 0.5 μΜ PMA for 2 h. The control samples were treated exactly the same way except for transfection with nontargeting control siRNA. F , SP600125 dose-dependently induced Epi-S' and aEpi-S in T47D cells. SP600125 was treated for 2 h. G , SP600125 time-dependently (with 5 μ m ) and dose-dependently (for 2 h) induced Epi-S' and aEpi-S in 4T1 cells. In all panels, Epi-S' collected from culture medium and other proteins, including Epi-S, from cell lysates were detected by Western blot analysis. Tubulin or β-actin was used for normalization. TM , transmembrane domain; SEA , sperm protein, enterokinase, and agrin domain; CUB1 , CUB2 , complement subcomponent C1r/C1s domain; 1, 2, 3, 4 LDLRA , low-density lipoprotein receptor class A repeats.

    Journal: The Journal of Biological Chemistry

    Article Title: A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CβII

    doi: 10.1074/jbc.RA119.011206

    Figure Lengend Snippet: SP600125 or PMA induce Prss14/epithin ectodomain shedding. A , diagram of Prss14/epithin domain structure and processed forms. Epi-S', Epi-S, aEpi-S are indicated. B , effects of MAP kinase inhibitors in Prss14/epithin shedding. 427.1.86 cells were treated with 10 μ m PD98059 ( PD ), 20 μ m SB203580 ( SB ), and 5 μ m SP600125 ( SP ) for 30 min and then with or without 0.5 μ m PMA for an additional 2 h. C , dose- and time-dependent profiles of Epi-S' and Epi-S. 427.1.86 cells were treated with the indicated concentration of SP600125 for 2 h ( left panel ) and 5 μ m SP600125 up to 2 h ( right panel ). D , 427.1.86 cells were pretreated with 10 μ m TAPI-0 ( TPI ) for 30 min, and then cells were treated with 5 μ m SP600125 or 0.5 μ m PMA for an additional 2 h. The TACE inhibitor abolished the appearance of Epi-S' while retaining Epi-S in the cell, regardless of shedding induction methods, PMA, and SP600125. E , removal of TACE with siRNA abolished shedding of Prss14/epithin. 427.1.86 cells were transfected with 200 n m TACE siRNA for 48 h, starved of serum for 4 h, and then treated with 5 μΜ SP600125 or 0.5 μΜ PMA for 2 h. The control samples were treated exactly the same way except for transfection with nontargeting control siRNA. F , SP600125 dose-dependently induced Epi-S' and aEpi-S in T47D cells. SP600125 was treated for 2 h. G , SP600125 time-dependently (with 5 μ m ) and dose-dependently (for 2 h) induced Epi-S' and aEpi-S in 4T1 cells. In all panels, Epi-S' collected from culture medium and other proteins, including Epi-S, from cell lysates were detected by Western blot analysis. Tubulin or β-actin was used for normalization. TM , transmembrane domain; SEA , sperm protein, enterokinase, and agrin domain; CUB1 , CUB2 , complement subcomponent C1r/C1s domain; 1, 2, 3, 4 LDLRA , low-density lipoprotein receptor class A repeats.

    Article Snippet: For drug treatments, 90% confluent cells were serum-starved for 4 h and then treated with 0.5 μm PMA (Sigma) or SP600125 (Merck Millipore, Billerica, MA).

    Techniques: Concentration Assay, Transfection, Western Blot

    JNK inhibition increases PKCβII activity, translocation into the membrane, and TACE phosphorylation. A , phosphorylation of PKCβII by the JNK inhibitor. 427.1.86 cells were pretreated with 10 μ m anisomycin ( AN ) for 30 min and then treated with 5 μ m SP600125 ( SP ) for an additional 1 h. SP600125 treatment induced PKCβII phosphorylation. Relative values of band intensity are expressed as means ± S.D. of four independent experiments. ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CβII

    doi: 10.1074/jbc.RA119.011206

    Figure Lengend Snippet: JNK inhibition increases PKCβII activity, translocation into the membrane, and TACE phosphorylation. A , phosphorylation of PKCβII by the JNK inhibitor. 427.1.86 cells were pretreated with 10 μ m anisomycin ( AN ) for 30 min and then treated with 5 μ m SP600125 ( SP ) for an additional 1 h. SP600125 treatment induced PKCβII phosphorylation. Relative values of band intensity are expressed as means ± S.D. of four independent experiments. ***, p

    Article Snippet: For drug treatments, 90% confluent cells were serum-starved for 4 h and then treated with 0.5 μm PMA (Sigma) or SP600125 (Merck Millipore, Billerica, MA).

    Techniques: Inhibition, Activity Assay, Translocation Assay

    PKCβII is critical for PMA- or SP600125-induced cell invasion. A , schematic of the serum induced invasion assay ( left panel ). Invasion of 4271.86 cells depends on TACE activity and Prss14/epithin expression. After serum starvation for 12 h, cells were plated in the upper chamber, and DMSO or 20 μ m TAPI-0 ( TPI ) was added to the lower chamber. Invaded cells on the underside of the membrane after 24 h were stained with crystal violet and counted. The left panel shows the -fold change of the average number of invaded cells in five randomly selected fields. All values are expressed as means ± S.D. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CβII

    doi: 10.1074/jbc.RA119.011206

    Figure Lengend Snippet: PKCβII is critical for PMA- or SP600125-induced cell invasion. A , schematic of the serum induced invasion assay ( left panel ). Invasion of 4271.86 cells depends on TACE activity and Prss14/epithin expression. After serum starvation for 12 h, cells were plated in the upper chamber, and DMSO or 20 μ m TAPI-0 ( TPI ) was added to the lower chamber. Invaded cells on the underside of the membrane after 24 h were stained with crystal violet and counted. The left panel shows the -fold change of the average number of invaded cells in five randomly selected fields. All values are expressed as means ± S.D. *, p

    Article Snippet: For drug treatments, 90% confluent cells were serum-starved for 4 h and then treated with 0.5 μm PMA (Sigma) or SP600125 (Merck Millipore, Billerica, MA).

