Journal: Cell reports

Article Title: Redox regulation of age-associated defects in generation and maintenance of T cell self-tolerance and immunity to foreign antigens

doi: 10.1016/j.celrep.2022.110363

Figure Lengend Snippet:

Article Snippet: CellsDirect one-Step RT-PCR kit (Invitrogen, Catalog number 11753–100) was used for reverse transcription and specific target amplification (RT-PCR) for each single-cell and universal mouse total RNA of 200 pg.

Techniques: Recombinant, Expressing, Microarray, Software

Journal: Nature Communications

Article Title: H3 ubiquitination by NEDD4 regulates H3 acetylation and tumorigenesis

doi: 10.1038/ncomms14799

Figure Lengend Snippet: ( a ) Summary of H3 ubiquitination sites identified in various large-scale quantitative proteomics studies. ( b ) Glucose deprivation abolished H3 ubiquitination. 293T cells were transfected with his-ubiquitin plasmid (His-Ub) for 36 h and treated with various stresses for 4 h before in vivo ubiquitination assay to access the H3 ubiquitination (see experimental procedures for details). ( c ) Add-back of glucose recovered H3 ubiquitination. 293T cells were transfected with his-ubiquitin plasmid for 36 h, then glucose-starved for 4 h and added-back glucose for indicated times (see experimental procedures for detail) before in vivo ubiquitination assay. ( d ) Screening of E3 ligases for H3 ubiquitination. 293T cells were transfected with his-ubiquitin plasmid and various E3 ligases constructs for in vivo ubiquitination assay. ( e ) NEDD4 E3 ligase dead mutant (CS mutant) failed to trigger H3 ubiquitination. 293T cells were transfected with his-ubiquitin plasmid and WT NEDD4 or NEDD4 CS mutant construct for in vivo ubiquitination assay. ( f ) NEDD4 knockdown abolished H3 ubiquitination. Control and NEDD4 knockdown 293T cells were transfected with his-ubiquitin plasmid for in vivo ubiquitination assay. ( g ) NEDD4 ubiquitinated H3 in vitro . In vitro ubiquitination assay was performed for recombinant NEDD4 and histone octamer (see experimental procedures for details). Reaction products were then assessed by western blotting using anti H3 antibody. H3 mono- and di-ubiquitination have predicted molecular weights of ∼25 kDa and ∼33 kDa. S.E. and L.E. are abbreviations for shorter exposure time and longer exposure time, respectively. ( h ) NEDD4 knockdown abolished glucose-induced H3 ubiquitination. Hep3B cells were glucose starved for 4 h and added-back glucose for 2 h before immunoprecipitation assay for endogenous ubiquitinated proteins (see experimental procedures for details). H3 ubiquitination was then visualized by western blotting. ( i ) Add-back of glucose recovered NEDD4 overexpression induced H3 ubiquitination. 293T cells were transfected with his-ubiquitin and NEDD4 plasmids for 36 h, then glucose-starved for 4 h and added-back glucose for indicated times before in vivo ubiquitination assay. ( j ) NEDD4 triggered monoubiquitination on H3. 293T cells were transfected with Flag-H3, HA-NEDD4, His-Ub WT and His-Ub K0 as indicated before in vivo ubiquitination assay. ( k ) Glucose-induced NEDD4 phosphorylation at Y43 and Y585. 293T cells transfected with WT or Y43/585F NEDD4 plasmids were treated with glucose and harvested for IP. ( l ) NEDD4 phosphorylation is required for H3 ubiquitination. 293T cells transfected with WT, Y43585F or Y43/585E NEDD4 plasmids were harvested for in vivo ubiquitination assay.

Article Snippet: The following antibodies were used in this study: anti-H3 (Abcam, ab12079, 1:5,000), anti-H3 pan-ac (Active Motif, 39139, 1:2,000), anti-H3 K4ac (Active Motif, 39381, 1:2,000), anti-H3 K9ac (Active Motif, 39917, 1:3,000), anti-H3 K14ac (Active Motif, 39697, 1:2,000), anti-H3 K18ac (Active Motif, 39755, 1:2,000), anti-H3 K23ac (Active Motif, 39131, 1:2,000), anti-H3 K27ac (Active Motif, 39133, 1:2,000), anti-H3 K36ac (Active Motif, 39379, 1:2,000), anti-H3 K56ac (Active Motif, 61061, 1:2,000), anti-H3 K4me3 (39915, 1:5,000), anti-H3 K9me3 (Active Motif, 39765, 1:5,000), anti-H3 K27me3 (Active Motif, 39155, 1:5,000), anti-H3 K4me2, K9me2, K27me2, K36me2, K79me2 (Cell Signaling Technology, 9847, 1:5,000), anti-H3s10p (Abcam, ab5176, 1:1,000), anti-H3.3 (EMD Millipore, 09-838, 1:1,000), anti-NEDD4 (Novus, NBP1-40112, 1:4,000), anti-GCN5 (Active Motif, 39975, 1:1,000), anti-USP22 (Abcam, ab4812, 1:2,000), anti-Flag (Sigma, 1:2,000), anti-HA (Covance, 1:2,000), anti-Actin (Sigma, 1:10,000), anti-IL1α (Abcam, ab17281), anti-IL1β (Abcam, ab2105) and anti-IgG heavy chain HRP (Sigma Aldrich, a1949, 1:1,000).

