Journal: Biomolecules

Article Title: The MAP Kinase Phosphatase MKP-1 Modulates Neurogenesis via Effects on BNIP3 and Autophagy

doi: 10.3390/biom11121871

Figure Lengend Snippet: Mkp-1 KO mice exhibit fewer DCX positive neurons in the dentate gyrus (DG). ( A , B ) Immunofluorescence images of the dentate gyrus in wild-type (WT) and Mkp-1 KO mice stained with doublecortin (DCX). ( C , D ) DAPI nuclear counterstain of the dentate gyrus. ( E , F ) Composite images with high-power view illustrating individual DCX+ cells (yellow arrow). ( G ) Results of DCX+ cell counts in WT and Mkp-1 KO cultures ( n = 3, t -test, ** p < 0.01).

Article Snippet: Quantitative PCR (qPCR) was performed with primers and exon spanning Taqman probes for mouse Mkp-1 and β-actin with FAM-MGB and VIC-MGB, respectively (Mm00457274_g1/4331182 and Mm02619580_g1/ 4448489, ThermoFisher Scientific). qPCR was performed on a StepOnePlus Real-Time PCR System (ThermoFisher Scientific), and analyses were performed using β-actin as an internal reference gene for normalization in the ∆∆CT analyses.

Techniques: Immunofluorescence, Staining

Journal: Biomolecules

Article Title: The MAP Kinase Phosphatase MKP-1 Modulates Neurogenesis via Effects on BNIP3 and Autophagy

doi: 10.3390/biom11121871

Figure Lengend Snippet: Loss of MKP-1 inhibits neurogenesis in differentiated NSC cultures. ( A ) Time course analysis of Mkp-1 mRNA fold-change in expression in differentiated wild-type neural stem cells compared with undifferentiated (UD) cultures ( n = 6, one-way ANOVA, *** p < 0.001, **** p < 0.0001). ( B ) Live-cell images of WT and Mkp-1 KO neural stem cultures 24 h post-differentiation. ( C ) Immunocytochemical staining of differentiated WT and Mkp-1 KO NSCs for Tuj1 (green) and GFAP (red) on day 3 (D3) and day 6 (D6) post-differentiation. ( D ) Absolute counts (#) per field and fraction (%) of Tuj1+ and GFAP+ cells relative to total cell # per field at D3 and D6 ( n = 15, two-way ANOVA, ** p < 0.01, **** p < 0.0001).

Article Snippet: Quantitative PCR (qPCR) was performed with primers and exon spanning Taqman probes for mouse Mkp-1 and β-actin with FAM-MGB and VIC-MGB, respectively (Mm00457274_g1/4331182 and Mm02619580_g1/ 4448489, ThermoFisher Scientific). qPCR was performed on a StepOnePlus Real-Time PCR System (ThermoFisher Scientific), and analyses were performed using β-actin as an internal reference gene for normalization in the ∆∆CT analyses.

Techniques: Expressing, Staining

Journal: Biomolecules

Article Title: The MAP Kinase Phosphatase MKP-1 Modulates Neurogenesis via Effects on BNIP3 and Autophagy

doi: 10.3390/biom11121871

Figure Lengend Snippet: Loss of MKP-1 induces autophagy in the CNS and neural stem cultures. ( A ) LC3 immunofluorescence with DAPI counterstaining within the dentate gyrus of WT and Mkp-1 KO mice. The inset illustrates the increased accumulation of LC3 puncta (yellow arrow) in Mkp-1 KO mice. The number of puncta was compared between WT and Mkp-1 samples ( n = 3, t -test, * p < 0.05). ( B ) Western blotting ( Left ) and quantitative densitometry ( Right ) for LC3-II levels in neural stem cultures 24, 48, and 72 h post-differentiation in both genotypes ( n = 3, two-way ANOVA, * p < 0.05). ( C ) Flow cytometry analyses of autophagy in NSCs stained with monodansylcadaverine (MDC). Representative MDC fluorescence plots from undifferentiated (UD) and differentiated (D) NSCs ( Left ) and results comparing the effect of genotype on the median of MDC fluorescence over time ( Right ) are shown ( n = 3, two-way ANOVA, * p < 0.05).

Article Snippet: Quantitative PCR (qPCR) was performed with primers and exon spanning Taqman probes for mouse Mkp-1 and β-actin with FAM-MGB and VIC-MGB, respectively (Mm00457274_g1/4331182 and Mm02619580_g1/ 4448489, ThermoFisher Scientific). qPCR was performed on a StepOnePlus Real-Time PCR System (ThermoFisher Scientific), and analyses were performed using β-actin as an internal reference gene for normalization in the ∆∆CT analyses.