    Techniques: Invasion Assay, Activity Assay, Expressing, Staining

    PKCβII is responsible for PMA- and SP600125-induced shedding of Prss14/epithin. A , 427.1.86 cells were pretreated with 5 μ m Go6976 ( Go ) or 1 μ m PKCβ-selective inhibitor (β i ) before treatment with 5 μ m SP600125 ( SP ) or 0.5 μ m PMA for 2 h. B , PKCα or PKCβ knockdown effects on SP600125- or PMA-induced Prss14/epithin shedding. 427.1.86 cells were transfected with 200 n m PKCα or PKCβ siRNA or nontargeting control siRNA for 48 h and then treated with 5 μ m SP600125 or 0.5 μ m of PMA for an additional 2 h. PKCβ knockdown abolished SP600125- and PMA-induced shedding of Prss14/epithin. C , 427.1.86 cells were transfected with 1 μg/ml of the DN form of PKCβI and PKC PKCβII for 72 h. Control cells were transfected with an empty vector. DN forms of PKCβII inhibited shedding. D , PKCβ inhibition of PRSS14 shedding in two human cell lines. PC3 prostate cancer cells and MCF7 breast cancer cells were maintained in serum-free medium overnight and then treated with 1 μ m PKCβ inhibitor for an additional 6 h before testing.

    Journal: The Journal of Biological Chemistry

    Article Title: A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CβII

    doi: 10.1074/jbc.RA119.011206

    Figure Lengend Snippet: PKCβII is responsible for PMA- and SP600125-induced shedding of Prss14/epithin. A , 427.1.86 cells were pretreated with 5 μ m Go6976 ( Go ) or 1 μ m PKCβ-selective inhibitor (β i ) before treatment with 5 μ m SP600125 ( SP ) or 0.5 μ m PMA for 2 h. B , PKCα or PKCβ knockdown effects on SP600125- or PMA-induced Prss14/epithin shedding. 427.1.86 cells were transfected with 200 n m PKCα or PKCβ siRNA or nontargeting control siRNA for 48 h and then treated with 5 μ m SP600125 or 0.5 μ m of PMA for an additional 2 h. PKCβ knockdown abolished SP600125- and PMA-induced shedding of Prss14/epithin. C , 427.1.86 cells were transfected with 1 μg/ml of the DN form of PKCβI and PKC PKCβII for 72 h. Control cells were transfected with an empty vector. DN forms of PKCβII inhibited shedding. D , PKCβ inhibition of PRSS14 shedding in two human cell lines. PC3 prostate cancer cells and MCF7 breast cancer cells were maintained in serum-free medium overnight and then treated with 1 μ m PKCβ inhibitor for an additional 6 h before testing.

    Article Snippet: For drug treatments, 90% confluent cells were serum-starved for 4 h and then treated with 0.5 μm PMA (Sigma) or SP600125 (Merck Millipore, Billerica, MA).

    Techniques: Transfection, Dominant Negative Mutation, Plasmid Preparation, Inhibition

    SP600125-induced Prss14/epithin shedding involves JNK activity and de novo synthesis of labile protein. A , 427.1.86 cells were pretreated with 10 μ m anisomycin ( AN ) for 30 min and then treated with 5 μ m SP600125 ( SP ) for an additional 2 h. Epi-S' collected from culture medium and Epi-S in the cells were detected by Western blot analysis. B , suppression of JNKs by transfection with JNK1- or JNK2-specific siRNA induced Prss14/epithin shedding. 427.1.86 cells were transfected with 100 n m of JNK siRNA or nontargeting control ( cont ) siRNA. After 48 h, the medium was replaced and incubated for an additional 2 h before harvesting medium and cells. Control siRNA designed not to target any genes was used as a negative control in knockdown experiments. The band intensities were scanned, and we estimated the degree of percent reduction or -fold increase relative to control samples as described in the text. C , effects of cycloheximide ( CHX ) in SP600125-induced shedding. 427.1.86 cells were pretreated with 10 μ m cycloheximide and then treated with 5 m m SP600125 for an additional 2 h before harvesting the samples. D , effects of pretreatment of actinomycin D ( AD , 5 μ m for 30 min) and/or α-amanitin (α -A , 5 μ m for 12 h) on SP600125-induced shedding was analyzed as in A .

    Journal: The Journal of Biological Chemistry

    Article Title: A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CβII

    doi: 10.1074/jbc.RA119.011206

    Figure Lengend Snippet: SP600125-induced Prss14/epithin shedding involves JNK activity and de novo synthesis of labile protein. A , 427.1.86 cells were pretreated with 10 μ m anisomycin ( AN ) for 30 min and then treated with 5 μ m SP600125 ( SP ) for an additional 2 h. Epi-S' collected from culture medium and Epi-S in the cells were detected by Western blot analysis. B , suppression of JNKs by transfection with JNK1- or JNK2-specific siRNA induced Prss14/epithin shedding. 427.1.86 cells were transfected with 100 n m of JNK siRNA or nontargeting control ( cont ) siRNA. After 48 h, the medium was replaced and incubated for an additional 2 h before harvesting medium and cells. Control siRNA designed not to target any genes was used as a negative control in knockdown experiments. The band intensities were scanned, and we estimated the degree of percent reduction or -fold increase relative to control samples as described in the text. C , effects of cycloheximide ( CHX ) in SP600125-induced shedding. 427.1.86 cells were pretreated with 10 μ m cycloheximide and then treated with 5 m m SP600125 for an additional 2 h before harvesting the samples. D , effects of pretreatment of actinomycin D ( AD , 5 μ m for 30 min) and/or α-amanitin (α -A , 5 μ m for 12 h) on SP600125-induced shedding was analyzed as in A .

