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anti neurl3 antibody  (Proteintech)


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    Proteintech anti neurl3 antibody
    Fig. 1 <t>NEURL3</t> is hypermethylated in NPC. (a) Heatmap clustering of the top 20 hypermethylated genes based on integrated analysis of two genome- wide DNA methylation microarray datasets (GSE52068 and GSE62336) in NPC and normal nasopharyngeal tissues. (b) The genome features of NEURL3 observed using the UCSC genome browser and schematic diagram of the CpG island sites at NEURL3 promoter region. (c) Methylation levels of NEURL3 in NPC tissues (n = 24) and normal tissues (n = 24) in the methylation microarray dataset GSE52068. (d) Methylation levels of NEURL3 in NPC tissues (n = 25) and normal tissues (n = 25) in HongKong methylation microarray dataset GSE62336. (e) Representative images of bisulfite pyrosequencing analysis of the NEURL3 promoter region. (f) Statistical analysis of methylation levels of NEURL3 in NPC (n = 8) and normal (n = 8) tissues. Data in c, d, and f are presented as the mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)
    Anti Neurl3 Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti neurl3 antibody/product/Proteintech
    Average 92 stars, based on 8 article reviews
    anti neurl3 antibody - by Bioz Stars, 2026-02
    92/100 stars

    Images

    1) Product Images from "The E3 ligase NEURL3 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma by promoting vimentin degradation."

    Article Title: The E3 ligase NEURL3 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma by promoting vimentin degradation.

    Journal: Journal of experimental & clinical cancer research : CR

    doi: 10.1186/s13046-024-02945-9

    Fig. 1 NEURL3 is hypermethylated in NPC. (a) Heatmap clustering of the top 20 hypermethylated genes based on integrated analysis of two genome- wide DNA methylation microarray datasets (GSE52068 and GSE62336) in NPC and normal nasopharyngeal tissues. (b) The genome features of NEURL3 observed using the UCSC genome browser and schematic diagram of the CpG island sites at NEURL3 promoter region. (c) Methylation levels of NEURL3 in NPC tissues (n = 24) and normal tissues (n = 24) in the methylation microarray dataset GSE52068. (d) Methylation levels of NEURL3 in NPC tissues (n = 25) and normal tissues (n = 25) in HongKong methylation microarray dataset GSE62336. (e) Representative images of bisulfite pyrosequencing analysis of the NEURL3 promoter region. (f) Statistical analysis of methylation levels of NEURL3 in NPC (n = 8) and normal (n = 8) tissues. Data in c, d, and f are presented as the mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)
    Figure Legend Snippet: Fig. 1 NEURL3 is hypermethylated in NPC. (a) Heatmap clustering of the top 20 hypermethylated genes based on integrated analysis of two genome- wide DNA methylation microarray datasets (GSE52068 and GSE62336) in NPC and normal nasopharyngeal tissues. (b) The genome features of NEURL3 observed using the UCSC genome browser and schematic diagram of the CpG island sites at NEURL3 promoter region. (c) Methylation levels of NEURL3 in NPC tissues (n = 24) and normal tissues (n = 24) in the methylation microarray dataset GSE52068. (d) Methylation levels of NEURL3 in NPC tissues (n = 25) and normal tissues (n = 25) in HongKong methylation microarray dataset GSE62336. (e) Representative images of bisulfite pyrosequencing analysis of the NEURL3 promoter region. (f) Statistical analysis of methylation levels of NEURL3 in NPC (n = 8) and normal (n = 8) tissues. Data in c, d, and f are presented as the mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)

    Techniques Used: Genome Wide, DNA Methylation Assay, Microarray, Methylation

    Fig. 3 NEURL3 suppresses NPC cell migration, invasion, and EMT in vitro. (a) In GSEA analysis using a public RNA-seq dataset (GSE102349), the gene sets associated with cell migration and metastasis pathways were significantly enriched in NPC samples with a low NEURL3 expression. (b) The migratory abili ties of HONE1, SUNE1, and HK1 cells transfected with HA-NEURL3 plasmid or its empty vector determined by wound healing assay. (c-d) The migratory (c) and invasive (d) abilities of HONE1, SUNE1, and HK1 cells transfected with HA-NEURL3 plasmid or its empty vector determined by Transwell assay. (e) IF staining showing the filopodia length on HONE1, SUNE1, and HK1 cells transfected with HA-NEURL3 plasmid or its empty vector. (f) The morphology changes induced by TGF-β1 in HONE1, and SUNE1 cells transfected with HA-NEURL3 plasmid or its empty vector. Scale bar, 50 μm. Data of b, c, d, and e are shown as mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)
    Figure Legend Snippet: Fig. 3 NEURL3 suppresses NPC cell migration, invasion, and EMT in vitro. (a) In GSEA analysis using a public RNA-seq dataset (GSE102349), the gene sets associated with cell migration and metastasis pathways were significantly enriched in NPC samples with a low NEURL3 expression. (b) The migratory abili ties of HONE1, SUNE1, and HK1 cells transfected with HA-NEURL3 plasmid or its empty vector determined by wound healing assay. (c-d) The migratory (c) and invasive (d) abilities of HONE1, SUNE1, and HK1 cells transfected with HA-NEURL3 plasmid or its empty vector determined by Transwell assay. (e) IF staining showing the filopodia length on HONE1, SUNE1, and HK1 cells transfected with HA-NEURL3 plasmid or its empty vector. (f) The morphology changes induced by TGF-β1 in HONE1, and SUNE1 cells transfected with HA-NEURL3 plasmid or its empty vector. Scale bar, 50 μm. Data of b, c, d, and e are shown as mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)

    Techniques Used: Migration, In Vitro, RNA Sequencing, Expressing, Transfection, Plasmid Preparation, Wound Healing Assay, Transwell Assay, Staining