Techniques: Immunofluorescence, Western Blot, Flow Cytometry, Staining, Fluorescence

Journal: Biomolecules

Article Title: The MAP Kinase Phosphatase MKP-1 Modulates Neurogenesis via Effects on BNIP3 and Autophagy

doi: 10.3390/biom11121871

Figure Lengend Snippet: Pharmacological manipulation of autophagy influences neurogenesis. ( A ) Experimental timeline illustrating the sequence of NSC differentiation and drug treatment, media exchange, and ICC endpoint analysis. ( B ) Autophagy level of WT NSC-D 24 h post differentiation with DMSO or rapamycin in DMSO (Rapa, 10 nM) using average MDC fluorescence by flow (left) and LC3II immunoblotting. ( C ) Changes of MDC fluorescence and LC3II level in Mkp-1 KO NSC-D 24 h after differentiation with or without the treatment of 3-MA (2 mM, dissolved in medium). ( D ) Effect of DMSO vs. rapamycin on the number (#) per field and fraction (%) of Tuj1+ neurons (green) relative to total cell # on day 6 in WT NSC-D cultures. ( E ) Effect of 3-MA treatment on the number (#) per field and fraction (%) of Tuj1+ neurons (green) relative to total cell # on day 6 of Mkp-1 KO NCS-D cultures ( n = 15, t -test, * p < 0.05, *** p < 0.001, **** p < 0.0001).

Article Snippet: Quantitative PCR (qPCR) was performed with primers and exon spanning Taqman probes for mouse Mkp-1 and β-actin with FAM-MGB and VIC-MGB, respectively (Mm00457274_g1/4331182 and Mm02619580_g1/ 4448489, ThermoFisher Scientific). qPCR was performed on a StepOnePlus Real-Time PCR System (ThermoFisher Scientific), and analyses were performed using β-actin as an internal reference gene for normalization in the ∆∆CT analyses.

Techniques: Sequencing, Fluorescence, Western Blot

Journal: Biomolecules

Article Title: The MAP Kinase Phosphatase MKP-1 Modulates Neurogenesis via Effects on BNIP3 and Autophagy

doi: 10.3390/biom11121871

Figure Lengend Snippet: BNIP3 induces autophagy and represses neurogenesis in Mkp-1 KO NSCs. ( A ) Relative activity of pGL3_Bnip3 vs. control pGL3_Basic reporter constructs in differentiated (24 h) WT and Mkp-1 KO NSCs ( n = 4, two-way ANOVA, **** p < 0.0001). ( B ) Protein blotting for BNIP3 expression in the cytoplasmic fraction of NSCs 24 h post-differentiation (Left) and the quantification by densitometry relative to the loading control HSP90 (right) ( n = 3, t -test, ** p < 0.01). ( C ) Experimental timeline for Bnip3 knockdown illustrating sequential siRNA treatment, differentiation, and various endpoint analyses. ( D ) Confirmation of BNIP3 knockdown in Mkp-1 KO cells 24 h post-differentiation by protein blotting (NT, non-targeting siRNA transfection; Bnip3, mouse Bnip3 siRNA transfection). ( E ) Changes of MDC and LC3II levels in Mkp-1 KO NSC-D with Bnip3 siRNA transfection using flow cytometry and immunoblotting, respectively, 24 h after differentiation ( n = 3, t -test, *** p < 0.001). ( F ) Absolute number (#) per field and fraction (%) of Tuj1+ neurons (green) relative to total cell # following Bnip3 siRNA transfection in Mkp-1 KO NSC-D on day 6 post differentiation ( n = 25, t -test, *** p < 0.001, **** p < 0.0001).

Article Snippet: Quantitative PCR (qPCR) was performed with primers and exon spanning Taqman probes for mouse Mkp-1 and β-actin with FAM-MGB and VIC-MGB, respectively (Mm00457274_g1/4331182 and Mm02619580_g1/ 4448489, ThermoFisher Scientific). qPCR was performed on a StepOnePlus Real-Time PCR System (ThermoFisher Scientific), and analyses were performed using β-actin as an internal reference gene for normalization in the ∆∆CT analyses.

Techniques: Activity Assay, Control, Construct, Expressing, Knockdown, Transfection, Flow Cytometry, Western Blot

Journal: British Journal of Pharmacology

Article Title: Corticosteroids and ? 2 -agonists upregulate mitogen-activated protein kinase phosphatase 1: in vitro mechanisms

doi: 10.1111/j.1476-5381.2012.01923.x

Figure Lengend Snippet: The corticosteroid dexamethasone and the β2-agonist formoterol, alone and in combination, increase MKP-1 mRNA expression and protein upregulation. To examine the temporal kinetics of MKP-1 upregulation induced by corticosteroids and β2-agonists, alone and in combination, growth-arrested ASM cells were treated for the indicated times with vehicle (X), formoterol (10 nM), dexamethasone (100 nM) or dexamethasone (100 nM) + formoterol (10 nM). (A) MKP-1 mRNA expression was quantified by real-time RT-PCR and results expressed as fold increase over 0 min (mean + SEM values from n= 3–7 replicates). (B, C) MKP-1 protein was quantified by Western blotting, using α-tubulin as the loading control, where (B) illustrates representative Western blots, and (C) demonstrates densitometric analysis (results are expressed as fold increase over 0 min) (mean + SEM values from n= 3–5 replicates). Statistical analysis was performed using two-way anova then Bonferroni's post-test [where * indicates a significant effect of formoterol or dexamethasone on MKP-1, compared with vehicle-treated cells, and § indicates a significant effect of formoterol on dexamethasone-induced MKP-1 (P < 0.05)].