    Article Snippet: For drug treatments, 90% confluent cells were serum-starved for 4 h and then treated with 0.5 μm PMA (Sigma) or SP600125 (Merck Millipore, Billerica, MA).

    Techniques: Activity Assay, Western Blot, Transfection, Incubation, Negative Control

    The JNK cascade is the principal pathway inhibited by Reversine and SP600125. ( A–C ) Gene Ontology analysis of the common target genes (22) of Reversine and SP600125 was done by the Cytoscape associated-plugin ‘ClueGO’. ‘Positive regulation of JNK cascade’ was the most associated term with both Reversine and SP600125 ( A ). Network view of target genes of Reversine and/or SP600125 was performed by Cytoscape and GeneMANIA associated-plugin. Red colors represent the inhibited proteins through the Kinase panel test and JNK related nodes were highlighted in green zones after Reversine ( B ) and SP600125 ( C ) treatment. ( D ) Overexpression and activation of JNK was investigated in RKO colon cancer cell line comparatively to the normal human colon mucosal epithelial cell line NCM460. ( E ) Inactivation of JNK by SP600125 or Reversine in RKO cells was confirmed by Western blot. ( F ) Downstream targets of JNK pathway after treatment with Reversine or SP600125 were checked by western blot.

    Journal: Scientific Reports

    Article Title: Reversine inhibits Colon Carcinoma Cell Migration by Targeting JNK1

    doi: 10.1038/s41598-018-30251-w

    Figure Lengend Snippet: The JNK cascade is the principal pathway inhibited by Reversine and SP600125. ( A–C ) Gene Ontology analysis of the common target genes (22) of Reversine and SP600125 was done by the Cytoscape associated-plugin ‘ClueGO’. ‘Positive regulation of JNK cascade’ was the most associated term with both Reversine and SP600125 ( A ). Network view of target genes of Reversine and/or SP600125 was performed by Cytoscape and GeneMANIA associated-plugin. Red colors represent the inhibited proteins through the Kinase panel test and JNK related nodes were highlighted in green zones after Reversine ( B ) and SP600125 ( C ) treatment. ( D ) Overexpression and activation of JNK was investigated in RKO colon cancer cell line comparatively to the normal human colon mucosal epithelial cell line NCM460. ( E ) Inactivation of JNK by SP600125 or Reversine in RKO cells was confirmed by Western blot. ( F ) Downstream targets of JNK pathway after treatment with Reversine or SP600125 were checked by western blot.

    Article Snippet: SP600125 (anthra[1,9-cd]pyrazol-6-(2H)-one), a reversible ATP-competitive inhibitor of MAPK-JNK, was identified as direct inhibitor of JNK activity in a high throughput screening of a private chemical library held by Celgene .

    Techniques: Over Expression, Activation Assay, Western Blot

    Inhibition of migration by Reversine and SP600125 is not due to treatment toxicity.( A–C ) The toxicity of Reversine and SP600125 at 24 h was evaluated by flow cytometry upon staining with the cell death–associated parameters dyes. ( A ) Human colorectal carcinoma RKO cells were left untreated or treated for 24 h with 10 μM SP600125 or Reversine and then co-stained with the vital dye propidium iodide (PI) and the FITC-conjugated Annexin V for the detection of phosphatidylserine exposure. Representative dot plot (SP600125 treatment) and quantitative data are reported. White columns depict the percentage of dying cells (PI − Annexin V + ) while black columns illustrate dead cells (PI + ). ( B ) Cells were co-stained with the vital dye propidium iodide (PI) and the mitochondrial membrane potential (Δψm)-sensing dye DiOC 6 (3). Representative plot of SP600125 treated cells is shown and quantitative data are represented. White and black columns depict the percentage of dying (PI − DiOC 6 (3) low ) and dead (PI + ) cells, respectively. ( C ) RKO cells left untreated or exposed for 24 h with SP60015 or Reversine were fixed with ethanol and labeled with the DNA dye PI, for the quantification of the subG1 apoptotic population. Quantitative data are reported. ( D–E ) The toxicity of Reversine and SP600125 on non-cancer cells line. ( D ) Non transformed fibroblast IMR90 and normal human colon mucosal epithelial cell line NCM460 were left untreated or treated for 24 h with 10 μM SP600125 or Reversine and then co-stained with the vital dye propidium iodide (PI) and the mitochondrial membrane potential (Δψm)-sensing dye DiOC 6 (3). Quantitative data are represented. White and black columns depict the percentage of dying (PI − DiOC 6 (3) low ) and dead (PI + ) cells, respectively. ( E ) IMR90 and NCM460 cells left untreated or exposed for 24 h with SP60015 or Reversine were fixed with ethanol and labeled with the DNA dye PI, for the quantification of the subG1 apoptotic population. Quantitative data are reported. ( F–G ) Reversine and SP600125 do not affect cells proliferation and induces polyploidization. ( F ) Cells were seeded in 96 well plates and cultured for 24 h with or without treatment. Nocodazole at 200 ng/ml was used as positive control. Cell proliferation was assessed using crystal violet assay. Quantitative data are shown. Grey bar depict the optic density at 0 h while dark grey bar represent the optic density at 24 h, respectively. ( G ) Cells left untreated or exposed for 24 h with SP60015 or Reversine were fixed with ethanol and stained with an antibody specific for phosphorylated histone 3 (pH3), which is a mitosis-specific marker. Representative scatter plots and quantitative results of pH3 are shown. Numbers indicate the percentage of cells found in each quadrant. Data are reported in SEM; n.s indicates non-statistical difference from the control (ANOVA). *(p