    Fig. 4 NEURL3 interacts with Vimentin to promote its degradation. (a) Silver staining of SDS-PAGE gels showing HA immunoprecipitates that were pulled down from SUNE1 cells transfected with HA-NEURL3 plasmid. The interest proteins are indicated. (b) Co-IP with anti-HA or Vimentin antibodies showing the interactions between HA-NEURL3 and endogenous Vimentin in HONE1 and SUNE1 cells. (c) IF staining showing co-localization of exogenous HA- NEURL3 and endogenous Vimentin in HONE1 and SUNE1 cells (Scale, 50 μm). (d) Schematic diagram showing the structure of Vimentin or its deletion mutant plasmids. (e) Co-IP revealing interactions of NEURL3 with different Vimentin mutants in 293T cells. (f-g) Vimentin protein levels in HONE1 and SUNE1 cells transfected with gradient concentrations of HA-tagged NEURL3 plasmid (f), as well as shNEURL3 plasmids or its control vector (g)(h) IF staining showing Vimentin protein levels in HONE1 and HK1 cells transfected with HA-NEURL3 plasmid or its vector control (Scale bar, 50 µM). (i-j) Rep resentative images and greyscale analyses of Vimentin protein levels in SUNE1 and 293T cells transfected with HA-NEURL3 plasmid or its vector control (i), as well as shNEURL3 plasmid or its control vector (j), after the CHX treatment. (k-l) Vimentin protein levels in HONE1 and SUNE1 cells transfected with HA-NEURL3 plasmid or its vector control after treated with MG132 (k) and CQ (l). Data of i and j are shown as mean ± SD, and the p-values were deter mined by Student’s t-test (*p < 0.05)
    Figure Legend Snippet: Fig. 4 NEURL3 interacts with Vimentin to promote its degradation. (a) Silver staining of SDS-PAGE gels showing HA immunoprecipitates that were pulled down from SUNE1 cells transfected with HA-NEURL3 plasmid. The interest proteins are indicated. (b) Co-IP with anti-HA or Vimentin antibodies showing the interactions between HA-NEURL3 and endogenous Vimentin in HONE1 and SUNE1 cells. (c) IF staining showing co-localization of exogenous HA- NEURL3 and endogenous Vimentin in HONE1 and SUNE1 cells (Scale, 50 μm). (d) Schematic diagram showing the structure of Vimentin or its deletion mutant plasmids. (e) Co-IP revealing interactions of NEURL3 with different Vimentin mutants in 293T cells. (f-g) Vimentin protein levels in HONE1 and SUNE1 cells transfected with gradient concentrations of HA-tagged NEURL3 plasmid (f), as well as shNEURL3 plasmids or its control vector (g)(h) IF staining showing Vimentin protein levels in HONE1 and HK1 cells transfected with HA-NEURL3 plasmid or its vector control (Scale bar, 50 µM). (i-j) Rep resentative images and greyscale analyses of Vimentin protein levels in SUNE1 and 293T cells transfected with HA-NEURL3 plasmid or its vector control (i), as well as shNEURL3 plasmid or its control vector (j), after the CHX treatment. (k-l) Vimentin protein levels in HONE1 and SUNE1 cells transfected with HA-NEURL3 plasmid or its vector control after treated with MG132 (k) and CQ (l). Data of i and j are shown as mean ± SD, and the p-values were deter mined by Student’s t-test (*p < 0.05)

    Techniques Used: Silver Staining, SDS Page, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Staining, Mutagenesis, Control

    Fig. 5 NEURL3 promotes K48-linked ubiquitination and degradation of Vimentin. (a) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with Myc-NEURL3 or its empty vector, together with Flag-Vimentin plasmid and a vector encoding HA-WT-Ub or its mutants (HA-K48O-Ub or HA-K63O-Ub). (b) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with shNEURL3 plasmid or its control vector, together with Flag-Vimentin plasmid and a vector encoding HA-WT-Ub or its mutants (HA-K48O-Ub or HA-K48R-Ub). (c) The ubiquitina tion level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with empty vector, wild-type (WT) Myc-NEURL3 or its C217Y mutant, together with Flag-Vimentin and HA-WT-Ub. (d-e) Representative images and greyscale analyses of Vimentin protein levels after CHX treatment in SUNE1 (d) and 293T (e) cells co-transfected with WT Myc-NEURL3 or its C217Y mutant, together with Flag-Vimentin. (f) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with Myc-NEURL3 or its empty vector, together with WT Flag-Vimentin or its K97R mutant and HA-WT-Ub. (g-h) Representative images and greyscale analyses of Vimentin protein levels after CHX treatment in SUNE1 (g) and 293T (h) cells co-transfected with Myc-NEURL3, together with WT Flag-Vimentin or its K97R mutant. Data of d, e, g, and h are shown as mean ± SD, and the p-values were determined by Student’s t-test (ns, no significance; *p < 0.05)
    Figure Legend Snippet: Fig. 5 NEURL3 promotes K48-linked ubiquitination and degradation of Vimentin. (a) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with Myc-NEURL3 or its empty vector, together with Flag-Vimentin plasmid and a vector encoding HA-WT-Ub or its mutants (HA-K48O-Ub or HA-K63O-Ub). (b) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with shNEURL3 plasmid or its control vector, together with Flag-Vimentin plasmid and a vector encoding HA-WT-Ub or its mutants (HA-K48O-Ub or HA-K48R-Ub). (c) The ubiquitina tion level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with empty vector, wild-type (WT) Myc-NEURL3 or its C217Y mutant, together with Flag-Vimentin and HA-WT-Ub. (d-e) Representative images and greyscale analyses of Vimentin protein levels after CHX treatment in SUNE1 (d) and 293T (e) cells co-transfected with WT Myc-NEURL3 or its C217Y mutant, together with Flag-Vimentin. (f) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with Myc-NEURL3 or its empty vector, together with WT Flag-Vimentin or its K97R mutant and HA-WT-Ub. (g-h) Representative images and greyscale analyses of Vimentin protein levels after CHX treatment in SUNE1 (g) and 293T (h) cells co-transfected with Myc-NEURL3, together with WT Flag-Vimentin or its K97R mutant. Data of d, e, g, and h are shown as mean ± SD, and the p-values were determined by Student’s t-test (ns, no significance; *p < 0.05)

    Techniques Used: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Control, Mutagenesis

    Fig. 6 Restoration of Vimentin reverses the tumor suppressive effect of NRURL3. HONE1, SUNE1, and HK1 cells were transiently co-transfected with HA- NEURL3 or empty vector together with Flag-Vimentin or empty vector. (a) The migratory abilities of transfected NPC cells were determined by wound healing assay. (b-c) The migratory (b) and invasive (c) abilities of transfected NPC cells were determined by Transwell assay. (d) IF staining showing the pseudopods on transfected NPC cells. (e) The morphology changes induced by TGF-β1 in transfected NPC cells. Scale bar, 50 μm. Data of a, b, and c are shown as mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)
    Figure Legend Snippet: Fig. 6 Restoration of Vimentin reverses the tumor suppressive effect of NRURL3. HONE1, SUNE1, and HK1 cells were transiently co-transfected with HA- NEURL3 or empty vector together with Flag-Vimentin or empty vector. (a) The migratory abilities of transfected NPC cells were determined by wound healing assay. (b-c) The migratory (b) and invasive (c) abilities of transfected NPC cells were determined by Transwell assay. (d) IF staining showing the pseudopods on transfected NPC cells. (e) The morphology changes induced by TGF-β1 in transfected NPC cells. Scale bar, 50 μm. Data of a, b, and c are shown as mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)