Techniques: Transfection, Plasmid Preparation, In Vivo, Ubiquitin Assay, Construct, Mutagenesis, In Vitro, Recombinant, Western Blot, Immunoprecipitation, Over Expression

Journal: Nature Communications

Article Title: H3 ubiquitination by NEDD4 regulates H3 acetylation and tumorigenesis

doi: 10.1038/ncomms14799

Figure Lengend Snippet: ( a – c ) NEDD4 regulates H3 K9ac at TSS of NEDD4 target genes. Shown were Venn diagram of genes with differential expression or differential H3 K9ac at TSS. GSEA was performed to evaluate the distribution of genes that show down-regulation of H3K9ac at TSS in NEDD4 knockdown cells in microarray-derived gene list, which is rank ordered either by T -test or fold change. ( d ) Heat map view of top and bottom gene list of microarray data sets. Microarray analysis for total RNA was performed for control and NEDD4 knockdown Hep3B cells. ( e ) NEDD4 knockdown impaired IL1α, IL1β and GCLM expression. qPCR was performed to analyse the mRNA level in control and NEDD4 knockdown Hep3B cells ( n =3, mean±s.e.m.). ( f ) IL1α, IL1β and GCLM were induced by glucose. Hep3B cells were glucose starved for 4 h and added-back glucose for 6 h before qPCR analysis ( n =3, mean±s.e.m.). ( g ) UCSC genome browser view of ChIP-seq H3 K9ac signals along the IL1B gene. ( h ) NEDD4 knockdown impaired H3 K9ac at TSS of IL1α IL1β and GCLM genes. ChIP-qPCR using anti-H3 K9ac antibody was performed for control and NEDD4 knockdown Hep3B cells ( n =3, mean±s.e.m.). ( i ) H3 K9ac was induced at TSS of IL1 α IL1β and GCLM genes by glucose. Hep3B cells were glucose-starved for 4 h and added-back glucose for 6 h before ChIP-qPCR analysis using anti-H3 K9ac antibody ( n =3, mean±s.e.m.). ( j ) NEDD4 knockdown impaired glucose-induced polymerase II (pol II) binding at TSS of IL1A and IL1B genes. Control and NEDD4 knockdown Hep3B cells were glucose-starved for 4 h and added-back glucose for 6 h before ChIP-qPCR analysis using anti-pol II antibody ( n =3, mean±s.e.m.). All asterisks (*) represent P <0.05, using Student's T -test.

Article Snippet: The following antibodies were used in this study: anti-H3 (Abcam, ab12079, 1:5,000), anti-H3 pan-ac (Active Motif, 39139, 1:2,000), anti-H3 K4ac (Active Motif, 39381, 1:2,000), anti-H3 K9ac (Active Motif, 39917, 1:3,000), anti-H3 K14ac (Active Motif, 39697, 1:2,000), anti-H3 K18ac (Active Motif, 39755, 1:2,000), anti-H3 K23ac (Active Motif, 39131, 1:2,000), anti-H3 K27ac (Active Motif, 39133, 1:2,000), anti-H3 K36ac (Active Motif, 39379, 1:2,000), anti-H3 K56ac (Active Motif, 61061, 1:2,000), anti-H3 K4me3 (39915, 1:5,000), anti-H3 K9me3 (Active Motif, 39765, 1:5,000), anti-H3 K27me3 (Active Motif, 39155, 1:5,000), anti-H3 K4me2, K9me2, K27me2, K36me2, K79me2 (Cell Signaling Technology, 9847, 1:5,000), anti-H3s10p (Abcam, ab5176, 1:1,000), anti-H3.3 (EMD Millipore, 09-838, 1:1,000), anti-NEDD4 (Novus, NBP1-40112, 1:4,000), anti-GCN5 (Active Motif, 39975, 1:1,000), anti-USP22 (Abcam, ab4812, 1:2,000), anti-Flag (Sigma, 1:2,000), anti-HA (Covance, 1:2,000), anti-Actin (Sigma, 1:10,000), anti-IL1α (Abcam, ab17281), anti-IL1β (Abcam, ab2105) and anti-IgG heavy chain HRP (Sigma Aldrich, a1949, 1:1,000).

Techniques: Expressing, Microarray, Derivative Assay, ChIP-sequencing, Binding Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: LGR5 Promotes Breast Cancer Progression and Maintains Stem-Like Cells Through Activation of Wnt/β-Catenin Signaling.

doi: 10.1002/stem.2083

Figure Lengend Snippet: Figure 3. LGR5 promotes cell migration, tumor metastasis, and activation of the Wnt/b-catenin pathway to induce epithelial- mesenchymal transition. (A): Transwell invasion assay demonstrating that LGR5 promotes cell invasion. Representative images of invaded cells are shown in the left panel, and the results are summarized in the right panel. The results are expressed as the mean 6 SD of three independent experiments (**, p < .01, independent Student’s t test). (B): Metastatic nodules (arrows) on the lung surface. The number of nodules was quantified on lungs of nude mice (n 5 6 per group) 8 weeks after tail vein injection of LGR5- and empty vector- transfected MCF-7 cells and sh-LGR5- and sh-ctr-transfected MDAMB231 cells (**, p < .01, independent Student’s t test). (C): Represen- tative images of hematoxylin and eosin stained sections derived from metastatic nodules at the lung surface. Original magnification, 3100. (D): Representative IF images showing expression of E-cadherin, vimentin, and b-catenin in LGR5-MCF7 cells compared with Vec- MCF7 cells (upper). sh-LGR5- and sh-ctr-transfected MDAMB231 cells (bottom). Nuclei were counterstained with DAPI. Original magnifi- cation, 3400. (E): Relative expressions of E-cadherin, b-catenin, vimentin, fibronectin, snail, and slug were compared between Vec- MCF7 and LGR5-MCF7 or shc-MDAMB231 and shLGR5-MDAMB231 cells by Western blotting (left). The Western blot image in the right panel illustrates that the Wnt inhibitor Dkk1 could effectively decrease b-catenin, snail, C-myc, and cyclinD1 expression induced by LGR5 in MCF-7 cells, whereas Wnt3a could reactivate expression of these genes in shLGR5-MDAMB231 cells.