Article Snippet: MKP-1 mRNA expression was quantified by real-time RT-PCR using an ABI Prism 7500 (Applied Biosystems, Foster City, CA, USA) and a MKP-1 primer set [Assays on Demand, dual specificity phosphatase 1 (DUSP1), Hs00610256_g1; Applied Biosystems] with a eukaryotic 18S rRNA endogenous control probe (Applied Biosystems) and subjected to the following amplification conditions: 50°C for 2 min, 1 cycle; 95°C for 10 min, 1 cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles.

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

Journal: British Journal of Pharmacology

Article Title: Corticosteroids and ? 2 -agonists upregulate mitogen-activated protein kinase phosphatase 1: in vitro mechanisms

doi: 10.1111/j.1476-5381.2012.01923.x

Figure Lengend Snippet: β2-agonist-induced MKP-1 mRNA and protein expression is upregulated in a cAMP-mediated manner via the β2-adrenoceptor-PKA pathway. To determine whether the β2-agonist formoterol increased MKP-1 mRNA and protein expression via the β2-adrenoceptor-PKA pathway, growth-arrested ASM cells were pretreated for 30 min with propranolol (0.1 µM), or 60 min with H-89 (10 µM) or vehicle, then treated with formoterol (10 nM) for 1 h. (A) MKP-1 mRNA was quantified by real-time RT-PCR and results expressed as fold increase over vehicle-treated cells. (B, C) MKP-1 protein was quantified by Western blotting (with α-tubulin as the loading control), where (B) illustrates a representative Western blot, and (C) demonstrates densitometric analysis (results expressed as fold increase over vehicle-treated cells). Statistical analysis was performed using Student's unpaired t test, where * denotes a significant effect on formoterol-induced MKP-1 (mean + SEM values from n= 4 replicates) (P < 0.05).

Article Snippet: MKP-1 mRNA expression was quantified by real-time RT-PCR using an ABI Prism 7500 (Applied Biosystems, Foster City, CA, USA) and a MKP-1 primer set [Assays on Demand, dual specificity phosphatase 1 (DUSP1), Hs00610256_g1; Applied Biosystems] with a eukaryotic 18S rRNA endogenous control probe (Applied Biosystems) and subjected to the following amplification conditions: 50°C for 2 min, 1 cycle; 95°C for 10 min, 1 cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles.

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

Journal: British Journal of Pharmacology

Article Title: Corticosteroids and ? 2 -agonists upregulate mitogen-activated protein kinase phosphatase 1: in vitro mechanisms

doi: 10.1111/j.1476-5381.2012.01923.x

Figure Lengend Snippet: Adenoviral overexpression of the selective PKA inhibitor, PKIα, represses formoterol-induced upregulation of MKP-1 mRNA and protein expression. To confirm the involvement of the PKA pathway in formoterol-induced MKP-1 mRNA expression and protein upregulation, ASM cells were infected at an MOI of 300 with either an empty Ad5 vector (Ad5-null) or a PKIα Ad5 expression vector (Ad5-PKIα). After 24 h, cells were fully confluent and were incubated in serum-free media overnight before the addition of vehicle, formoterol (form: 10 nM), dexamethasone (dex: 100 nM) or dex (100 nM) + form (10 nM) in combination for 1 h. (A) confirms overexpression of PKIα mRNA in cells infected with the PKIα-Ad5 expression vector, compared with null vector (results expressed as PKIα 18 s−1). (B) Demonstrates the repression of formoterol-induced MKP-1 mRNA by PKIα (results are expressed as percentage of formoterol-induced MKP-1 mRNA). (C) Confirms the overexpression of PKIα protein in cells infected with Ad5-PKIα, compared with Ad5-null. (D, E) Demonstrate the repression of formoterol-induced MKP-1 protein upregulation by PKIα, where (D) is a representative Western blot, and (E) shows densitometric analysis expressed as percentage of formoterol-induced MKP-1 protein. Statistical analysis was performed using Student's unpaired t-test, where § denotes a significant repressive effect of PKIα on formoterol-induced MKP-1 expression, both alone and in combination with dexamethasone [mean + SEM values from n= 6 replicates (mRNA) and n= 8 replicates (protein)] (P < 0.05).

Article Snippet: MKP-1 mRNA expression was quantified by real-time RT-PCR using an ABI Prism 7500 (Applied Biosystems, Foster City, CA, USA) and a MKP-1 primer set [Assays on Demand, dual specificity phosphatase 1 (DUSP1), Hs00610256_g1; Applied Biosystems] with a eukaryotic 18S rRNA endogenous control probe (Applied Biosystems) and subjected to the following amplification conditions: 50°C for 2 min, 1 cycle; 95°C for 10 min, 1 cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles.