    Techniques Used: Transfection, Plasmid Preparation, Wound Healing Assay, Transwell Assay, Staining

    Fig. 7 NEURL3 inhibits NPC metastasis in vivo. SUNE1 cells stably expressing HA-NEURL3 or its empty vector were subcutaneously injected into foot pads of mice to establish an inguinal lymph node metastasis model. (a) Representative images of the formed footpad tumors and inguinal lymph nodes. (b-c) Representative images of inguinal lymph nodes (b), and statistical analysis of the volume of lymph nodes (c) between the two groups. Data is shown as mean ± SD, and the p-value was determined by Student’s t-test (*p < 0.05). (d) H&E staining showing the infiltration of tumor cells into skin and muscle of footpad tumors (Scale bar, 100 μm). (e) The infiltration of cancer cells into inguinal lymph nodes as determined by a positive of pan-cytokeratin IHC staining (Scale bar, 2 mm or 20 μm). (f) Comparison of the positive ratios of the inguinal lymph node metastasis between the two groups. P-value was determined by Chi-square test (*p < 0.05). SUNE1 cells stably expressing HA-NEURL3 or its empty vector were inoculating into tail veins of mice to estab lish a lung metastatic colonization model. (g) Representative images of macroscopic metastatic nodules formed on the lung surfaces of mice in the two groups. (h-i) Representative images (h; Scale bars, 5 mm, 2 mm, and 20 μm) and statistical analysis (i) of metastatic nodules formed in the lungs of mice determined by H&E staining. Data is shown as mean ± SD, and the p-value was determined by Student’s t-test (*p < 0.05). (j) Proposed working model. NEURL3 promotes the degradation of Vimentin protein in a ubiquitin-proteasome pathway, inhibiting NPC metastasis (left). In NPC, promoter hypermeth ylation of NEURL3 causes its downregulation, enabling increased expression of Vimentin to promote NPC metastasis (right)
    Figure Legend Snippet: Fig. 7 NEURL3 inhibits NPC metastasis in vivo. SUNE1 cells stably expressing HA-NEURL3 or its empty vector were subcutaneously injected into foot pads of mice to establish an inguinal lymph node metastasis model. (a) Representative images of the formed footpad tumors and inguinal lymph nodes. (b-c) Representative images of inguinal lymph nodes (b), and statistical analysis of the volume of lymph nodes (c) between the two groups. Data is shown as mean ± SD, and the p-value was determined by Student’s t-test (*p < 0.05). (d) H&E staining showing the infiltration of tumor cells into skin and muscle of footpad tumors (Scale bar, 100 μm). (e) The infiltration of cancer cells into inguinal lymph nodes as determined by a positive of pan-cytokeratin IHC staining (Scale bar, 2 mm or 20 μm). (f) Comparison of the positive ratios of the inguinal lymph node metastasis between the two groups. P-value was determined by Chi-square test (*p < 0.05). SUNE1 cells stably expressing HA-NEURL3 or its empty vector were inoculating into tail veins of mice to estab lish a lung metastatic colonization model. (g) Representative images of macroscopic metastatic nodules formed on the lung surfaces of mice in the two groups. (h-i) Representative images (h; Scale bars, 5 mm, 2 mm, and 20 μm) and statistical analysis (i) of metastatic nodules formed in the lungs of mice determined by H&E staining. Data is shown as mean ± SD, and the p-value was determined by Student’s t-test (*p < 0.05). (j) Proposed working model. NEURL3 promotes the degradation of Vimentin protein in a ubiquitin-proteasome pathway, inhibiting NPC metastasis (left). In NPC, promoter hypermeth ylation of NEURL3 causes its downregulation, enabling increased expression of Vimentin to promote NPC metastasis (right)

    Techniques Used: In Vivo, Stable Transfection, Expressing, Plasmid Preparation, Injection, Staining, Immunohistochemistry, Comparison, Ubiquitin Proteomics



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    Fig. 1 <t>NEURL3</t> is hypermethylated in NPC. (a) Heatmap clustering of the top 20 hypermethylated genes based on integrated analysis of two genome- wide DNA methylation microarray datasets (GSE52068 and GSE62336) in NPC and normal nasopharyngeal tissues. (b) The genome features of NEURL3 observed using the UCSC genome browser and schematic diagram of the CpG island sites at NEURL3 promoter region. (c) Methylation levels of NEURL3 in NPC tissues (n = 24) and normal tissues (n = 24) in the methylation microarray dataset GSE52068. (d) Methylation levels of NEURL3 in NPC tissues (n = 25) and normal tissues (n = 25) in HongKong methylation microarray dataset GSE62336. (e) Representative images of bisulfite pyrosequencing analysis of the NEURL3 promoter region. (f) Statistical analysis of methylation levels of NEURL3 in NPC (n = 8) and normal (n = 8) tissues. Data in c, d, and f are presented as the mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)
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    <t>NEURL3</t> is induced by HCV infection. (A) NEURL3 was upregulated in the mRNA microarray analysis of Huh7-MAVSR cells infected with HCVcc (JFH1 strain) at an MOI of 5 at 24, 48, and 72 h postinfection. (B and C) RT-qPCR analysis of NEURL3, IFN-β, and HCV RNA levels in Huh7-MAVSR cells infected with HCVcc at an MOI of 5 at 24, 48, and 72 h postinfection (B) and in primary human hepatocytes infected with HCVcc at an MOI of 2 at 48 h postinfection (C). Values represent means ± standard deviations (SD) from 2 independent experiments. (D to F) RT-qPCR analysis of NEURL3 (D), IFN-β (E), and HCV (F) RNA levels in the MDA5 knockout Huh7-MAVSR cells as well as control cells that were infected with HCVcc at an MOI of 5. (G to I) The LGP2 knockout Huh7-MAVSR cells as well as control cells were infected with HCVcc at an MOI of 5. The cells were collected at the indicated times postinfection and analyzed by RT-qPCR for the intracellular NEURL3 (G), IFN-β (H), and HCV (I) RNA levels. Values are presented as mRNA fold induction relative to the noninfection control and represent means ± SD from 3 independent experiments. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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    Fig. 1 NEURL3 is hypermethylated in NPC. (a) Heatmap clustering of the top 20 hypermethylated genes based on integrated analysis of two genome- wide DNA methylation microarray datasets (GSE52068 and GSE62336) in NPC and normal nasopharyngeal tissues. (b) The genome features of NEURL3 observed using the UCSC genome browser and schematic diagram of the CpG island sites at NEURL3 promoter region. (c) Methylation levels of NEURL3 in NPC tissues (n = 24) and normal tissues (n = 24) in the methylation microarray dataset GSE52068. (d) Methylation levels of NEURL3 in NPC tissues (n = 25) and normal tissues (n = 25) in HongKong methylation microarray dataset GSE62336. (e) Representative images of bisulfite pyrosequencing analysis of the NEURL3 promoter region. (f) Statistical analysis of methylation levels of NEURL3 in NPC (n = 8) and normal (n = 8) tissues. Data in c, d, and f are presented as the mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)