Article Snippet: Cells were incubated overnight at 48C with the following primary antibodies: LGR5 (Abcam, 1:100); E-cadherin, vimentin, and b-catenin (Cell Signaling Technology, Danvers, MA, 1:100); cytokeratin14 and cytokeratin18 (Bioss People’s Republic of China; 1:100).

Techniques: Migration, Activation Assay, Transwell Invasion Assay, Injection, Plasmid Preparation, Transfection, Staining, Derivative Assay, Expressing, Western Blot

Journal: Stem cells (Dayton, Ohio)

Article Title: LGR5 Promotes Breast Cancer Progression and Maintains Stem-Like Cells Through Activation of Wnt/β-Catenin Signaling.

doi: 10.1002/stem.2083

Figure Lengend Snippet: Figure 5. LGR5 enhances Wnt/b-catenin pathway activity to induce stemness. (A): Cellular morphology is shown at the second day after passaging the LGR5high cells (0 hour, upper) and culturing in the absence of other factors (control) or the presence of either Wnt3a-conditioned medium (Wnt3a) or Dickkopf1 (Dkk1) (96 hours, lower). (B): Cells treated with R-spondin1 and Wnt3a generated sig- nificantly more spheres, and treatment with DKK1 significantly reduced the number of spheres. The data represent the mean 6 SD of three independent experiments, *, p < .05; **, p < .01. (C): The spheroid formed from LGR5high cells expressed different levels of b-catenin in the control medium or when treated with Wnt3a or DKK1. Nuclei were stained with DAPI. Original magnification: 3400. (D): Breast cancer specimens were analyzed by immunohistochemical staining, and the representative LGR5, b-catenin, c-myc, and cyclinD1 expression are shown. Scale bar 5 50 mm. (E): Analysis of immunohistochemistry tissue microarray data showing linear regres- sions and significant Pearson correlations of LGR5 with b-catenin (n 5 129), LGR5 with C-myc (n 5 123), and LGR5 with cyclinD1 (n 5 109) in breast cancer.

Article Snippet: Cells were incubated overnight at 48C with the following primary antibodies: LGR5 (Abcam, 1:100); E-cadherin, vimentin, and b-catenin (Cell Signaling Technology, Danvers, MA, 1:100); cytokeratin14 and cytokeratin18 (Bioss People’s Republic of China; 1:100).

Techniques: Activity Assay, Passaging, Control, Generated, Staining, Immunohistochemical staining, Expressing, Immunohistochemistry, Microarray

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The differential expression and pan-cancer analysis of FGL1 in multiple databases, confirmed by tissue microarray immunohistochemical staining. (A,B) The mRNA expression of 19 ligands (A) and the corresponding 17 receptors (B) were explored in LUAD; the differential expression of FGL1 is significant and selected as the target gene. (C) The expression of FGL1 in various lung cancer subtypes, the number in the brackets referred to the number of articles corresponding to the tumor. (D) The pan-cancer analysis of FGL1 were made by the Oncomine, TIMER, and UALCAN databases, the number in the brackets referred to the number of articles corresponding to the tumor. (E) 3 GSE data sets (GSE 10072, GSE 33532, and GSE 32863) and Oncomine meta-analysis were used to explore the expression of FGL1 in LUAD. (F) 70 LUAD, 70 LUSC tissues, and 140 paired normal tissues were stained as a tissue microarray by immunohistochemical staining, and we confirmed the expression level of FGL1. **, P<0.01; ***, P<0.001; ****, P<0.0001. FGL1, fibrinogen-like protein 1; LUAD, lung adenocarcinoma.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques: Quantitative Proteomics, Microarray, Immunohistochemical staining, Staining, Expressing

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The interactive network of FGL1. (A) The gene-gene interactive network of FGL1 was constructed by the GeneMANIA database. (B) The protein-protein interactive network was constructed by the STRING database. FGL1, fibrinogen-like protein 1.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques: Construct

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The genetic alteration of FGL1 was presented by the cBioPortal database. +, the article included provides the corresponding data; −, the article included not provides the corresponding data; 5%*, the alteration frequency of deep deletion is 5%, significant in all alterations. FGL1, fibrinogen-like protein 1.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques:

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: FGL1-related immune infiltration and the correlation analysis with PD-L1. (A) Immune infiltration related to FGL1 was analyzed by the TIMER database. (B) The correlation analysis of FGL1 and PD-L1 (CD274) (GEPIA). FGL1, fibrinogen-like protein 1; PD-L1, programmed death-ligand 1; GEPIA, Gene Expression Profiling Interactive Analysis.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques: Gene Expression

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The survival analysis of FGL1 in LUAD. (A,B) The survival analysis between FGL1 expression and DFS (A), OS (B) in the GEPIA database. (C) The expression plot and survival analysis of GSE 31210, analyzed by PrognoScan database. (D) The survival analysis between FGL1 deep deletion mutation and OS. (E) The expression of FGL1 in various clinical stages. FGL1, fibrinogen-like protein 1; LUAD, lung adenocarcinoma; DFS, disease-free survival; OS, overall survival.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques: Expressing, Mutagenesis