    Journal: Scientific Reports

    Article Title: Reversine inhibits Colon Carcinoma Cell Migration by Targeting JNK1

    doi: 10.1038/s41598-018-30251-w

    Figure Lengend Snippet: Inhibition of migration by Reversine and SP600125 is not due to treatment toxicity.( A–C ) The toxicity of Reversine and SP600125 at 24 h was evaluated by flow cytometry upon staining with the cell death–associated parameters dyes. ( A ) Human colorectal carcinoma RKO cells were left untreated or treated for 24 h with 10 μM SP600125 or Reversine and then co-stained with the vital dye propidium iodide (PI) and the FITC-conjugated Annexin V for the detection of phosphatidylserine exposure. Representative dot plot (SP600125 treatment) and quantitative data are reported. White columns depict the percentage of dying cells (PI − Annexin V + ) while black columns illustrate dead cells (PI + ). ( B ) Cells were co-stained with the vital dye propidium iodide (PI) and the mitochondrial membrane potential (Δψm)-sensing dye DiOC 6 (3). Representative plot of SP600125 treated cells is shown and quantitative data are represented. White and black columns depict the percentage of dying (PI − DiOC 6 (3) low ) and dead (PI + ) cells, respectively. ( C ) RKO cells left untreated or exposed for 24 h with SP60015 or Reversine were fixed with ethanol and labeled with the DNA dye PI, for the quantification of the subG1 apoptotic population. Quantitative data are reported. ( D–E ) The toxicity of Reversine and SP600125 on non-cancer cells line. ( D ) Non transformed fibroblast IMR90 and normal human colon mucosal epithelial cell line NCM460 were left untreated or treated for 24 h with 10 μM SP600125 or Reversine and then co-stained with the vital dye propidium iodide (PI) and the mitochondrial membrane potential (Δψm)-sensing dye DiOC 6 (3). Quantitative data are represented. White and black columns depict the percentage of dying (PI − DiOC 6 (3) low ) and dead (PI + ) cells, respectively. ( E ) IMR90 and NCM460 cells left untreated or exposed for 24 h with SP60015 or Reversine were fixed with ethanol and labeled with the DNA dye PI, for the quantification of the subG1 apoptotic population. Quantitative data are reported. ( F–G ) Reversine and SP600125 do not affect cells proliferation and induces polyploidization. ( F ) Cells were seeded in 96 well plates and cultured for 24 h with or without treatment. Nocodazole at 200 ng/ml was used as positive control. Cell proliferation was assessed using crystal violet assay. Quantitative data are shown. Grey bar depict the optic density at 0 h while dark grey bar represent the optic density at 24 h, respectively. ( G ) Cells left untreated or exposed for 24 h with SP60015 or Reversine were fixed with ethanol and stained with an antibody specific for phosphorylated histone 3 (pH3), which is a mitosis-specific marker. Representative scatter plots and quantitative results of pH3 are shown. Numbers indicate the percentage of cells found in each quadrant. Data are reported in SEM; n.s indicates non-statistical difference from the control (ANOVA). *(p

    Article Snippet: SP600125 (anthra[1,9-cd]pyrazol-6-(2H)-one), a reversible ATP-competitive inhibitor of MAPK-JNK, was identified as direct inhibitor of JNK activity in a high throughput screening of a private chemical library held by Celgene .

    Techniques: Inhibition, Migration, Flow Cytometry, Cytometry, Staining, Labeling, Transformation Assay, Cell Culture, Positive Control, Crystal Violet Assay, Marker

    SP600125 reduces the metastasis of the human colon carcinoma RKO cells in vivo . ( A ) NSG mice were pre-treated i.p. (Intra peritoneal) with a control vehicle (n = 8) or 10 mg/kg SP600125 (n = 7). The day after, RKO cells were injected intravenously (i.v.). Treatment was performed every 3 days. Mice were sacrificed after 8 weeks and tumors were counted. ( B ) Representative pictures of liver in vehicle (C1, C2 and C3) and SP600125 treated mice (T1, T2, and T3) respectively. ( C–E ) The number of tumors per liver in the mice treated with the vehicle or SP600125 is depicted in ( C ) while liver weight and the ratio of the liver weight per mouse are showed in ( D ) and ( E ), respectively. ( F ) HE staining for quantification of tumor incidence in the lung of mice treated with the vehicle or SP600125. Stars indicate tumors. ( G ) Number of tumors per lung. ( H ) The weight of mice in control vs SP600125. n.s indicates non-statistical difference from the control (T-test).

    Journal: Scientific Reports

    Article Title: Reversine inhibits Colon Carcinoma Cell Migration by Targeting JNK1

    doi: 10.1038/s41598-018-30251-w

    Figure Lengend Snippet: SP600125 reduces the metastasis of the human colon carcinoma RKO cells in vivo . ( A ) NSG mice were pre-treated i.p. (Intra peritoneal) with a control vehicle (n = 8) or 10 mg/kg SP600125 (n = 7). The day after, RKO cells were injected intravenously (i.v.). Treatment was performed every 3 days. Mice were sacrificed after 8 weeks and tumors were counted. ( B ) Representative pictures of liver in vehicle (C1, C2 and C3) and SP600125 treated mice (T1, T2, and T3) respectively. ( C–E ) The number of tumors per liver in the mice treated with the vehicle or SP600125 is depicted in ( C ) while liver weight and the ratio of the liver weight per mouse are showed in ( D ) and ( E ), respectively. ( F ) HE staining for quantification of tumor incidence in the lung of mice treated with the vehicle or SP600125. Stars indicate tumors. ( G ) Number of tumors per lung. ( H ) The weight of mice in control vs SP600125. n.s indicates non-statistical difference from the control (T-test).

    Article Snippet: SP600125 (anthra[1,9-cd]pyrazol-6-(2H)-one), a reversible ATP-competitive inhibitor of MAPK-JNK, was identified as direct inhibitor of JNK activity in a high throughput screening of a private chemical library held by Celgene .