    Journal: Journal of experimental & clinical cancer research : CR

    Article Title: The E3 ligase NEURL3 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma by promoting vimentin degradation.

    doi: 10.1186/s13046-024-02945-9

    Figure Lengend Snippet: Fig. 1 NEURL3 is hypermethylated in NPC. (a) Heatmap clustering of the top 20 hypermethylated genes based on integrated analysis of two genome- wide DNA methylation microarray datasets (GSE52068 and GSE62336) in NPC and normal nasopharyngeal tissues. (b) The genome features of NEURL3 observed using the UCSC genome browser and schematic diagram of the CpG island sites at NEURL3 promoter region. (c) Methylation levels of NEURL3 in NPC tissues (n = 24) and normal tissues (n = 24) in the methylation microarray dataset GSE52068. (d) Methylation levels of NEURL3 in NPC tissues (n = 25) and normal tissues (n = 25) in HongKong methylation microarray dataset GSE62336. (e) Representative images of bisulfite pyrosequencing analysis of the NEURL3 promoter region. (f) Statistical analysis of methylation levels of NEURL3 in NPC (n = 8) and normal (n = 8) tissues. Data in c, d, and f are presented as the mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)

    Article Snippet: Finally, sections were incubated with anti-NEURL3 antibody (1:200, 16648-1-AP; Proteintech) overnight at 4 °C, followed by biotinylated secondary antibody (Boster, Wuhan, China).

    Techniques: Genome Wide, DNA Methylation Assay, Microarray, Methylation

    Fig. 3 NEURL3 suppresses NPC cell migration, invasion, and EMT in vitro. (a) In GSEA analysis using a public RNA-seq dataset (GSE102349), the gene sets associated with cell migration and metastasis pathways were significantly enriched in NPC samples with a low NEURL3 expression. (b) The migratory abili ties of HONE1, SUNE1, and HK1 cells transfected with HA-NEURL3 plasmid or its empty vector determined by wound healing assay. (c-d) The migratory (c) and invasive (d) abilities of HONE1, SUNE1, and HK1 cells transfected with HA-NEURL3 plasmid or its empty vector determined by Transwell assay. (e) IF staining showing the filopodia length on HONE1, SUNE1, and HK1 cells transfected with HA-NEURL3 plasmid or its empty vector. (f) The morphology changes induced by TGF-β1 in HONE1, and SUNE1 cells transfected with HA-NEURL3 plasmid or its empty vector. Scale bar, 50 μm. Data of b, c, d, and e are shown as mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)

    Journal: Journal of experimental & clinical cancer research : CR

    Article Title: The E3 ligase NEURL3 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma by promoting vimentin degradation.

    doi: 10.1186/s13046-024-02945-9

    Figure Lengend Snippet: Fig. 3 NEURL3 suppresses NPC cell migration, invasion, and EMT in vitro. (a) In GSEA analysis using a public RNA-seq dataset (GSE102349), the gene sets associated with cell migration and metastasis pathways were significantly enriched in NPC samples with a low NEURL3 expression. (b) The migratory abili ties of HONE1, SUNE1, and HK1 cells transfected with HA-NEURL3 plasmid or its empty vector determined by wound healing assay. (c-d) The migratory (c) and invasive (d) abilities of HONE1, SUNE1, and HK1 cells transfected with HA-NEURL3 plasmid or its empty vector determined by Transwell assay. (e) IF staining showing the filopodia length on HONE1, SUNE1, and HK1 cells transfected with HA-NEURL3 plasmid or its empty vector. (f) The morphology changes induced by TGF-β1 in HONE1, and SUNE1 cells transfected with HA-NEURL3 plasmid or its empty vector. Scale bar, 50 μm. Data of b, c, d, and e are shown as mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)

    Article Snippet: Finally, sections were incubated with anti-NEURL3 antibody (1:200, 16648-1-AP; Proteintech) overnight at 4 °C, followed by biotinylated secondary antibody (Boster, Wuhan, China).

    Techniques: Migration, In Vitro, RNA Sequencing, Expressing, Transfection, Plasmid Preparation, Wound Healing Assay, Transwell Assay, Staining

    Fig. 4 NEURL3 interacts with Vimentin to promote its degradation. (a) Silver staining of SDS-PAGE gels showing HA immunoprecipitates that were pulled down from SUNE1 cells transfected with HA-NEURL3 plasmid. The interest proteins are indicated. (b) Co-IP with anti-HA or Vimentin antibodies showing the interactions between HA-NEURL3 and endogenous Vimentin in HONE1 and SUNE1 cells. (c) IF staining showing co-localization of exogenous HA- NEURL3 and endogenous Vimentin in HONE1 and SUNE1 cells (Scale, 50 μm). (d) Schematic diagram showing the structure of Vimentin or its deletion mutant plasmids. (e) Co-IP revealing interactions of NEURL3 with different Vimentin mutants in 293T cells. (f-g) Vimentin protein levels in HONE1 and SUNE1 cells transfected with gradient concentrations of HA-tagged NEURL3 plasmid (f), as well as shNEURL3 plasmids or its control vector (g)(h) IF staining showing Vimentin protein levels in HONE1 and HK1 cells transfected with HA-NEURL3 plasmid or its vector control (Scale bar, 50 µM). (i-j) Rep resentative images and greyscale analyses of Vimentin protein levels in SUNE1 and 293T cells transfected with HA-NEURL3 plasmid or its vector control (i), as well as shNEURL3 plasmid or its control vector (j), after the CHX treatment. (k-l) Vimentin protein levels in HONE1 and SUNE1 cells transfected with HA-NEURL3 plasmid or its vector control after treated with MG132 (k) and CQ (l). Data of i and j are shown as mean ± SD, and the p-values were deter mined by Student’s t-test (*p < 0.05)