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The knockdown of FGL1 and RNA-seq results. (A) The knockdown of FGL1 confirmed by western blot. (B) The knockdown of FGL1 confirmed by immunofluorescent staining. (C) Volcano map of differential gene changes in PC9 cells after FGL1 knockdown. (D) Volcano map of differential gene changes in Jurkat T cells after FGL1 knockdown. (E) The enrichment of differential genes in PC9 cells, analyzed by GSEA. **, P<0.01; ***, P<0.001. NC, negative control; KD, knockdown; LUAD, lung adenocarcinoma; FGL1, fibrinogen-like protein 1.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques: Knockdown, RNA Sequencing, Western Blot, Staining, Negative Control

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The experiments related to cell proliferation. (A,B) Effects of FGL1 knockdown on the cycle of PC9 cells and Jurkat T cells. (C) Real-time cell analyzer presents the real-time condition of cell proliferation. (D) Colony formation confirms the cell proliferation of PC9 cells and HCC827 cells, the magnifications in the figure are 40× and 200×, respectively, both of them were stained with crystal violet. **, P<0.01; ***, P<0.001; ****, P<0.0001. NC, negative control; KD, knockdown; LUAD, lung adenocarcinoma; FGL1, fibrinogen-like protein 1.

Article Snippet: The microarray was put into an autoclaved citric acid buffer (pH 6.0) to boil for 15 minutes; 3% hydrogen peroxide was used to block the activity of peroxidase for a 20-minute incubation, and an anti- FGL1 polyclonal antibody (1:100 dilution; Proteintech, Wuhan, China) was used to incubate the microarray overnight at 4 °C.

Techniques: Knockdown, Staining, Negative Control

Journal: PLoS Genetics

Article Title: Antagonistic Cross-Regulation between Sox9 and Sox10 Controls an Anti-tumorigenic Program in Melanoma

doi: 10.1371/journal.pgen.1004877

Figure Lengend Snippet: A , Scheme showing the localization of epidermal melanocytes (in red) in the human skin. B, C , Immunostaining for MITF (green, left panel) and SOX9 (red, right panel) in the human skin demonstrating the lack of SOX9 expression in the epidermal melanocytes. Inserts show higher magnification images of MITF and SOX9 immunostainings. Scale bars, 25 μm. D , Scheme showing the localization of melanocytes (in red) within the hair follicle. E , Immunostaining for MITF (green) and SOX9 (red) in the human skin reveals the expression of SOX9 in the cells of outer root sheath but not in the MITF-positive melanoblasts/melanocytes. Scale bar 100 μm. F, G , High magnification images of immunostaining for MITF and SOX9 in the upper part of human hair follicle ( F ) and the follicular bulb ( G ). H , Analysis of SOX9 (red, left panel) and SOX10 (red, right panel) expression in the patients with human giant congenital naevi demonstrates the lack of SOX9 expression in the SOX10-positive giant congenital naevi cells. Inserts show higher magnification. I , Representative examples of immunostaining for SOX9 (green) and SOX10 (red) in a tissue microarray of primary melanoma samples are shown. J-K , Distribution of SOX10 vs. SOX9 expression in human melanoma (based on TCGA database). 334 melanoma patients were divided in two groups, namely SOX10 High/ SOX9 Low and SOX10 Low / SOX9 high based on SOX10 and SOX9 expression levels. DP, dermal papilla; HF, hair follicle; M, melanocytes; ORS, outer root sheath. Scale bars, 25 μm.

Article Snippet: The following primary antibodies were used: anti-Sox10 (goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox10 (mouse, 1:200, R&D), anti-Sox9 (rabbit, 1:100, sc-20095, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox9 (rabbit, 1:100, ab36748, Abcam), anti-Sox9 (M00006662, Abnova), anti-Sox9 (AB5535, Millipore), anti-Sox9 (GTX 109661, GenTex), anti-MITF (mouse, clone 6D3, 1:500) was a kind gift from Heinz Arnheiter (NIH, USA).

Techniques: Immunostaining, Expressing, Microarray

Journal: PLoS Genetics

Article Title: Antagonistic Cross-Regulation between Sox9 and Sox10 Controls an Anti-tumorigenic Program in Melanoma

doi: 10.1371/journal.pgen.1004877

Figure Lengend Snippet: A , A schematic representation of the experimental strategy used to analyze the expression of Sox9, Sox10, Mitf and Tyr genes in the melanocytic lineage in vivo . B , Results of RNA-seq analysis demonstrating high Sox10 and low Sox9 expression in melanocytic cells at various stages of development. C , A schematic representation of the anatomical location of the melanocyte stem cells, melanoblasts and differentiated melanocytes within the hair follicle in the mouse skin. D , X-Gal staining (blue) combined with Sox9 immunostaining (red) in skin sections of Dct::LacZ mice demonstrating the lack of the Sox9 expression in the melanocyte stem cells located in the bulge region of the hair follicle (upper panels) and in the differentiated melanocytes located in the hair follicular bulb (lower panels). E , Skin sections of Dct::LacZ mice stained for Sox10 (red) in combination with X-Gal staining (blue) reveal the expression of Sox10 in the melanocyte stem cells (upper panels) as well as in the differentiated melanocytes (lower panels). Dashed lines demarcate HFs. Insets show high magnification views. F-I , Experimental strategy used to analyze the effect of the lack of Sox9 ( F ) and Sox10 ( H ) expression in the mouse melanocytic lineage. Pictures of two representative mice at 6 months of age lacking Sox9 gene ( G ) and Sox10 gene ( I ) demonstrating the effects on hair graying. Bg, bulge; HF, hair follicle; MSCs, melanocyte stem cells; Mo, months; E 15.5, embryonic day 15.5; P0, postnatal day 0; SG, sebaceous gland. Scale bars, 25 μm.