Techniques: Over Expression, Expressing, Infection, Plasmid Preparation, Incubation, Western Blot

Journal: British Journal of Pharmacology

Article Title: Corticosteroids and ? 2 -agonists upregulate mitogen-activated protein kinase phosphatase 1: in vitro mechanisms

doi: 10.1111/j.1476-5381.2012.01923.x

Figure Lengend Snippet: Dexamethasone activates MKP-1 transcription in ASM cells via a corticosteroid-responsive region located between −1380 and −1266 bp of the human MKP-1 promoter. To identify the MKP-1 promoter region responsible for corticosteroid responsiveness, ASM cells were transiently transfected with a pGL3 basic luciferase reporter containing ∼3 kb of the human MKP-1 promoter (−2975 to +247 bp) and a series of 5′-promoter deletion constructs. Cells were treated with vehicle or dexamethasone (100 nM) for 24 h and results expressed as MKP-1 promoter luciferase activity, relative to vehicle-treated cells (fold increase). Statistical analysis was performed using Student's unpaired t-test where * denotes a significant effect of the promoter deletion on dexamethasone-induced luciferase activity (mean + SEM values from n= 8 replicates) (P < 0.05).

Article Snippet: MKP-1 mRNA expression was quantified by real-time RT-PCR using an ABI Prism 7500 (Applied Biosystems, Foster City, CA, USA) and a MKP-1 primer set [Assays on Demand, dual specificity phosphatase 1 (DUSP1), Hs00610256_g1; Applied Biosystems] with a eukaryotic 18S rRNA endogenous control probe (Applied Biosystems) and subjected to the following amplification conditions: 50°C for 2 min, 1 cycle; 95°C for 10 min, 1 cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles.

Techniques: Transfection, Luciferase, Construct, Activity Assay

Journal: British Journal of Pharmacology

Article Title: Corticosteroids and ? 2 -agonists upregulate mitogen-activated protein kinase phosphatase 1: in vitro mechanisms

doi: 10.1111/j.1476-5381.2012.01923.x

Figure Lengend Snippet: Additive effects of β2-agonists and corticosteroids occur at the transcriptional, rather than the post-transcriptional, level of MKP-1 gene expression. (A) To examine the stability of MKP-1 mRNA induced by the corticosteroid dexamethasone and the β2-agonist formoterol, alone and in combination, growth-arrested ASM cells were treated with vehicle, dexamethasone (100 nM), formoterol (10 nM) or dexamethasone (100 nM) + formoterol (10 nM) for 1 h. MKP-1 mRNA stability was measured by actinomycin D chase using real-time RT-PCR and results expressed as % mRNA remaining over time. Data are mean + SEM values from n= 4 replicates. (B) To measure dexamethasone-induced translocation of HuR, growth-arrested ASM cells were treated with vehicle or dexamethasone (100 nM) for 1 h. Cytoplasmic and nuclear fractions were prepared and HuR protein measured by Western blotting, using α-tubulin and lamin A/C as the loading controls for the cytoplasmic or nuclear fractions respectively. (B) Illustrates a representative Western blot of n= 3 replicates. (C) To determine the effects of the corticosteroid dexamethasone and the β2-agonist formoterol, alone and in combination, on MKP-1 promoter activity, ASM cells were transfected with the ∼3 kb human MKP-1 gene promoter (−2975 to +247 bp) and treated for 24 h with vehicle, formoterol (10 nM), dexamethasone (100 nM) or dexamethasone (100 nM) + formoterol (10 nM) in combination. Luciferase activity was measured and results expressed as MKP-1 promoter luciferase activity, relative to vehicle-treated cells (fold increase). Statistical analysis was performed using Student's unpaired t-test where * denotes a significant effect of the asthma therapeutics on luciferase activity, compared with vehicle-treated cells, and § indicates a significant effect of formoterol + dexamethasone in combination on luciferase activity, compared with formoterol or dexamethasone treatment alone (mean + SEM values from n= 12 replicates for formoterol, n= 21 replicates for dexamethasone and n= 12 replicates for formoterol + dexamethasone) (P < 0.05).

Article Snippet: MKP-1 mRNA expression was quantified by real-time RT-PCR using an ABI Prism 7500 (Applied Biosystems, Foster City, CA, USA) and a MKP-1 primer set [Assays on Demand, dual specificity phosphatase 1 (DUSP1), Hs00610256_g1; Applied Biosystems] with a eukaryotic 18S rRNA endogenous control probe (Applied Biosystems) and subjected to the following amplification conditions: 50°C for 2 min, 1 cycle; 95°C for 10 min, 1 cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles.

Techniques: Gene Expression, Quantitative RT-PCR, Translocation Assay, Western Blot, Activity Assay, Transfection, Luciferase

Journal: Molecular Pain

Article Title: Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

doi: 10.1186/1744-8069-8-34

Figure Lengend Snippet: MKP-1 expression in spinal cord following peripheral nerve injury. Quantification of MKP-1 protein ( a ) and representative Western blot of MKP-1 expression ( b ) in L5–L6 spinal cord of naïve rats and of rats that have undergone sham (Sh) or L5 nerve transection (L5NT or NT) surgery ( n = 3–6) on postoperative days 1 (D1) and 4 (D4) . * P < 0.05 for sham versus naïve on postoperative days 1 and 4, also between sham and L5 nerve transection groups on postoperative day 4. All data are expressed as mean ± s.e.m.