    Journal: Journal of experimental & clinical cancer research : CR

    Article Title: The E3 ligase NEURL3 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma by promoting vimentin degradation.

    doi: 10.1186/s13046-024-02945-9

    Figure Lengend Snippet: Fig. 4 NEURL3 interacts with Vimentin to promote its degradation. (a) Silver staining of SDS-PAGE gels showing HA immunoprecipitates that were pulled down from SUNE1 cells transfected with HA-NEURL3 plasmid. The interest proteins are indicated. (b) Co-IP with anti-HA or Vimentin antibodies showing the interactions between HA-NEURL3 and endogenous Vimentin in HONE1 and SUNE1 cells. (c) IF staining showing co-localization of exogenous HA- NEURL3 and endogenous Vimentin in HONE1 and SUNE1 cells (Scale, 50 μm). (d) Schematic diagram showing the structure of Vimentin or its deletion mutant plasmids. (e) Co-IP revealing interactions of NEURL3 with different Vimentin mutants in 293T cells. (f-g) Vimentin protein levels in HONE1 and SUNE1 cells transfected with gradient concentrations of HA-tagged NEURL3 plasmid (f), as well as shNEURL3 plasmids or its control vector (g)(h) IF staining showing Vimentin protein levels in HONE1 and HK1 cells transfected with HA-NEURL3 plasmid or its vector control (Scale bar, 50 µM). (i-j) Rep resentative images and greyscale analyses of Vimentin protein levels in SUNE1 and 293T cells transfected with HA-NEURL3 plasmid or its vector control (i), as well as shNEURL3 plasmid or its control vector (j), after the CHX treatment. (k-l) Vimentin protein levels in HONE1 and SUNE1 cells transfected with HA-NEURL3 plasmid or its vector control after treated with MG132 (k) and CQ (l). Data of i and j are shown as mean ± SD, and the p-values were deter mined by Student’s t-test (*p < 0.05)

    Article Snippet: Finally, sections were incubated with anti-NEURL3 antibody (1:200, 16648-1-AP; Proteintech) overnight at 4 °C, followed by biotinylated secondary antibody (Boster, Wuhan, China).

    Techniques: Silver Staining, SDS Page, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Staining, Mutagenesis, Control

    Fig. 5 NEURL3 promotes K48-linked ubiquitination and degradation of Vimentin. (a) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with Myc-NEURL3 or its empty vector, together with Flag-Vimentin plasmid and a vector encoding HA-WT-Ub or its mutants (HA-K48O-Ub or HA-K63O-Ub). (b) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with shNEURL3 plasmid or its control vector, together with Flag-Vimentin plasmid and a vector encoding HA-WT-Ub or its mutants (HA-K48O-Ub or HA-K48R-Ub). (c) The ubiquitina tion level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with empty vector, wild-type (WT) Myc-NEURL3 or its C217Y mutant, together with Flag-Vimentin and HA-WT-Ub. (d-e) Representative images and greyscale analyses of Vimentin protein levels after CHX treatment in SUNE1 (d) and 293T (e) cells co-transfected with WT Myc-NEURL3 or its C217Y mutant, together with Flag-Vimentin. (f) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with Myc-NEURL3 or its empty vector, together with WT Flag-Vimentin or its K97R mutant and HA-WT-Ub. (g-h) Representative images and greyscale analyses of Vimentin protein levels after CHX treatment in SUNE1 (g) and 293T (h) cells co-transfected with Myc-NEURL3, together with WT Flag-Vimentin or its K97R mutant. Data of d, e, g, and h are shown as mean ± SD, and the p-values were determined by Student’s t-test (ns, no significance; *p < 0.05)

    Journal: Journal of experimental & clinical cancer research : CR

    Article Title: The E3 ligase NEURL3 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma by promoting vimentin degradation.

    doi: 10.1186/s13046-024-02945-9

    Figure Lengend Snippet: Fig. 5 NEURL3 promotes K48-linked ubiquitination and degradation of Vimentin. (a) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with Myc-NEURL3 or its empty vector, together with Flag-Vimentin plasmid and a vector encoding HA-WT-Ub or its mutants (HA-K48O-Ub or HA-K63O-Ub). (b) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with shNEURL3 plasmid or its control vector, together with Flag-Vimentin plasmid and a vector encoding HA-WT-Ub or its mutants (HA-K48O-Ub or HA-K48R-Ub). (c) The ubiquitina tion level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with empty vector, wild-type (WT) Myc-NEURL3 or its C217Y mutant, together with Flag-Vimentin and HA-WT-Ub. (d-e) Representative images and greyscale analyses of Vimentin protein levels after CHX treatment in SUNE1 (d) and 293T (e) cells co-transfected with WT Myc-NEURL3 or its C217Y mutant, together with Flag-Vimentin. (f) The ubiquitination level of exogenous Vimentin in HONE1 and SUNE1 cells co-transfected with Myc-NEURL3 or its empty vector, together with WT Flag-Vimentin or its K97R mutant and HA-WT-Ub. (g-h) Representative images and greyscale analyses of Vimentin protein levels after CHX treatment in SUNE1 (g) and 293T (h) cells co-transfected with Myc-NEURL3, together with WT Flag-Vimentin or its K97R mutant. Data of d, e, g, and h are shown as mean ± SD, and the p-values were determined by Student’s t-test (ns, no significance; *p < 0.05)

    Article Snippet: Finally, sections were incubated with anti-NEURL3 antibody (1:200, 16648-1-AP; Proteintech) overnight at 4 °C, followed by biotinylated secondary antibody (Boster, Wuhan, China).

    Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Control, Mutagenesis

    Fig. 6 Restoration of Vimentin reverses the tumor suppressive effect of NRURL3. HONE1, SUNE1, and HK1 cells were transiently co-transfected with HA- NEURL3 or empty vector together with Flag-Vimentin or empty vector. (a) The migratory abilities of transfected NPC cells were determined by wound healing assay. (b-c) The migratory (b) and invasive (c) abilities of transfected NPC cells were determined by Transwell assay. (d) IF staining showing the pseudopods on transfected NPC cells. (e) The morphology changes induced by TGF-β1 in transfected NPC cells. Scale bar, 50 μm. Data of a, b, and c are shown as mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)

    Journal: Journal of experimental & clinical cancer research : CR

    Article Title: The E3 ligase NEURL3 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma by promoting vimentin degradation.

    doi: 10.1186/s13046-024-02945-9

    Figure Lengend Snippet: Fig. 6 Restoration of Vimentin reverses the tumor suppressive effect of NRURL3. HONE1, SUNE1, and HK1 cells were transiently co-transfected with HA- NEURL3 or empty vector together with Flag-Vimentin or empty vector. (a) The migratory abilities of transfected NPC cells were determined by wound healing assay. (b-c) The migratory (b) and invasive (c) abilities of transfected NPC cells were determined by Transwell assay. (d) IF staining showing the pseudopods on transfected NPC cells. (e) The morphology changes induced by TGF-β1 in transfected NPC cells. Scale bar, 50 μm. Data of a, b, and c are shown as mean ± SD, and the p-values were determined by Student’s t-test (*p < 0.05)

    Article Snippet: Finally, sections were incubated with anti-NEURL3 antibody (1:200, 16648-1-AP; Proteintech) overnight at 4 °C, followed by biotinylated secondary antibody (Boster, Wuhan, China).