Article Snippet: The following primary antibodies were used: anti-Sox10 (goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox10 (mouse, 1:200, R&D), anti-Sox9 (rabbit, 1:100, sc-20095, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox9 (rabbit, 1:100, ab36748, Abcam), anti-Sox9 (M00006662, Abnova), anti-Sox9 (AB5535, Millipore), anti-Sox9 (GTX 109661, GenTex), anti-MITF (mouse, clone 6D3, 1:500) was a kind gift from Heinz Arnheiter (NIH, USA).

Techniques: Expressing, In Vivo, RNA Sequencing Assay, Staining, Immunostaining

Journal: PLoS Genetics

Article Title: Antagonistic Cross-Regulation between Sox9 and Sox10 Controls an Anti-tumorigenic Program in Melanoma

doi: 10.1371/journal.pgen.1004877

Figure Lengend Snippet: A-D , Immunostaining for Sox9 ( A, C ) and Sox10 ( B, D ) in the skin sections of Tyr::Nras Q61K and Tyr::Nras Q61K INK4a −/− mice. E-H , Experimental strategy used to abrogate the expression of Sox9 (E) and Sox10 (G) in the mouse melanocytic lineage. Pictures of two representative mice 1 year after tamoxifen injections reveal no reduction in the skin hyperpigmentation in Tyr::Nras Q61K Sox9 fl/+ Tyr-CreERT2 mice as compared to their Tyr::Nras Q61K littermates (F) in contrast to a pronounced skin whitening observed upon Sox10 loss (H). BF, bright field; HF, hair follicle; mo, months; P0, postnatal day 0. Scale bars, 25 μm.

Article Snippet: The following primary antibodies were used: anti-Sox10 (goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox10 (mouse, 1:200, R&D), anti-Sox9 (rabbit, 1:100, sc-20095, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox9 (rabbit, 1:100, ab36748, Abcam), anti-Sox9 (M00006662, Abnova), anti-Sox9 (AB5535, Millipore), anti-Sox9 (GTX 109661, GenTex), anti-MITF (mouse, clone 6D3, 1:500) was a kind gift from Heinz Arnheiter (NIH, USA).

Techniques: Immunostaining, Expressing

Journal: PLoS Genetics

Article Title: Antagonistic Cross-Regulation between Sox9 and Sox10 Controls an Anti-tumorigenic Program in Melanoma

doi: 10.1371/journal.pgen.1004877

Figure Lengend Snippet: A , Experimental design used to investigate the level of SOX9 and SOX10 expression in vitro . Cultured human keratinocytes, melanocytes, cells derived from biopsies of patients with giant congential naevi and melanoma cells (M010817 cell line) were subjected to RNA isolation and subsequent Q-RT-PCR analysis. Keratinocytes were used as a control. B, C , Quantitative real-time PCR analysis showing the decline of SOX9 expression ( C ) and increase of SOX10 expression ( B ) that correlate with the acquisition of malignant state by human NRAS Q61K -mutated cells. Data are presented as the mean fold change and are normalized over levels found in melanocytes. D, E , SOX10 and SOX9 expression in a large set of proliferative and invasive cell lines analysed by gene expression using microarrays ( D ) and Western blot ( E ) techniques. F , Experimental design used to deregulate SOX10 expression in human cells derived from the biopsy of a patient with NRAS Q61K -mutated giant congenital naevus. G, H , Quantitative real-time PCR analysis of SOX10 ( G ) and SOX9 ( H ) expression after the knockdown of SOX10. I , Experimental design used to analyze the expression of Sox9 in the melanocytic lineage from Tyr::Nras Q61K and Tyr::Nras Q61K Sox10 LacZ/+ mice. K, L , Cells were isolated from the trunk skin of Tyr::Nras Q61K and Tyr::Nras Q61K Sox10 LacZ/+ mice and stained for Melan-a and c-Kit antibodies. FACS-sorted cells were subsequently used for the RNA isolation and quantitative real-time PCR with primers specific for the coding regions of Sox9 gene. Data are presented as the mean fold change and are normalized to the control. Kerat, keratinocytes; M, melanocytes; Nev, naevus cells; Mel, melanoma cells; KD, knock down.

Article Snippet: The following primary antibodies were used: anti-Sox10 (goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox10 (mouse, 1:200, R&D), anti-Sox9 (rabbit, 1:100, sc-20095, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox9 (rabbit, 1:100, ab36748, Abcam), anti-Sox9 (M00006662, Abnova), anti-Sox9 (AB5535, Millipore), anti-Sox9 (GTX 109661, GenTex), anti-MITF (mouse, clone 6D3, 1:500) was a kind gift from Heinz Arnheiter (NIH, USA).

Techniques: Expressing, In Vitro, Cell Culture, Derivative Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Staining

Journal: PLoS Genetics

Article Title: Antagonistic Cross-Regulation between Sox9 and Sox10 Controls an Anti-tumorigenic Program in Melanoma

doi: 10.1371/journal.pgen.1004877

Figure Lengend Snippet: A , SOX9 overexpression in human melanoma cells closely resembles the gene expression signature of SOX10 knockdown as revealed by unsupervised hierarchical clustering of control M010817 melanoma cells, SOX9 overexpressing M010817 cells and SOX10 knock down M010817 cells. Microarray gene expression accession number: GSE37059. B , Western blot analysis showing that SOX10 expression is downregulated upon overexpression of SOX9 in two independent human melanoma cell lines (A375 and M010817). C , Chromatin immunoprecipitation assay demonstrating the binding of SOX9 to the promoter of SOX10 in human melanoma M010817 cells. D, E , Quantitative real-time PCR analysis of SOX10 ( E ) and SOX9 ( F ) expression after the knockdown of SOX10 and after the double knockdown of SOX10 and SOX9 in M010817 cell line. F , Quantification of number of Annexin V-positive cells based on the FACS analysis in the melanoma M010817 cells upon SOX9 KD, SOX10 KD or double SOX9/SOX10 KD. OE, overexpression; KD, knock down; ChIP, chromatin immunoprecipitation; prom, promoter.