Article Snippet: Quantitative RT-PCR was performed using a 96-well plate with the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster city, CA) according to the following conditions: 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min, then 40 cycles at 95°C for 15 sec, 60°C for 1 min. All samples were run in duplicate using Taqman gene expression assays (Applied Biosystems, Foster City, CA) with rat MKP-1 (Rn00678341_g1) predesigned and preformulated primer and probe.

Techniques: Expressing, Western Blot

Journal: Molecular Pain

Article Title: Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

doi: 10.1186/1744-8069-8-34

Figure Lengend Snippet: In vitro characterization of rat MKP-1 overexpression. MKP-1 mRNA levels ( a , qRT-PCR) and Western blot of MKP-1 protein ( b ) in normal BV-2 cells and stably MKP-1 overexpressing BV-2 cells. ( c ) Representative Western blots of p-p38, and p-JNK expression in BV-2 cells stably overexpressing MKP-1 following 1 or 2 h incubation in medium (control alone) or lipopolysaccharide (LPS) stimulation (1 μg/ml). IL-6 ( d ), TNF-α ( e ), nitrite ( f ), and MCP-1) ( g ) release in control (normal) and stably expressing MKP-1 BV-2 cells challenged with LPS (1 μg/ml) for 6 or 12 h. Results are expressed as mean ± s.e.m. of three experiments in triplicate. * P < 0.05 vs. control BV-2 cells.

Article Snippet: Quantitative RT-PCR was performed using a 96-well plate with the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster city, CA) according to the following conditions: 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min, then 40 cycles at 95°C for 15 sec, 60°C for 1 min. All samples were run in duplicate using Taqman gene expression assays (Applied Biosystems, Foster City, CA) with rat MKP-1 (Rn00678341_g1) predesigned and preformulated primer and probe.

Techniques: In Vitro, Over Expression, Quantitative RT-PCR, Western Blot, Stable Transfection, Expressing, Incubation

Journal: Molecular Pain

Article Title: Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

doi: 10.1186/1744-8069-8-34

Figure Lengend Snippet: Reversal effects of BV-2 cell MKP-1 induction in cytokine production by siRNA MKP-1 knockdown. ( a ) Efficient siRNA BV-2 cell transfection using Alexa-555 red fluorescent-labeled siRNA. ( b ) MKP-1 mRNA level in BV-2 cells stably expressing MKP-1 after 24 h transfection with MKP-1 siRNA compared to negative control siRNA and vehicle treated cells. IL-6 ( c ), TNF-α ( d ), and IL-1β ( e ) mRNA quantification (qRT-PCR) in control (normal) and stably expressing MKP-1 BV-2 cells transfected 24 h with a negative siRNA, or a siRNA against MKP-1 and challenged with LPS (1 μg/ml) for 6 h (IL-6 and TNF-α) or 12 h (IL-1β). Since MKP-1 siRNA at 10 nM displayed an opposite effect than expected in TNF-α mRNA (potential non-specific effect), 1 nM was used for protein release experiments. Monocyte chemoattractant protein (MCP)-1 ( f ), IL-6 ( g ), TNF-α ( h ), and nitrite ( i ) release in control (normal) and stably expressing MKP-1 BV-2 cells transfected 24 h with a negative siRNA, or a siRNA against MKP-1 and challenged with LPS (1 μg/ml) for 6 h (MCP-1, IL-6, and TNF-α) or 12 h (nitrite). Results are expressed as mean ± s.e.m. of three experiments in duplicate. * P < 0.05 vs. BV-2 cells stably expressing MKP-1 (pMKP1 +).

Article Snippet: Quantitative RT-PCR was performed using a 96-well plate with the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster city, CA) according to the following conditions: 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min, then 40 cycles at 95°C for 15 sec, 60°C for 1 min. All samples were run in duplicate using Taqman gene expression assays (Applied Biosystems, Foster City, CA) with rat MKP-1 (Rn00678341_g1) predesigned and preformulated primer and probe.

Techniques: Transfection, Labeling, Stable Transfection, Expressing, Negative Control, Quantitative RT-PCR

Journal: Molecular Pain

Article Title: Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

doi: 10.1186/1744-8069-8-34

Figure Lengend Snippet: In vivo induction of spinal MKP-1. Quantification of MKP-1 mRNA ( n = 3) ( a ) and of MKP-1 protein ( n = 3) ( b ), as well as a representative Western blot of MKP-1 expression ( c ), in L5–L6 spinal cord in rats that have undergone L5 nerve transection surgery and been treated i.t. with PEI only, PEI + empty cDNA, and PEI + MKP-1 cDNA four days after treatment and surgery. * P < 0.05 versus PEI only or PEI + empty vector. All data are expressed as mean ± s.e.m. ( d ) Representative images obtained by confocal microscopy and immunofluorescence depicting MKP-1 (in green) and the microglial cell marker Iba-1 (in red), the neuronal marker NeuN (in red), and the astrocytic cell markers GFAP (in red) and S100b (in red) in spinal cord of rats four days after L5 nerve transection and treated with PEI + MKP-1 cDNA or PEI only. Arrows indicate co-regionalization (in yellow/orange). Arrowheads show punctate co-regionalization in NeuN-expressing neurons.