    Techniques: Transfection, Plasmid Preparation, Wound Healing Assay, Transwell Assay, Staining

    Fig. 7 NEURL3 inhibits NPC metastasis in vivo. SUNE1 cells stably expressing HA-NEURL3 or its empty vector were subcutaneously injected into foot pads of mice to establish an inguinal lymph node metastasis model. (a) Representative images of the formed footpad tumors and inguinal lymph nodes. (b-c) Representative images of inguinal lymph nodes (b), and statistical analysis of the volume of lymph nodes (c) between the two groups. Data is shown as mean ± SD, and the p-value was determined by Student’s t-test (*p < 0.05). (d) H&E staining showing the infiltration of tumor cells into skin and muscle of footpad tumors (Scale bar, 100 μm). (e) The infiltration of cancer cells into inguinal lymph nodes as determined by a positive of pan-cytokeratin IHC staining (Scale bar, 2 mm or 20 μm). (f) Comparison of the positive ratios of the inguinal lymph node metastasis between the two groups. P-value was determined by Chi-square test (*p < 0.05). SUNE1 cells stably expressing HA-NEURL3 or its empty vector were inoculating into tail veins of mice to estab lish a lung metastatic colonization model. (g) Representative images of macroscopic metastatic nodules formed on the lung surfaces of mice in the two groups. (h-i) Representative images (h; Scale bars, 5 mm, 2 mm, and 20 μm) and statistical analysis (i) of metastatic nodules formed in the lungs of mice determined by H&E staining. Data is shown as mean ± SD, and the p-value was determined by Student’s t-test (*p < 0.05). (j) Proposed working model. NEURL3 promotes the degradation of Vimentin protein in a ubiquitin-proteasome pathway, inhibiting NPC metastasis (left). In NPC, promoter hypermeth ylation of NEURL3 causes its downregulation, enabling increased expression of Vimentin to promote NPC metastasis (right)

    Journal: Journal of experimental & clinical cancer research : CR

    Article Title: The E3 ligase NEURL3 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma by promoting vimentin degradation.

    doi: 10.1186/s13046-024-02945-9

    Figure Lengend Snippet: Fig. 7 NEURL3 inhibits NPC metastasis in vivo. SUNE1 cells stably expressing HA-NEURL3 or its empty vector were subcutaneously injected into foot pads of mice to establish an inguinal lymph node metastasis model. (a) Representative images of the formed footpad tumors and inguinal lymph nodes. (b-c) Representative images of inguinal lymph nodes (b), and statistical analysis of the volume of lymph nodes (c) between the two groups. Data is shown as mean ± SD, and the p-value was determined by Student’s t-test (*p < 0.05). (d) H&E staining showing the infiltration of tumor cells into skin and muscle of footpad tumors (Scale bar, 100 μm). (e) The infiltration of cancer cells into inguinal lymph nodes as determined by a positive of pan-cytokeratin IHC staining (Scale bar, 2 mm or 20 μm). (f) Comparison of the positive ratios of the inguinal lymph node metastasis between the two groups. P-value was determined by Chi-square test (*p < 0.05). SUNE1 cells stably expressing HA-NEURL3 or its empty vector were inoculating into tail veins of mice to estab lish a lung metastatic colonization model. (g) Representative images of macroscopic metastatic nodules formed on the lung surfaces of mice in the two groups. (h-i) Representative images (h; Scale bars, 5 mm, 2 mm, and 20 μm) and statistical analysis (i) of metastatic nodules formed in the lungs of mice determined by H&E staining. Data is shown as mean ± SD, and the p-value was determined by Student’s t-test (*p < 0.05). (j) Proposed working model. NEURL3 promotes the degradation of Vimentin protein in a ubiquitin-proteasome pathway, inhibiting NPC metastasis (left). In NPC, promoter hypermeth ylation of NEURL3 causes its downregulation, enabling increased expression of Vimentin to promote NPC metastasis (right)

    Article Snippet: Finally, sections were incubated with anti-NEURL3 antibody (1:200, 16648-1-AP; Proteintech) overnight at 4 °C, followed by biotinylated secondary antibody (Boster, Wuhan, China).

    Techniques: In Vivo, Stable Transfection, Expressing, Plasmid Preparation, Injection, Staining, Immunohistochemistry, Comparison, Ubiquitin Proteomics

    NEURL3 is induced by HCV infection. (A) NEURL3 was upregulated in the mRNA microarray analysis of Huh7-MAVSR cells infected with HCVcc (JFH1 strain) at an MOI of 5 at 24, 48, and 72 h postinfection. (B and C) RT-qPCR analysis of NEURL3, IFN-β, and HCV RNA levels in Huh7-MAVSR cells infected with HCVcc at an MOI of 5 at 24, 48, and 72 h postinfection (B) and in primary human hepatocytes infected with HCVcc at an MOI of 2 at 48 h postinfection (C). Values represent means ± standard deviations (SD) from 2 independent experiments. (D to F) RT-qPCR analysis of NEURL3 (D), IFN-β (E), and HCV (F) RNA levels in the MDA5 knockout Huh7-MAVSR cells as well as control cells that were infected with HCVcc at an MOI of 5. (G to I) The LGP2 knockout Huh7-MAVSR cells as well as control cells were infected with HCVcc at an MOI of 5. The cells were collected at the indicated times postinfection and analyzed by RT-qPCR for the intracellular NEURL3 (G), IFN-β (H), and HCV (I) RNA levels. Values are presented as mRNA fold induction relative to the noninfection control and represent means ± SD from 3 independent experiments. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 is induced by HCV infection. (A) NEURL3 was upregulated in the mRNA microarray analysis of Huh7-MAVSR cells infected with HCVcc (JFH1 strain) at an MOI of 5 at 24, 48, and 72 h postinfection. (B and C) RT-qPCR analysis of NEURL3, IFN-β, and HCV RNA levels in Huh7-MAVSR cells infected with HCVcc at an MOI of 5 at 24, 48, and 72 h postinfection (B) and in primary human hepatocytes infected with HCVcc at an MOI of 2 at 48 h postinfection (C). Values represent means ± standard deviations (SD) from 2 independent experiments. (D to F) RT-qPCR analysis of NEURL3 (D), IFN-β (E), and HCV (F) RNA levels in the MDA5 knockout Huh7-MAVSR cells as well as control cells that were infected with HCVcc at an MOI of 5. (G to I) The LGP2 knockout Huh7-MAVSR cells as well as control cells were infected with HCVcc at an MOI of 5. The cells were collected at the indicated times postinfection and analyzed by RT-qPCR for the intracellular NEURL3 (G), IFN-β (H), and HCV (I) RNA levels. Values are presented as mRNA fold induction relative to the noninfection control and represent means ± SD from 3 independent experiments. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Microarray, Quantitative RT-PCR, Knock-Out