Article Snippet: The following primary antibodies were used: anti-Sox10 (goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox10 (mouse, 1:200, R&D), anti-Sox9 (rabbit, 1:100, sc-20095, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox9 (rabbit, 1:100, ab36748, Abcam), anti-Sox9 (M00006662, Abnova), anti-Sox9 (AB5535, Millipore), anti-Sox9 (GTX 109661, GenTex), anti-MITF (mouse, clone 6D3, 1:500) was a kind gift from Heinz Arnheiter (NIH, USA).

Techniques: Over Expression, Expressing, Microarray, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: Up-regulated p53 target gene in MEG3 microarray.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Microarray

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: Down-regulated p53 target gene in MEG3 microarray.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Microarray

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) Reporter assays detected stimulation of p53-mediatd transactivation by MEG3 deletion mutants M1, M1+M2, M3, M2+M3 in HepG2 cells. The value are means of three independent experiments ±S.D, * P<0.05. (B) Cropped blots show the increased level of p53 protein 48h after transfection of the pcDNA-MEG3 in HepG2 cells. (C) Analysis of the effect of MEG3 overexpression on the half-life of p53. Cropped blots show the relative abundance of p53 after treated with translation inhibitor CHX for various amount of time in HepG2 cells (0, 0.5,1, 2h) overexpressing MEG3. (D) Analysis of the effect of MEG3 overexpression on the half-life of p53. Cropped blots show the relative abundance of p53 after treated with translation inhibitor CHX for various amount of time in doxo-induced HepG2 cells (0, 2,4, 6h)overexpressing MEG3.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Transfection, Over Expression

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) Western blot of p53 protein bound to in vitro transcribed biotinylated MEG3 incubated with HEK293 cell extract transfected with Flag-p53 vector. (B) Western blot of p53 protein bound to in vitro transcribed biotinylated MEG3 and deletion mutations RNA incubated with doxo-induced HepG2 cell extract (MEG3 deletion mutants M1, M1+M2, M3, M2+M3 are shown in ). (C) In vitro transcribed biotinylated MEG3 retrieved purified GST-p53 but not GST. (D) RIP experiments were performed using an antibody against the p53 on extracts from doxo-induced HepG2 cells. The purified RNA was used for qRT-PCR, and the enrichment of the lncRNA MEG3 was normalized to GAPDH (upper, western blot of p53 protein after immunoprecipitation). Data was relative to mock-IP (IgG). (E) A series of p53 deletion mutants which were flag-tagged was treated as in (A), and association was detected by anti-Flag. Up represents successful expression of p53 deletion mutants. Down represents in vitro transcribed biotinylated MEG3 RNA was incubated with HEK293 cell extract which was transfected with p53 deletion mutants and associated proteins were detected by anti-Flag.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Western Blot, In Vitro, Incubation, Transfection, Plasmid Preparation, Purification, Quantitative RT-PCR, Immunoprecipitation, Expressing

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) SK-Hep–1 hepatocellular carcinoma cell lines with stably expression of MEG3 were determined by qRT-PCR. The value are means of three independent experiments ±SD, *** P<0.001. (B) Expression of GADD45A, EGR1, SESN2 and TGFA was examined by qRT-PCR and normalized to GAPDH. The bars represent the relative fold change of these genes in SK-Hep–1 after transfection with pcDNA3.0-MEG3 compared with blank vector pcDNA3.0. (C) Expression of GADD45A, EGR1, SESN2 and TGFA was examined by qRT-PCR and normalized to GAPDH after knockdown p53 level in HepG2 cells. The bars represent the relative fold change of these genes in HepG2 cells after co-transfection of pcDNA3.0 and NC, pcDNA3.0-MEG3 and NC, pcDNA3.0-MEG3 and sip53, pcDNA3.0 and sip53, respectively. (D) A schematic diagram illustrating how MEG3 can function as tumor suppressor through interactions with p53.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Stable Transfection, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Knockdown, Cotransfection