    Techniques: In Vivo, Mouse Assay, Injection, Staining, T-Test

    Identification of Reversine and SP600125 common targets. ( A–C ). A kinase profiler screen of cells treated with 10 μM Reversine (Rev) or 10 μM SP600125 (SP) was analyzed. The heat map in panel ( A ) shows the residual kinase activity (% of control). The scale ranges from 0% (blue) to 100% (red). The Venn diagram in panel ( B ) displays the proteins that were inhibited more than 90% by either 10 μM of Reversine or SP600125. The 22 common target proteins are listed in panel ( C ).

    Journal: Scientific Reports

    Article Title: Reversine inhibits Colon Carcinoma Cell Migration by Targeting JNK1

    doi: 10.1038/s41598-018-30251-w

    Figure Lengend Snippet: Identification of Reversine and SP600125 common targets. ( A–C ). A kinase profiler screen of cells treated with 10 μM Reversine (Rev) or 10 μM SP600125 (SP) was analyzed. The heat map in panel ( A ) shows the residual kinase activity (% of control). The scale ranges from 0% (blue) to 100% (red). The Venn diagram in panel ( B ) displays the proteins that were inhibited more than 90% by either 10 μM of Reversine or SP600125. The 22 common target proteins are listed in panel ( C ).

    Article Snippet: SP600125 (anthra[1,9-cd]pyrazol-6-(2H)-one), a reversible ATP-competitive inhibitor of MAPK-JNK, was identified as direct inhibitor of JNK activity in a high throughput screening of a private chemical library held by Celgene .

    Techniques: Activity Assay

    Reversine and SP600125 inhibit human colon carcinoma RKO cells migration. (A) Wound-Healing assay. The human colon carcinoma RKO cell lines were plated in a 6-well plate and kept to monolayer confluence and then a wound-healing assay was performed. Representative photomicrographs are shown. The yellow broken lines delimit the cell-free area. Quantitative data of anti-migration % comparatively to control are presented in the right of the panel. ( B ) Two-dimensional migration assay using the Oris™ cell assay. Cells were allowed to migrate for 24 h after the removal of cell seeding stoppers and treatment, fixed and stained with phalloidin and DAPI to evaluate their motile potential. Representative photomicrographs are shown. The yellow broken lines delimit the cell-free area after 24 h of migration while the purple broken lines delimit the cell-free area at time 0 h. Quantitative data are presented in the right of the panel. ( C ) Individual cell tracking assay. Cells were grown in non-confluent conditions and imaged for 24 hours, using time-lapse microscopy, to evaluate respective migration potential of individual cells. Representative photomicrographs are shown with the trajectories reconstituted using Image J software of some cells with or without treatment. Quantitative data of cells speed are presented in the right of the panel. ( D ) Boyden chamber assay. Cells were collected, washed and suspended in free serum medium. Cells were then added to the upper compartments of the Boyden chamber in the presence or absence of Reversine or SP600125. Representative photomicrographs of cells that had migrated 24 h later to the lower side of the filter are shown. Quantitative data are presented in the right of the panel. Data are reported in SEM; n = 3. ***(p

    Journal: Scientific Reports

    Article Title: Reversine inhibits Colon Carcinoma Cell Migration by Targeting JNK1

    doi: 10.1038/s41598-018-30251-w

    Figure Lengend Snippet: Reversine and SP600125 inhibit human colon carcinoma RKO cells migration. (A) Wound-Healing assay. The human colon carcinoma RKO cell lines were plated in a 6-well plate and kept to monolayer confluence and then a wound-healing assay was performed. Representative photomicrographs are shown. The yellow broken lines delimit the cell-free area. Quantitative data of anti-migration % comparatively to control are presented in the right of the panel. ( B ) Two-dimensional migration assay using the Oris™ cell assay. Cells were allowed to migrate for 24 h after the removal of cell seeding stoppers and treatment, fixed and stained with phalloidin and DAPI to evaluate their motile potential. Representative photomicrographs are shown. The yellow broken lines delimit the cell-free area after 24 h of migration while the purple broken lines delimit the cell-free area at time 0 h. Quantitative data are presented in the right of the panel. ( C ) Individual cell tracking assay. Cells were grown in non-confluent conditions and imaged for 24 hours, using time-lapse microscopy, to evaluate respective migration potential of individual cells. Representative photomicrographs are shown with the trajectories reconstituted using Image J software of some cells with or without treatment. Quantitative data of cells speed are presented in the right of the panel. ( D ) Boyden chamber assay. Cells were collected, washed and suspended in free serum medium. Cells were then added to the upper compartments of the Boyden chamber in the presence or absence of Reversine or SP600125. Representative photomicrographs of cells that had migrated 24 h later to the lower side of the filter are shown. Quantitative data are presented in the right of the panel. Data are reported in SEM; n = 3. ***(p

    Article Snippet: SP600125 (anthra[1,9-cd]pyrazol-6-(2H)-one), a reversible ATP-competitive inhibitor of MAPK-JNK, was identified as direct inhibitor of JNK activity in a high throughput screening of a private chemical library held by Celgene .

    Techniques: Migration, Wound Healing Assay, Staining, Cell Tracking Assay, Time-lapse Microscopy, Software, Boyden Chamber Assay

    Roles of MAPKs in FE-induced apoptosis. (A) MCF-7 cells were treated with 820 µg/mL FE for the indicated time periods. JNK, p38, and ERK1/2 MAPKs and their phosphorylated forms were determined by western blotting using specific antibodies. (B) Effects of MAPK inhibitors on apoptosis induced by FE. MCF-7 cells were pretreated with 10 µM SP600125, SB203580, or PD98059 for 1 h and then exposed to 820 µg/mL FE for 48 h (controls were not exposed to FE). After the treatment, annexin V-PI double staining was used to analyze apoptotic cell death using the IN Cell Analyzer 1000. All results were obtained from 3 independent experiments. Differences with p