    NEURL3 is not an IFN-stimulated gene. (A to D) RT-qPCR analysis of NEURL3 and ISG (MXA or IP-10) mRNA levels in Huh7-MAVSR cells treated with 100 IU/ml of IFN-α (A), 100 IU/ml of IFN-β (B), 100 ng/ml of IFN-γ (C), or 100 ng/ml of IFN-λ3 (IL-28B) (D). (E to H) RT-qPCR analysis of NEURL3 and ISG (MXA or IP-10) mRNA levels in Huh7 cells treated with 100 IU/ml of IFN-α (E), 100 IU/ml of IFN-β (F), 100 ng/ml of IFN-γ (G), or 100 ng/ml of IFN-λ3 (IL-28B) (H). Values are presented as fold induction relative to the mock control and represent means ± SD from 2 independent experiments.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 is not an IFN-stimulated gene. (A to D) RT-qPCR analysis of NEURL3 and ISG (MXA or IP-10) mRNA levels in Huh7-MAVSR cells treated with 100 IU/ml of IFN-α (A), 100 IU/ml of IFN-β (B), 100 ng/ml of IFN-γ (C), or 100 ng/ml of IFN-λ3 (IL-28B) (D). (E to H) RT-qPCR analysis of NEURL3 and ISG (MXA or IP-10) mRNA levels in Huh7 cells treated with 100 IU/ml of IFN-α (E), 100 IU/ml of IFN-β (F), 100 ng/ml of IFN-γ (G), or 100 ng/ml of IFN-λ3 (IL-28B) (H). Values are presented as fold induction relative to the mock control and represent means ± SD from 2 independent experiments.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Quantitative RT-PCR

    NEURL3 inhibits HCV infection. (A) Western blotting of Huh7 cells that were transduced with lentivirus expressing NEURL3 or empty vector control. (B and C) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc (strain JFH1) at an MOI of 0.01. The intracellular HCV RNA levels were determined by RT-qPCR (B), and the infectivity titers in the culture supernatants were determined by titration assay (C) at 24, 48, 72, and 96 h postinfection. Assays were performed in duplicates, and error bars indicate the means ± SD. (D) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVpp (strain H77). The infection was quantified by luciferase assay. Assays were performed in triplicates, and error bars indicate the means ± SD. (E) JFH-1 subgenomic replicon cells were transfected with NEURL3-expressing plasmid, and intracellular HCV NS3 and NEURL3 protein levels at 24, 48, and 72 h posttransfection were analyzed by Western blotting. (F) RT-qPCR analysis of Huh7-Vector or Huh7-NEURL3 cells that were infected with HCVΔE1 virus (JFH1 with the E1 deletion) (36), and intracellular HCV RNA levels at day 3 postinfection were analyzed by RT-qPCR and normalized to the cellular actin mRNA levels. Assays were performed in duplicates, and error bars indicate the means ± SD. (G to I) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc at an MOI of 2, and intracellular HCV RNA (G), extracellular infectivity titers (H), and intracellular infectivity titers (I) at 24 h postinfection were determined by RT-qPCR or titration assay, respectively. Assays were performed in triplicates, and error bars indicate the means ± SD. ns, non significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 inhibits HCV infection. (A) Western blotting of Huh7 cells that were transduced with lentivirus expressing NEURL3 or empty vector control. (B and C) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc (strain JFH1) at an MOI of 0.01. The intracellular HCV RNA levels were determined by RT-qPCR (B), and the infectivity titers in the culture supernatants were determined by titration assay (C) at 24, 48, 72, and 96 h postinfection. Assays were performed in duplicates, and error bars indicate the means ± SD. (D) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVpp (strain H77). The infection was quantified by luciferase assay. Assays were performed in triplicates, and error bars indicate the means ± SD. (E) JFH-1 subgenomic replicon cells were transfected with NEURL3-expressing plasmid, and intracellular HCV NS3 and NEURL3 protein levels at 24, 48, and 72 h posttransfection were analyzed by Western blotting. (F) RT-qPCR analysis of Huh7-Vector or Huh7-NEURL3 cells that were infected with HCVΔE1 virus (JFH1 with the E1 deletion) (36), and intracellular HCV RNA levels at day 3 postinfection were analyzed by RT-qPCR and normalized to the cellular actin mRNA levels. Assays were performed in duplicates, and error bars indicate the means ± SD. (G to I) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc at an MOI of 2, and intracellular HCV RNA (G), extracellular infectivity titers (H), and intracellular infectivity titers (I) at 24 h postinfection were determined by RT-qPCR or titration assay, respectively. Assays were performed in triplicates, and error bars indicate the means ± SD. ns, non significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Western Blot, Transduction, Expressing, Plasmid Preparation, Quantitative RT-PCR, Titration, Luciferase, Transfection

    NEURL3 has no antiviral effect against ZIKV, DENV, and VSV. Huh7-Vector or Huh7-NEURL3 cells were infected with ZIKV (A to C) or DENV (D to F) at an MOI of 0.1 or with VSV (G to I) at an MOI of 1. The intracellular (A, D, G) and extracellular (B, E, H) RNA levels as well as extracellular virus titers (C, F, I) at 24, 48, 72, and 96 h postinfection were determined by RT-qPCR and plaque/titration assay, respectively. Assays were performed in duplicate or triplicate, and error bars indicate the means ± SD. ns, not significant (P > 0.05).