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Hyperlipidemia stimuli induce IL-17 and its receptor subunit expression. A, the plasma IL-17 concentration was determined by ELISA. WT and ApoE−/− mice were fed with normal chow or high fat diet (HFD) for 8 weeks starting at 8 weeks of age. Plasma from mice was collected for ELISA analysis. B, IL-17RA mRNA expression in MAECs was determined by qRT-PCR. MAECs were treated with ox-LDL (50 μg/ml) for 24 h, and the IL-17RA mRNA expression was quantified. IL-17RA expression was significantly increased with ox-LDL treatment relative to the control cells. Data were normalized to β-actin. C and D, the protein expressions of IL-17RA and IL-17RC in HAECs were quantified by flow cytometry. HAECs were treated with LDL (50 μg/ml) or ox-LDL (50 μg/ml) for 24 h. C, IL-17RA expression was increased with ox-LDL treatment in HAECs. D, ox-LDL treatment significantly increased IL-17RC expression in HAECs. Data are presented as means ± S.E. (error bars) (n = 3). *, p < 0.05; **, p < 0.01 versus untreated control cells.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Treatment of HAECs with cytokine IL-17 increases the non-treated monocyte adhesion to HAECs. A, IL-17-treated HAECs have increased THP-1 cell adhesion. HAECs were treated with IL-17 (100 ng/ml) for the indicated time, and untreated labeled monocytes were allowed to adhere onto the endothelial monolayer. IL-17 treatment increased THP-1 monocytic cell adhesion onto HAECs in a time-dependent manner. The adhesion is shown as a percentage of the control group. B, untreated primary human monocyte adhesion to HAECs treated with IL-17 gradually increased up to 24 h. C, fluorescence microscopic images of labeled primary human monocyte adhered to HAECs. Data are presented as means ± S.E. (error bars) (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Labeling, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Depletion of IL-17 gene in ApoE−/− mice significantly decreases in vivo leukocyte rolling and adhesion to endothelium. ApoE−/− mice and ApoE−/−/IL-17A−/− mice were fed with a high fat diet for 3 weeks before intravital microscopy analysis was performed. A, intravital microscopy was used to determine the endothelial activation by determining leukocyte rolling and adhesion to endothelium in vivo. CCD, charge-coupled device. B, mean vessel diameters of venules were not significantly different between ApoE−/− and ApoE−/−/IL-17A−/− mice used for the intravital microscopy study. C, number of cells that rolled past an imaginary line that was perpendicular to the vessels during a 1-min period were not significant in both animal groups used in the study. D, the number of cells adhered to 100 μm of endothelium along the vessel for 1 min was significantly reduced in IL-17-null mice relative to the controls. Data are presented as means ± S.E. (error bars) (n = 7–8; five vessels per mouse were recorded). *, p < 0.05 (significant); n.s., not significant.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: In Vivo, Intravital Microscopy, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: IL-17 induces the gene expression of four proinflammatory cytokines and chemokines, namely IL-6, GM-CSF, CXCL1, and CXCL2, in mouse and human aortic endothelial cells. The expressions of 84 genes related to EC biology were profiled by using the Mouse Endothelial Cell Biology RT2 Profiler PCR Array. MAECs were treated with IL-17 for 24 h and used in the Mouse Endothelial Cell Biology RT2 Profiler PCR Array. A, 84 genes assessed by the array. B, scatter plot of the changes of gene expression in IL-17-treated MAECs. Expressions of four genes were dramatically induced by IL-17 treatment; blue lines indicate a -fold change of 4. C, IL-17-mediated increases in the expressions of CXCL1, CXCL2, IL-6, and CSF2 (GM-CSF) were further confirmed in MAECs by quantitative PCR. Data are presented as means ± S.E. (n = 5). *, p < 0.05; **, p < 0.01; ***p < 0.001 versus control. D, human IL-17 treatment significantly enhanced the expressions of CXCL1, CXCL2, IL-6, and CSF2 in HAECs treated with IL-17 (100 ng/ml) for 12 h as detected by qRT-PCR. Data were normalized to β-actin expression and are presented as means ± S.E. (n = 3). *, p < 0.05 versus control. E, the blockage of proinflammatory cytokines IL-6 and GM-CSF and chemokines CXCL1 and CXCL2 with specific antibodies decreased non-treated monocyte adhesion to IL-17-stimulated endothelial cells. Endothelial cells were treated with IL-17, and the blocking antibodies against CXCL1/2, IL-6, and/or GM-CSF were added 30 min prior to the adhesion assay. Antibodies against CXCL1/2, IL-6, and GM-CSF reversed the effect of IL-17 on increasing monocyte adhesion to endothelial cells relative to the IgG control. F, fluorescence microscopic image of labeled non-treated THP-1 cells adhered to endothelial cells. Data are presented as means ± S.E. (error bars) (n = 3). *, p < 0.05 versus control; #, p < 0.05 versus IL-17-treated group.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Blocking Assay, Cell Adhesion Assay, Fluorescence, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: IL-17 and IL-6 synergistically induce ICAM-1 up-regulation in HAECs. HAECs were treated with IL-6 (20 ng/ml), IL-17 (50 ng/ml), or IL-6 (20 ng/ml) + IL-17 (50 ng/ml) for 6 h. After the treatment, cells were collected, and ICAM-1 expression was quantified by a flow cytometry method with fluorescence-conjugated anti-ICAM-1 antibody. A, histograms showing an increase of ICAM-1 expression after IL-17 (50 ng/ml) treatment that was further increased when the cells were treated with IL-6 (20 ng/ml) plus IL-17 (50 ng/ml). B, bar graph showing quantification of ICAM-1 expression in HAECs. Data are presented as mean ± S.E. (error bars) (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Mouse aortic microarray results indicate that IL-17 up-regulates the expressions of many genes in mouse aortas including IL-17RA, endothelial cell adhesion molecules, and MAPK pathway components. A, mouse aortic RNAs were isolated from ApoE−/−mice and IL-17−/−/ApoE−/− mice fed with a high fat diet for 3 weeks and subjected to Affymetrix microarray analysis. B, volcano plot (scatter plot) of -fold change versus p value for all gene transcripts determined by the Affymetrix GeneChip Mouse Gene 2.0ST Arrays in mouse aortas from ApoE−/−mice and IL-17−/−/ApoE−/− mice fed with a high fat diet for 3 weeks. The volcano plot shows that there are numerous genes that were modified with statistical significance in IL-17−/−/ApoE−/− mouse aortas relative to ApoE−/− mouse aortas. C, the heat map and -fold changes of IL-17 receptor signaling genes. D, heat map and -fold changes of endothelial adhesion molecule genes. E, heat map and -fold changes of p38 MAPK (MAPK14) pathway genes. The values with p < 0.05 are highlighted in red.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Microarray, Isolation, Modification