Article Snippet: Quantitative RT-PCR was performed using a 96-well plate with the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster city, CA) according to the following conditions: 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min, then 40 cycles at 95°C for 15 sec, 60°C for 1 min. All samples were run in duplicate using Taqman gene expression assays (Applied Biosystems, Foster City, CA) with rat MKP-1 (Rn00678341_g1) predesigned and preformulated primer and probe.

Techniques: In Vivo, Western Blot, Expressing, Plasmid Preparation, Confocal Microscopy, Immunofluorescence, Marker

Journal: Molecular Pain

Article Title: Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

doi: 10.1186/1744-8069-8-34

Figure Lengend Snippet: Effects of in vivo induction of spinal MKP-1 in behavioral hypersensitivity and spinal p-p38 expression. Withdrawal threshold to mechanical stimulation ( a ) in rats undergone L5 nerve transection surgery and treated i.t. with PEI only ( n = 9), PEI + empty cDNA ( n = 4), and PEI + MKP-1 cDNA ( n = 9) four days after treatment and surgery. Quantification of p-p38 protein ( b ) and representative Western blot of p-p38 ( c ) expression from L5–L6 spinal cord of rats that have undergone L5 nerve transection surgery and been treated i.t. with PEI only ( n = 3), PEI + empty cDNA ( n = 3) and PEI + MKP-1 cDNA ( n = 3) four days after treatment and surgery. * P < 0.05 versus PEI only or PEI + empty vector. All data are expressed as mean ± s.e.m. ( d ) Representative images obtained by confocal microscopy and immunofluorescence depicting p-p38 (in green) and the microglial marker Iba-1 (in red), the neuronal marker NeuN (in red), and the astrocytic marker S100b (in red) in spinal cord of rats four days after L5 nerve transection and treated with PEI + MKP-1 cDNA or PEI only. Arrows indicate co-regionalization (in yellow/orange).

Article Snippet: Quantitative RT-PCR was performed using a 96-well plate with the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster city, CA) according to the following conditions: 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min, then 40 cycles at 95°C for 15 sec, 60°C for 1 min. All samples were run in duplicate using Taqman gene expression assays (Applied Biosystems, Foster City, CA) with rat MKP-1 (Rn00678341_g1) predesigned and preformulated primer and probe.

Techniques: In Vivo, Expressing, Western Blot, Plasmid Preparation, Confocal Microscopy, Immunofluorescence, Marker

Journal: Molecular Pain

Article Title: Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

doi: 10.1186/1744-8069-8-34

Figure Lengend Snippet: Effects of in vivo induction of spinal MKP-1 in pro-inflammatory/algesic effectors. Concentrations of IL-1β ( a ), IL-6 ( b ), TNF-α ( c ), and MCP-1 ( d ) from L5–L6 spinal cord of rats undergone L5 nerve transection surgery and treated i.t. with PEI only ( n = 5), PEI + empty cDNA ( n = 4), and PEI + MKP-1 cDNA ( n = 9) four days after treatment and surgery. * P < 0.05 versus PEI only or PEI + empty vector. All data are expressed as mean ± s.e.m.

Article Snippet: Quantitative RT-PCR was performed using a 96-well plate with the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster city, CA) according to the following conditions: 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min, then 40 cycles at 95°C for 15 sec, 60°C for 1 min. All samples were run in duplicate using Taqman gene expression assays (Applied Biosystems, Foster City, CA) with rat MKP-1 (Rn00678341_g1) predesigned and preformulated primer and probe.

Techniques: In Vivo, Plasmid Preparation

Journal: Oncotarget

Article Title: Isorhapontigenin (ISO) inhibited cell transformation by inducing G0/G1 phase arrest via increasing MKP-1 mRNA stability

doi:

Figure Lengend Snippet: (A and B) Cl41 cells were treated with TPA/EGF (40 ng /ml) alone, or pretreated with ISO (50 μM) for 30 min and then co-incubated with ISO and TPA/EGF for indicated time periods. The cell extracts were subjected to Western blot analysis with specific antibodies against PHLPP1, PP2A-A, PP2A-B, p-PP2A, PTEN, and MKP-1. (C and D) Cl41 cells were transfected with the siMKP-1 or nonsense constructs. Stable transfectants were established and cell extracts were subjected to Western blot analysis with specific antibody against MKP-1(C). The indicated transfectants were treated with medium or TPA/EGF (40 ng /ml) alone, or pretreated with ISO (50 μM) for 30 min and then co-incubated with ISO and TPA/EGF for 24 hours. The cell extracts were subjected to Western blot analysis with specific antibodies against MKP-1, cyclin D1, p-c-JunSer63, p-c-JunSer73, c-Jun, p-JNK, JNK, p-MKK7, and MKK7 (D). (E and G) Cl41 cells were treated with medium or TPA/EGF (40 ng /ml) alone, or pretreated with 50 μM of ISO for 30 min and then co-incubated with ISO and TPA/EGF for 48 hours as described in “Materials and Methods”. Data represent one of three different experiments as indicated in Flow cytometric analysis of cell cycle distribution (E) and percentage of cell-cycle phase (G). (F and H) The cell transformation was determined using the indicated Cl41 stable transfectants in the presence of TPA/EGF (40 ng /ml)with or without ISO (50 μM). Representative images of colonies of transformed cells in soft agar assay were presented (F), and the colonies were counted under microscopy and presented as colonies per 10,000 cells from three independent experiments (H). The symbol (*) indicates a significant increase as compared with that in nonsense transfectants treated with TPA/EGF alone ( P <0.05).

