Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: (A) Schematic depicts bioinformatics approach to compare Egr2 and Myrf binding sites in Schwann cells versus oligodendrocytes, respectively. Egr2 binding sites in Schwann cell-rich P15 rat sciatic nerve and Myrf sites in cultured oligodendrocytes were recently published (Lopez-Anido et al. 2015, Srinivasan et al. 2012, Bujalka et al. 2013). (B) Overlap analysis of genes with Egr2 and Myrf binding sites nearby identifies 304 genes that may be shared targets in Schwann cells and oligodendrocytes. (C) Gene ontology (GO) terms clustering and Kegg pathway analysis on Egr2/Myrf shared target genes reveals enrichment in signaling pathways and other processes. (D) Scatter plot represents gene expression for a given gene (represented by dots) in control versus Egr2-deficient sciatic nerve. Gene expression values were obtained from a previous microarray study on mice with a hypomorphic allele of Egr2 (Le et al. 2005a, Le et al. 2005b). Dark grey dots are either upregulated in Egr2-deficient nerve by >1.25-fold or downregulated by <0.8-fold. The black dots represent genes that are expressed in control nerve and also have an Egr2/Myrf binding event nearby, and these include (highlighted in red) the well-studied myelin gene Myelin-associated glycoprotein (Mag) as well as Dual specificity phosphatase 15 (Dusp15), a gene with an unknown role in peripheral nerve myelination. (E) ChIP-Seq analysis profiles depict genomic regions enriched with transcriptional regulators and enhancers in P15 rat sciatic nerve and spinal cord. Included are binding profiles of Sox10 and Egr2 (Lopez-Anido et al. 2015, Srinivasan et al. 2012). Profiles of H3K27ac and the oligodendrocyte-specific regulator Myrf in cultured oligodendrocytes were also obtained from published datasets (Yu et al. 2013, Bujalka et al. 2013, Lopez-Anido et al. 2015). Egr2- versus Myrf-enriched enhancers specific to Schwann cells versus oligodendrocytes are indicated by grey boxes.

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: Binding Assay, Cell Culture, Protein-Protein interactions, Gene Expression, Control, Microarray, ChIP-sequencing

Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: (A) ChIP-Seq analysis of P15 sciatic nerve reveals Sox10 and Egr2 enrichment at the Dusp15 gene, with a grey box highlighting the promoter region. (B) ChIP-Seq analysis on P25 rat sham sciatic nerve show the H3K27ac-marked enhancer (grey box), which is diminished in cut nerve (3 d post-injury). (C) Luciferase assays were performed in S16 Schwann cells with constructs containing the promoter region of Dusp15 (Pro_Dusp15) and treated with Sox10 siRNA. Relative light units (RLU) are shown relative to siControl treatment after normalizing to β-galactosidase activity from a co-transfected CMV-lacZ plasmid. Quantitative RT-PCR was used to determine relative mRNA expression levels in (D) S16 rat Schwann cells treated with siRNA against Sox10. (E) S16 Schwann cells were treated with siRNA against Egr2 and quantitative RT-PCR was used to measure mRNA levels of Sox10, Egr2 and Dusp15. (F) Dusp15 levels were measured in uncut (sham) or cut sciatic nerve (1 and 4 d post-injury) using quantitative RT-PCR. 18S housekeeping gene was used to normalize all quantitative RT-PCR assays and error bars indicate the standard deviation of three biological replicates (n=7 for 1 d post-injury and 3 for 4 d post-injury). (*P<0.05)

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: ChIP-sequencing, Luciferase, Construct, Activity Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: (A) Dusp15 levels were measured using quantitative RT-PCR in RT4 Schwann cells treated with siRNA against Dusp15. (B) Western Blot analysis of Erk1/2 phosphorylation was performed and (C) quantified in RT4 Schwann cells treated with siRNA against Dusp15. (D) Dusp15 mRNA levels were compared between S16 and RT4 Schwann cells using quantitative RT-PCR. (E) Diagram depicting guide RNA (red) target sites for CRISPR/Cas9 deletion of the Dusp15 gene. (F) Genotyping of Cas9 treated cells was determined by PCR using primers (orange pair) flanking the deleted genomic and the uncut 3′UTR region in both lines (green). (G) Dusp15 mRNA levels in WT and KO cell lines were determined by quantitative RT-PCR. (H) Erk1/2 phosphorylation was measured in WT versus KO lines and compared to total levels of Erk1/2 by western blot analysis. (I) Band signals obtained from western blot analysis were normalized to Total-Erk (T-Erk) levels. All experiments represent average values of biological triplicates. (*P<0.05)

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: Quantitative RT-PCR, Western Blot, Phospho-proteomics, CRISPR

Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: Quantitative RT-PCR was used to determine Ccl2, Vegfc and Pmp2 mRNA levels in (A) RT4 Schwann cells treated with siRNA against Dusp15 and (B) Dusp15-KO-RT4 Schwann cell line. (C) Ccl2 mRNA levels were measured in Dusp15-KO Schwann cells transfected with an expression plasmid for Dusp15 using quantitative RT-PCR. (*P<0.05)

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation

Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: Mag, Mbp, Cx32, Pmp22 and Srebp-1c mRNA levels were measured by quantitative RT-PCR in (A) RT4 Schwann cells, Dusp15 and Mag in (B) Primary Schwann cells treated with siRNA against Dusp15 and in (C and D) Dusp15-KO Schwann cell line compared to parent RT4 Schwann cells. (E) Mbp and (F) Pmp22 mRNA levels were measured in Dusp15-KO Schwann cells treated with ectopic expression levels of Dusp15. All experiments represent biological triplicates, and expression levels were normalized to 18S rRNA. (*P<0.05)

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: Quantitative RT-PCR, Expressing

Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: Mbp, Mag, Pmp22, Mpz, Pmp2, Vegfc and Ccl2 mRNA levels were measured in RT4 Schwann cells treated with (A and B) Trametinib 18nM, or with (C, D and F) PMA using quantitative RT-PCR. Samples were normalized to the level of 18S rRNA. Error bars represent the standard deviation from biological triplicates.

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: Quantitative RT-PCR, Standard Deviation

Journal: Journal of neurochemistry

Article Title: Dual specificity phosphatase 15 regulates Erk activation in Schwann cells

doi: 10.1111/jnc.13911

Figure Lengend Snippet: Mag, Mpz, Mbp, Pmp22, Sox10, Egr2, Dusp15, Pmp2 and Ccl2 were measured in Primary Schwann cells when these were treated with Trametinib (A) and PMA (B) as before. Genes were measured using quantitative RT-PCR and normalized to 18S rRNA. Error bars represent the standard deviation from biological triplicates. (*P<0.05)

Article Snippet: S16 ( Toda et al. 1994 ) and RT4-D6P2T ( Hai et al. 2002 ) (referred to as RT4, obtained from ATCC) rat Schwann cells were grown in supplemented DMEM (Corning cellgro, 10-017-CV) with penicillin, streptomycin and 5% bovine growth serum (Hyclone, SH30541.03HI).

Techniques: Quantitative RT-PCR, Standard Deviation