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Depletion of IL-17 in ApoE−/− mice decreases the phosphorylation of p38 MAPK but not the phosphorylation of JNK MAPK nor the phosphorylation of eNOS in mouse aortas, and IL-17 also induces the phosphorylation of p38 MAPK in vitro. A, mouse aortic proteins were isolated from ApoE−/−mice and IL-17−/−/ApoE−/− mice fed with a high fat diet for 3 weeks. Representative Western blotting analyses of phosphorylated and total p38, phosphorylated and total JNK, and phosphorylated and total eNOS, and β-actin expressions are shown. B, quantification of total p38. C, quantification of total JNK. D, quantification of the phosphorylated p38. E, quantification of phosphorylated JNK. F, schematic representation of how IL-17 transcriptionally and post-translationally activates the p38 MAPK pathway but not JNK MAPK pathway. G, quantitation of phosphorylated eNOS (Ser-1177). H, quantification of phosphorylated eNOS (Thr-495). I, IL-17 treatment induced the phosphorylation of p38 MAPK and its upstream kinase Map2k3 in HAECs. Representative Western blotting analyses of phosphorylated p38 MAPK, total p38 MAPK, phosphorylated Map2k3, and total Map2k3 are shown. J, IL-17 treatment did not induce JNK and its upstream kinase Map2k4 in HAECs. Western blotting analyses of phosphorylated JNK, total JNK, phosphorylated Map2k4, and total Map2k4 are represented. Data are presented as means ± S.E. (error bars). *, p < 0.05 (significant); n.s., not significant.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: In Vitro, Isolation, Western Blot, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Inhibition of IL-17-mediated phosphorylation of p38 MAPK attenuates the inflammation and endothelial activation in human aortic endothelial cells in vitro. A, p38 MAPK deficiency decreased IL-6 and ICAM-1 gene expressions. Microarray database mining analysis was carried out by analyzing the National Institutes of Health NCBI GEO data set GSE7342, which studied the role of p38α in regulating the gene expression in mouse embryonic development. B, inhibition of p38 MAPK decreased the expression of ICAM-1 in IL-17-treated HAECs. HAECs were pretreated for 1 h with p38 inhibitor (SB203580; 10 μm) followed by IL-17 (100 ng/ml) treatment for 6 h. The cells were stained with fluorescent dye allophycocyanin-conjugated ICAM-1 antibody for 30 min and analyzed by flow cytometry with IgG isotype control. C and D, the HAECs were pretreated with SB203580 (10 μm) for 1 h followed by IL-17 treatment (100 ng/ml) for 24 h. The CXCL1/2 gene expression was measured by quantitative PCR. C, p38 MAPK inhibitor significantly ameliorated IL-17-induced CXCL1 gene expression. D, p38 MAPK inhibitor significantly ameliorated CXCL2 gene expression mediated by IL-17. E, p38 MAPK inhibitor significantly attenuated the IL-17-induced non-treated monocyte adhesion to HAECs. The HAECs were pretreated with SB203580 (10 μm) for 1 h prior to incubation with IL-17 (100 ng/ml) for 24 h. Data are presented as means ± S.E. (error bars). ***, p < 0.001; **, p < 0.01; *, p < 0.05.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Inhibition, Activation Assay, In Vitro, Microarray, Expressing, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: Deficiency of IL-17A in ApoE−/− mice modulates the lipid profile but does not significantly affect atherosclerotic lesion formation. A, mouse experimental design. Male ApoE−/− mice and IL-17−/−/ApoE−/− mice were put on either a high fat diet or regular chow diet at 8 weeks of age for 3 weeks. B, body weight, heart weight, and spleen weight of ApoE−/− mice and IL-17−/−/ApoE−/− mice that were on either a normal chow diet or a high fat diet for 3 weeks. Data are presented as means ± S.E. (n = 7–10). C, the plasma lipid profiles of mice that were fed a high fat (HF) diet or normal chow diet for 3 weeks. FFA, free fatty acids. Data are presented as means ± S.E. (n = 7–10). *, p < 0.05. D, atherosclerotic plaque formation in the whole aorta was assessed with Sudan IV staining. The upper panel represents the images of 3-week-diet mouse group aortas stained with Sudan IV. The lower panel indicates the quantification of atherosclerotic area percentage in the whole aorta of the 3-week-diet mouse group. Data are presented as means ± S.E. (n = 4–11). E, atherosclerotic plaque in aortic sinus was determined by Oil Red O staining of aortic cross-sections. The upper panel represents the images of aortic sinus cross-section stained with Oil Red O in 3-week-diet mouse groups. The lower panel shows the quantification of atherosclerotic area percentage in the aortic sinus of 3-week-diet mouse groups. Data are presented as means ± S.E. (error bars) (n = 5).

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Interleukin-17A Promotes Aortic Endothelial Cell Activation via Transcriptionally and Post-translationally Activating p38 Mitogen-activated Protein Kinase (MAPK) Pathway *

doi: 10.1074/jbc.M115.690081

Figure Lengend Snippet: The proposed novel working model. IL-17 promotes endothelial cell activation by inducing p38 MAPK, which leads to up-regulation of proinflammatory cytokines, chemokines, and endothelial cell adhesion molecules.

Article Snippet: HAECs (passage 9) were cultured in 24-well plates and treated with human IL-17 (100 ng/ml; R&D Systems).

Techniques: Activation Assay