Article Snippet: The specific small-interference RNA (siRNA) vector targeted mouse mkp1 (dusp1) was purchased from Sigma-Aldrich Chemical (St. Louis, MO).

Techniques: Incubation, Western Blot, Transfection, Construct, Transformation Assay, Soft Agar Assay, Microscopy

Journal: Oncotarget

Article Title: Isorhapontigenin (ISO) inhibited cell transformation by inducing G0/G1 phase arrest via increasing MKP-1 mRNA stability

doi:

Figure Lengend Snippet: (A) Cl41 cells were treated with medium or TPA/EGF (40 ng /ml) alone, or pretreated with 50μM of ISO for 30 min and then co-incubated with ISO and TPA/EGF for 12 hours. RT-PCR was performed to determine mkp-1 mRNA levels. The gapdh mRNA levels were used as loading control. (B) Cl41 cells stably transfected with MKP-1 promoter luciferase reporter were treated with medium or TPA/EGF (40 ng /ml) alone, or pretreated with 50 μM of ISO for 30 min followed by co-incubation with ISO and TPA/EGF for 24 hours. The luciferase activity was measured as described in “Materials and Methods”. The results were presented as relative MKP-1 promoter activity compared with that of the cells treated with TPA/EGF. The symbol (*) indicates a significant decrease as compared with cells treated with TPA/EGF alone ( P <0.05). (C) Cl41 cells were pretreated with ISO (50 μM) for 6 hours and then co-incubated with ISO and actinomycin D (20 μM) for indicated time periods. Total RNA was isolated and RT-PCR was then performed to determine mkp-1 mRNA levels. The result was a representative one from three independent experiments. (D) The relative mRNA level of mkp-1 was determined by ImageQuant 5.2 (GE Healthcare). Natural logarithm of ratio [ mkp-1 ] t /[ mkp-1 ] 0 was plotted against the time and the half life of mkp-1 mRNA was calculated via linear regression. (E) Cl41 cells were treated with ISO (50 μM) for indicated time periods. The total cell extracts were subjected to Western blot analysis with specific antibodies against HNRPD, VHL, Hur, Nucleolin, and MKP-1. (F) The diagram indicates mechanisms responsible for ISO inhibition of cell transformation.

Article Snippet: The specific small-interference RNA (siRNA) vector targeted mouse mkp1 (dusp1) was purchased from Sigma-Aldrich Chemical (St. Louis, MO).

Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Transfection, Luciferase, Activity Assay, Isolation, Western Blot, Inhibition, Transformation Assay

Journal: Frontiers in Oncology

Article Title: Siglec-15 Promotes Tumor Progression in Osteosarcoma via DUSP1/MAPK Pathway

doi: 10.3389/fonc.2021.710689

Figure Lengend Snippet: Siglec-15 promotes DUSP1 expression in OS cells. (A) Identification of genes regulated by Siglec-15 in OS cells using RNA-Seq assay. (B, C) A heat map and a volcano plot were constructed from the DEGs between siSiglec-15 group and NC group. (D) The top 10 significantly downregulated genes were analyzed by RT-PCR. (E) DUSP1 gene expression in siSiglec-15 group and NC group. (F) Western blotting assay showed that compared with NC group, DUSP1 and p-DUSP1 expression deceased, but p-JNK/MAPK, JNK/MAPK, p-P38/MAPK and P38/MAPK activation increased in siSiglec-15 group. ** P < 0.01, *** P < 0.001, NS, no significance.

Article Snippet: PCNA (10205-2-AP, rabbit, 1/1000), and GAPDH (60004-1-IG, mouse, 1/50000) were purchased from ProteinTech (Wuhan, China); and antibodies against p-DUSP1 (#2857, rabbit, 1/1000), JNK/MAPK(#9252, rabbit, 1/1000), p-JNK/MAPK (#4668, rabbit, 1/1000), P38/MAPK (#8690, rabbit, 1/1000), p-P38/MAPK (#4511, rabbit, 1/1000) were purchased from Cell Signaling Technology (Boston, USA).