    Journal: PLoS ONE

    Article Title: Fucoidan Extract Induces Apoptosis in MCF-7 Cells via a Mechanism Involving the ROS-Dependent JNK Activation and Mitochondria-Mediated Pathways

    doi: 10.1371/journal.pone.0027441

    Figure Lengend Snippet: Roles of MAPKs in FE-induced apoptosis. (A) MCF-7 cells were treated with 820 µg/mL FE for the indicated time periods. JNK, p38, and ERK1/2 MAPKs and their phosphorylated forms were determined by western blotting using specific antibodies. (B) Effects of MAPK inhibitors on apoptosis induced by FE. MCF-7 cells were pretreated with 10 µM SP600125, SB203580, or PD98059 for 1 h and then exposed to 820 µg/mL FE for 48 h (controls were not exposed to FE). After the treatment, annexin V-PI double staining was used to analyze apoptotic cell death using the IN Cell Analyzer 1000. All results were obtained from 3 independent experiments. Differences with p

    Article Snippet: The JNK inhibitor SP600125 and the p38 inhibitor SB203580 were purchased from Enzo Life Sciences International, Inc. (Plymouth Meeting, PA).

    Techniques: Western Blot, Double Staining

    Effect of ROS on FE-induced apoptosis. (A and B) Cells were pretreated for 1 h with or without 2 mM NAC followed by 820 µg/mL FE for 15–360 min. The cells were labeled with 5 µM DCFH-DA, and the fluorescence was measured using the IN Cell Analyzer 1000 as explained in the methods section. (C) Cells were treated with 820 µg/mL FE for 48 h after incubation with 2 mM NAC for 1 h and then the apoptotic cells were analyzed using the IN Cell Analyzer 1000. (D) MCF-7 cells were treated with 2 mM NAC for 1 h before treatment with 820 µg/mL FE for 6 h. Phosphorylation of JNK, p38, and ERK1/2 MAPKs was detected by western blotting. (E) Cells were pretreated with 2 mM NAC and 10 µM SP600125 for 1 h and then incubated with 820 µg/mL FE for 48 h. Disruption of ΔΨm was detected by Rh123 staining by using the IN Cell Analyzer 1000. All results were obtained from 3 independent experiments. Differences with p

    Journal: PLoS ONE

    Article Title: Fucoidan Extract Induces Apoptosis in MCF-7 Cells via a Mechanism Involving the ROS-Dependent JNK Activation and Mitochondria-Mediated Pathways

    doi: 10.1371/journal.pone.0027441

    Figure Lengend Snippet: Effect of ROS on FE-induced apoptosis. (A and B) Cells were pretreated for 1 h with or without 2 mM NAC followed by 820 µg/mL FE for 15–360 min. The cells were labeled with 5 µM DCFH-DA, and the fluorescence was measured using the IN Cell Analyzer 1000 as explained in the methods section. (C) Cells were treated with 820 µg/mL FE for 48 h after incubation with 2 mM NAC for 1 h and then the apoptotic cells were analyzed using the IN Cell Analyzer 1000. (D) MCF-7 cells were treated with 2 mM NAC for 1 h before treatment with 820 µg/mL FE for 6 h. Phosphorylation of JNK, p38, and ERK1/2 MAPKs was detected by western blotting. (E) Cells were pretreated with 2 mM NAC and 10 µM SP600125 for 1 h and then incubated with 820 µg/mL FE for 48 h. Disruption of ΔΨm was detected by Rh123 staining by using the IN Cell Analyzer 1000. All results were obtained from 3 independent experiments. Differences with p

    Article Snippet: The JNK inhibitor SP600125 and the p38 inhibitor SB203580 were purchased from Enzo Life Sciences International, Inc. (Plymouth Meeting, PA).

    Techniques: Labeling, Fluorescence, Incubation, Western Blot, Staining

    Antiviral activity of type I IFN in combination with MAPK inhibitors. HCC (data for HepG2 cells are shown as representative data), PH5CH8, and PHH cells were treated with IFN-α/β (100 IU/ml) in combination with 25 μM SP600125 (JNKi)

    Journal: Journal of Virology

    Article Title: Posttranslational Modification of Vesicular Stomatitis Virus Glycoprotein, but Not JNK Inhibition, Is the Antiviral Mechanism of SP600125

    doi: 10.1128/JVI.06649-11

    Figure Lengend Snippet: Antiviral activity of type I IFN in combination with MAPK inhibitors. HCC (data for HepG2 cells are shown as representative data), PH5CH8, and PHH cells were treated with IFN-α/β (100 IU/ml) in combination with 25 μM SP600125 (JNKi)

    Article Snippet: Chemicals (SB203580 [10 μM], SP600125 [25 μM], and U0126 [10 μM]) were purchased from Calbiochem-Merck (Gibbstown, NJ).

    Techniques: Activity Assay

    SP600125 induces a posttranslational modification of the VSV-G protein. (A) Levels of viral glycoprotein (VSV-G) from isolated viral particles (top) or infected-cell lysates (bottom) were analyzed by Western blotting. Huh-7 cells, used as representative

    Journal: Journal of Virology

    Article Title: Posttranslational Modification of Vesicular Stomatitis Virus Glycoprotein, but Not JNK Inhibition, Is the Antiviral Mechanism of SP600125

    doi: 10.1128/JVI.06649-11

    Figure Lengend Snippet: SP600125 induces a posttranslational modification of the VSV-G protein. (A) Levels of viral glycoprotein (VSV-G) from isolated viral particles (top) or infected-cell lysates (bottom) were analyzed by Western blotting. Huh-7 cells, used as representative

    Article Snippet: Chemicals (SB203580 [10 μM], SP600125 [25 μM], and U0126 [10 μM]) were purchased from Calbiochem-Merck (Gibbstown, NJ).