Techniques: Expressing, RNA Sequencing, Construct, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Western Blot, Activation Assay

Journal: Frontiers in Oncology

Article Title: Siglec-15 Promotes Tumor Progression in Osteosarcoma via DUSP1/MAPK Pathway

doi: 10.3389/fonc.2021.710689

Figure Lengend Snippet: DUSP1 reverses the effects of shSiglec-15 on the proliferation, migration and invasion of OS cells. (A–C) NC+DUSP1 group increased the proliferation and clone formation of OS cells compared with NC+Vector group, and shSiglec-15+DUSP1 group also increased the proliferation and clone formation OS cells compared with shSiglec-15+Vector group. (D–G) NC+DUSP1 group promoted the migration and invasion of OS cells compared with NC+Vector group, and shSiglec-15+DUSP1 group rescued the migration and invasion of OS cells compared with shSiglec-15+Vector group. * P < 0.05, ** P < 0.01, *** P < 0.001, NS, no significance. Scale bars, 400 µm and 200 µm.

Article Snippet: PCNA (10205-2-AP, rabbit, 1/1000), and GAPDH (60004-1-IG, mouse, 1/50000) were purchased from ProteinTech (Wuhan, China); and antibodies against p-DUSP1 (#2857, rabbit, 1/1000), JNK/MAPK(#9252, rabbit, 1/1000), p-JNK/MAPK (#4668, rabbit, 1/1000), P38/MAPK (#8690, rabbit, 1/1000), p-P38/MAPK (#4511, rabbit, 1/1000) were purchased from Cell Signaling Technology (Boston, USA).

Techniques: Migration, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: Siglec-15 Promotes Tumor Progression in Osteosarcoma via DUSP1/MAPK Pathway

doi: 10.3389/fonc.2021.710689

Figure Lengend Snippet: Siglec-15 promotes OS growth in vivo by inducing EMT. (A) The mice were sacrificed for tumor harvesting after 24 days and tumor image. (B) The tumor growth curves were plotted in shSiglec-15 group and shCtrl group. (C) Tumor weight was measured in shSiglec-15 group and shCtrl group. (D) Western blotting assay showed the expression of N-cadherin, Slug, Vimentin, and MMP-9 in shSiglec-15 group and shCtrl group. (E) Western blotting assay showed DUSP1, p-DUSP1, MAPK/JNK, p-MAPK/JNK, MAPK/P38, and p-MAPK/P38 in shSiglec-15 group and shCtrl group. (F) IHC staining for Siglec-15, DUSP1, Vimentin, P38/MAPK, PCNA and Ki-67 in in shSiglec-15 group and shCtrl group. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: PCNA (10205-2-AP, rabbit, 1/1000), and GAPDH (60004-1-IG, mouse, 1/50000) were purchased from ProteinTech (Wuhan, China); and antibodies against p-DUSP1 (#2857, rabbit, 1/1000), JNK/MAPK(#9252, rabbit, 1/1000), p-JNK/MAPK (#4668, rabbit, 1/1000), P38/MAPK (#8690, rabbit, 1/1000), p-P38/MAPK (#4511, rabbit, 1/1000) were purchased from Cell Signaling Technology (Boston, USA).

Techniques: In Vivo, Western Blot, Expressing, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: Siglec-15 Promotes Tumor Progression in Osteosarcoma via DUSP1/MAPK Pathway

doi: 10.3389/fonc.2021.710689

Figure Lengend Snippet: Both Siglec-15 and DUSP1 are expressed in OS specimens. (A) Representative image of Siglec-15 protein and DUSP1 protein in OS tissue at different stages. (B) The IHC scores of Siglec-15 and DUSP1 protein in high expression group and low expression group. (C, D) The IHC scores of DUSP1 for the Enneking stage and the IHC scores of Siglec-15 for lung metastasis. (E) Kaplan-Meier analysis between low Siglec-15 expression group and high Siglec-15 expression group. (F) The correlation between Siglec-15 expression and DUSP1 expression by the Pearson’s correlation analysis. * P < 0.05, ** P < 0.01 and *** P < 0.001. Scale bars, 100 µm, in the lower right of photos.

Article Snippet: PCNA (10205-2-AP, rabbit, 1/1000), and GAPDH (60004-1-IG, mouse, 1/50000) were purchased from ProteinTech (Wuhan, China); and antibodies against p-DUSP1 (#2857, rabbit, 1/1000), JNK/MAPK(#9252, rabbit, 1/1000), p-JNK/MAPK (#4668, rabbit, 1/1000), P38/MAPK (#8690, rabbit, 1/1000), p-P38/MAPK (#4511, rabbit, 1/1000) were purchased from Cell Signaling Technology (Boston, USA).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Siglec-15 Promotes Tumor Progression in Osteosarcoma via DUSP1/MAPK Pathway

doi: 10.3389/fonc.2021.710689

Figure Lengend Snippet: Relationship between clinicopathologic parameters and Siglec-15 and DUSP1 expression in human osteosarcoma tissues.

Article Snippet: PCNA (10205-2-AP, rabbit, 1/1000), and GAPDH (60004-1-IG, mouse, 1/50000) were purchased from ProteinTech (Wuhan, China); and antibodies against p-DUSP1 (#2857, rabbit, 1/1000), JNK/MAPK(#9252, rabbit, 1/1000), p-JNK/MAPK (#4668, rabbit, 1/1000), P38/MAPK (#8690, rabbit, 1/1000), p-P38/MAPK (#4511, rabbit, 1/1000) were purchased from Cell Signaling Technology (Boston, USA).

Techniques: Expressing