    Techniques: Modification, Isolation, Infection, Western Blot

    Viral transcription/translation and budding are not affected by SP600125. HepG2 cells and PHH were infected with VSV-GFP, and RNA was extracted at the indicated time points. (A and B) Viral mRNA for the N protein (A) and genome RNA (B) were quantified

    Journal: Journal of Virology

    Article Title: Posttranslational Modification of Vesicular Stomatitis Virus Glycoprotein, but Not JNK Inhibition, Is the Antiviral Mechanism of SP600125

    doi: 10.1128/JVI.06649-11

    Figure Lengend Snippet: Viral transcription/translation and budding are not affected by SP600125. HepG2 cells and PHH were infected with VSV-GFP, and RNA was extracted at the indicated time points. (A and B) Viral mRNA for the N protein (A) and genome RNA (B) were quantified

    Article Snippet: Chemicals (SB203580 [10 μM], SP600125 [25 μM], and U0126 [10 μM]) were purchased from Calbiochem-Merck (Gibbstown, NJ).

    Techniques: Infection

    SP600125 decreases the fusion activity of VSV-G. (A) Huh-7 cells were transfected with an expression vector for the VSV glycoprotein (pCMV-VSV-G). At 8 h posttransfection, supernatants were replaced with fresh medium containing either DMSO or SP600125

    Journal: Journal of Virology

    Article Title: Posttranslational Modification of Vesicular Stomatitis Virus Glycoprotein, but Not JNK Inhibition, Is the Antiviral Mechanism of SP600125

    doi: 10.1128/JVI.06649-11

    Figure Lengend Snippet: SP600125 decreases the fusion activity of VSV-G. (A) Huh-7 cells were transfected with an expression vector for the VSV glycoprotein (pCMV-VSV-G). At 8 h posttransfection, supernatants were replaced with fresh medium containing either DMSO or SP600125

    Article Snippet: Chemicals (SB203580 [10 μM], SP600125 [25 μM], and U0126 [10 μM]) were purchased from Calbiochem-Merck (Gibbstown, NJ).

    Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation

    The JNK inhibitor SP600125 interferes with VSV infectivity. (A) Viral titers obtained from DMSO-, SP600125 (JNKi)-, and IFN-treated cells were compared to the corresponding genome copy numbers. Infection was performed for 16 h, and viral genomic RNA in

    Journal: Journal of Virology

    Article Title: Posttranslational Modification of Vesicular Stomatitis Virus Glycoprotein, but Not JNK Inhibition, Is the Antiviral Mechanism of SP600125

    doi: 10.1128/JVI.06649-11

    Figure Lengend Snippet: The JNK inhibitor SP600125 interferes with VSV infectivity. (A) Viral titers obtained from DMSO-, SP600125 (JNKi)-, and IFN-treated cells were compared to the corresponding genome copy numbers. Infection was performed for 16 h, and viral genomic RNA in

    Article Snippet: Chemicals (SB203580 [10 μM], SP600125 [25 μM], and U0126 [10 μM]) were purchased from Calbiochem-Merck (Gibbstown, NJ).

    Techniques: Infection

    SP600125 alters VSV-G posttranslationally and hampers its fusogenic activity.

    Journal: Journal of Virology

    Article Title: Posttranslational Modification of Vesicular Stomatitis Virus Glycoprotein, but Not JNK Inhibition, Is the Antiviral Mechanism of SP600125

    doi: 10.1128/JVI.06649-11

    Figure Lengend Snippet: SP600125 alters VSV-G posttranslationally and hampers its fusogenic activity.

    Article Snippet: Chemicals (SB203580 [10 μM], SP600125 [25 μM], and U0126 [10 μM]) were purchased from Calbiochem-Merck (Gibbstown, NJ).

    Techniques: Activity Assay

    VSV-G* is not the result of cross-linking or hyperglycosylation processes. (A) Semipurified virions were exposed to increasing concentrations of SP600125 (JNKi) for 1 h on ice. As a control, virions were cross-linked with PFA (2%) on ice for 15 min. One-tenth

    Journal: Journal of Virology

    Article Title: Posttranslational Modification of Vesicular Stomatitis Virus Glycoprotein, but Not JNK Inhibition, Is the Antiviral Mechanism of SP600125

    doi: 10.1128/JVI.06649-11

    Figure Lengend Snippet: VSV-G* is not the result of cross-linking or hyperglycosylation processes. (A) Semipurified virions were exposed to increasing concentrations of SP600125 (JNKi) for 1 h on ice. As a control, virions were cross-linked with PFA (2%) on ice for 15 min. One-tenth

    Article Snippet: Chemicals (SB203580 [10 μM], SP600125 [25 μM], and U0126 [10 μM]) were purchased from Calbiochem-Merck (Gibbstown, NJ).

    Techniques:

    ERK signaling regulates c-Fos expression and posttranslational modifications. (A) PDGF induction of c-Fos expression in NIH 3T3 cells. Cells were grown overnight in the absence of serum and then stimulated with PDGF (20 ng/ml) for the indicated times. Nuclear fractions were collected, and c-Fos was detected by Western blotting. (B) ERK dependency of c-Fos expression. Cells were serum starved overnight, incubated for 30 min in the absence (c) or presence of U0126 (U), SP600125 (SP), or SB203580 (SB) (10 μM each), and stimulated with PDGF (20 ng/ml; 2 h), and c-Fos expression in nuclear fractions was determined as described for panel A. (C) ERK dependency of c- fos mRNA expression. Cells were serum starved overnight, incubated for 30 min in the absence or presence of U0126, and stimulated with PDGF (20 ng/ml) for the indicated times. c- fos transcript levels were detected by Northern blotting as described in Materials and Methods. (D) ERK dependency of c-Fos posttranslational modifications. Cells were transiently transfected with pCEFL-AU5-c-Fos (1 μg/well) and grown in serum-free medium overnight after transfection. Cells were stimulated with PDGF (20 ng/ml; 30 min), and total lysates were processed for Western blotting using anti-c-Fos antibodies (top panel). Pretreatment with MAPK inhibitors was performed as described above. Endogenous active (middle panel) and total (bottom panel) ERK expression from the same samples served as controls for PDGF and U0126 effects on ERK phosphorylation. (E) c-Fos phosphorylation induced by PDGF. NIH 3T3 cells were transfected with pCEFL-AU5-c-Fos (1 μg/well), cultured in serum-free medium overnight, and stimulated with PDGF for 10 or 30 min. Cell lysates were subjected to PP2A digestion followed by immunoblotting using anti-c-Fos antibodies (see Materials and Methods). (F) SP600125 (SP) and SB203580 (SB) inhibitory action on JNK and p38 activities. HEK-293T cells were transfected with HA-JNK1 or HA-p38α alone or in combination with their upstream activating kinases (MEKK and MEK3EE, respectively). In vitro kinase assays were performed as described in Materials and Methods in the absence or presence of the indicated final concentrations of the inhibitors. 32 P-labeled substrate (P-ATF2) is indicated. NT, nontransfected cells.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of the Carboxyl-Terminal Transactivation Domain of c-Fos by Extracellular Signal-Regulated Kinase Mediates the Transcriptional Activation of AP-1 and Cellular Transformation Induced by Platelet-Derived Growth Factor

    doi: 10.1128/MCB.23.19.7030-7043.2003

    Figure Lengend Snippet: ERK signaling regulates c-Fos expression and posttranslational modifications. (A) PDGF induction of c-Fos expression in NIH 3T3 cells. Cells were grown overnight in the absence of serum and then stimulated with PDGF (20 ng/ml) for the indicated times. Nuclear fractions were collected, and c-Fos was detected by Western blotting. (B) ERK dependency of c-Fos expression. Cells were serum starved overnight, incubated for 30 min in the absence (c) or presence of U0126 (U), SP600125 (SP), or SB203580 (SB) (10 μM each), and stimulated with PDGF (20 ng/ml; 2 h), and c-Fos expression in nuclear fractions was determined as described for panel A. (C) ERK dependency of c- fos mRNA expression. Cells were serum starved overnight, incubated for 30 min in the absence or presence of U0126, and stimulated with PDGF (20 ng/ml) for the indicated times. c- fos transcript levels were detected by Northern blotting as described in Materials and Methods. (D) ERK dependency of c-Fos posttranslational modifications. Cells were transiently transfected with pCEFL-AU5-c-Fos (1 μg/well) and grown in serum-free medium overnight after transfection. Cells were stimulated with PDGF (20 ng/ml; 30 min), and total lysates were processed for Western blotting using anti-c-Fos antibodies (top panel). Pretreatment with MAPK inhibitors was performed as described above. Endogenous active (middle panel) and total (bottom panel) ERK expression from the same samples served as controls for PDGF and U0126 effects on ERK phosphorylation. (E) c-Fos phosphorylation induced by PDGF. NIH 3T3 cells were transfected with pCEFL-AU5-c-Fos (1 μg/well), cultured in serum-free medium overnight, and stimulated with PDGF for 10 or 30 min. Cell lysates were subjected to PP2A digestion followed by immunoblotting using anti-c-Fos antibodies (see Materials and Methods). (F) SP600125 (SP) and SB203580 (SB) inhibitory action on JNK and p38 activities. HEK-293T cells were transfected with HA-JNK1 or HA-p38α alone or in combination with their upstream activating kinases (MEKK and MEK3EE, respectively). In vitro kinase assays were performed as described in Materials and Methods in the absence or presence of the indicated final concentrations of the inhibitors. 32 P-labeled substrate (P-ATF2) is indicated. NT, nontransfected cells.

    Article Snippet: The JNK inhibitor SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one], was from Biomol (Plymouth Meeting, Pa.).

    Techniques: Expressing, Western Blot, Incubation, Northern Blot, Transfection, Cell Culture, In Vitro, Labeling

    JNK and ERK pathways mediate AP-1 activation by PDGF and serum. NIH 3T3 cells were transfected with pAP-1-Luc and pRL-null, cultured under serum-free conditions, and pretreated for 30 min with inhibitors for each MAPK pathway (U0126, SP600125, and SB203580; 10 μM each) before PDGF (20 ng/ml) or FBS (10%) stimulation. Dual luciferase activities were determined 4 h after treatment, as described in Materials and Methods. The data represent the firefly luciferase activity normalized by Renilla luciferase activity present in each sample, expressed as absolute counts. Values are the average ± standard deviation of triplicate samples from a typical experiment. Nearly identical results were obtained in three additional experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Phosphorylation of the Carboxyl-Terminal Transactivation Domain of c-Fos by Extracellular Signal-Regulated Kinase Mediates the Transcriptional Activation of AP-1 and Cellular Transformation Induced by Platelet-Derived Growth Factor

    doi: 10.1128/MCB.23.19.7030-7043.2003

    Figure Lengend Snippet: JNK and ERK pathways mediate AP-1 activation by PDGF and serum. NIH 3T3 cells were transfected with pAP-1-Luc and pRL-null, cultured under serum-free conditions, and pretreated for 30 min with inhibitors for each MAPK pathway (U0126, SP600125, and SB203580; 10 μM each) before PDGF (20 ng/ml) or FBS (10%) stimulation. Dual luciferase activities were determined 4 h after treatment, as described in Materials and Methods. The data represent the firefly luciferase activity normalized by Renilla luciferase activity present in each sample, expressed as absolute counts. Values are the average ± standard deviation of triplicate samples from a typical experiment. Nearly identical results were obtained in three additional experiments.

    Article Snippet: The JNK inhibitor SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one], was from Biomol (Plymouth Meeting, Pa.).

    Techniques: Activation Assay, Transfection, Cell Culture, Luciferase, Activity Assay, Standard Deviation