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 has no antiviral effect against ZIKV, DENV, and VSV. Huh7-Vector or Huh7-NEURL3 cells were infected with ZIKV (A to C) or DENV (D to F) at an MOI of 0.1 or with VSV (G to I) at an MOI of 1. The intracellular (A, D, G) and extracellular (B, E, H) RNA levels as well as extracellular virus titers (C, F, I) at 24, 48, 72, and 96 h postinfection were determined by RT-qPCR and plaque/titration assay, respectively. Assays were performed in duplicate or triplicate, and error bars indicate the means ± SD. ns, not significant (P > 0.05).

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Plasmid Preparation, Infection, Quantitative RT-PCR, Titration

    Interaction between NEURL3 and HCV structural and nonstructural proteins. HEK293T cells were cotransfected with plasmids expressing NEURL3 together with Flag-tagged E1 (A), core (B), E2 (C), NS2 (D), NS3 (E), NS4B (F), NS5A (G), or NS5B (H), and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies. (I) HEK293T cells were cotransfected with plasmids expressing E1-A4 and NEURL3-Flag, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-A4 and anti-Flag antibodies.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: Interaction between NEURL3 and HCV structural and nonstructural proteins. HEK293T cells were cotransfected with plasmids expressing NEURL3 together with Flag-tagged E1 (A), core (B), E2 (C), NS2 (D), NS3 (E), NS4B (F), NS5A (G), or NS5B (H), and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies. (I) HEK293T cells were cotransfected with plasmids expressing E1-A4 and NEURL3-Flag, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-A4 and anti-Flag antibodies.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Expressing, Immunoprecipitation, Western Blot

    NEURL3 specifically colocalizes with HCV E1 protein. (A) Huh7-Vector or Huh7-NEURL3-Flag cells were transfected with plasmids expressing E1, E2, or NS2 for 48 h and subjected to immunofluorescence staining of NEURL3 (green) and E1, E2, or NS2 (red). The colocalization of NEURL3 and HCV protein was analyzed in pixel dot plots on the right, with the x axis representing red signal intensity (E1, E2, or NS2) and the y axis representing green signal (NEURL3). (B) Pearson's coefficient, the indicator of colocalization between NEURL3 and HCV protein (E1, E2, and NS2), was determined by regression of the pixel dot plots. Ten individual images were analyzed for each group. Error bars indicate the means ± SD. ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 specifically colocalizes with HCV E1 protein. (A) Huh7-Vector or Huh7-NEURL3-Flag cells were transfected with plasmids expressing E1, E2, or NS2 for 48 h and subjected to immunofluorescence staining of NEURL3 (green) and E1, E2, or NS2 (red). The colocalization of NEURL3 and HCV protein was analyzed in pixel dot plots on the right, with the x axis representing red signal intensity (E1, E2, or NS2) and the y axis representing green signal (NEURL3). (B) Pearson's coefficient, the indicator of colocalization between NEURL3 and HCV protein (E1, E2, and NS2), was determined by regression of the pixel dot plots. Ten individual images were analyzed for each group. Error bars indicate the means ± SD. ***, P < 0.001.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Plasmid Preparation, Transfection, Expressing, Immunofluorescence, Staining

    NEURL3 interacts with HCV envelope glycoprotein E1 in the context of HCV infection. (A) Western blot of Huh7 cells that were transduced with lentivirus expressing Flag-tagged NEURL3 or empty vector control. The anti-Flag antibody was used in the blot. (B, C) Huh7-Vector or Huh7-NEURL3-Flag cells were infected with HCVcc (strain JFH1-E1A4) at an MOI of 0.01. The intracellular HCV RNA levels (B) and extracellular infectivity titers (C) at day 6 postinfection were determined by RT-qPCR and titration assay. Assays were performed in duplicates, and error bars indicate the mean ± SD. **, P < 0.01; ***, P < 0.001. (D) Huh7 cells expressing NEURL3-Flag or empty vector control were infected with JFH1 virus containing an A4 epitope in E1 at an MOI of 1. Cell lysates collected at day 7 postinfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting using anti-A4, anti-Flag, anti-core, and anti-E2 antibodies.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 interacts with HCV envelope glycoprotein E1 in the context of HCV infection. (A) Western blot of Huh7 cells that were transduced with lentivirus expressing Flag-tagged NEURL3 or empty vector control. The anti-Flag antibody was used in the blot. (B, C) Huh7-Vector or Huh7-NEURL3-Flag cells were infected with HCVcc (strain JFH1-E1A4) at an MOI of 0.01. The intracellular HCV RNA levels (B) and extracellular infectivity titers (C) at day 6 postinfection were determined by RT-qPCR and titration assay. Assays were performed in duplicates, and error bars indicate the mean ± SD. **, P < 0.01; ***, P < 0.001. (D) Huh7 cells expressing NEURL3-Flag or empty vector control were infected with JFH1 virus containing an A4 epitope in E1 at an MOI of 1. Cell lysates collected at day 7 postinfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting using anti-A4, anti-Flag, anti-core, and anti-E2 antibodies.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Western Blot, Transduction, Expressing, Plasmid Preparation, Quantitative RT-PCR, Titration, Immunoprecipitation

    NEURL3 blocks the E1 and E2 interaction. (A) HEK293T cells were cotransfected with plasmids expressing Flag-tagged E1, E2, and NEURL3, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-E2 and anti-Flag antibodies. (B) Quantification of coimmunoprecipitated E2 relative to input E2 by Image J. The values were normalized to coimmunoprecipitated E1 and expressed as a relative value to the control. Error bars indicate the means ± SD from 3 individual experiments. ***, P < 0.001. (C) Huh7-Vector or Huh7-NEURL3-Flag cells were infected with HCVcc (strain JFH1-E1A4) at an MOI of 1 for 48 h and subjected to immunofluorescence staining of NEURL3 (blue), E1 (green), and E2 (red). The colocalization of E1 and E2 was analyzed in pixel dot plots on the right, with the x axis representing red signal intensity (E2) and the y axis representing green signal (E1). (D) Pearson's coefficient, the indicator of colocalization between E1 and E2, was determined by regression of the pixel dot plots. Ten individual images were analyzed for each group. Error bars indicate the means ± SD. ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 blocks the E1 and E2 interaction. (A) HEK293T cells were cotransfected with plasmids expressing Flag-tagged E1, E2, and NEURL3, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-E2 and anti-Flag antibodies. (B) Quantification of coimmunoprecipitated E2 relative to input E2 by Image J. The values were normalized to coimmunoprecipitated E1 and expressed as a relative value to the control. Error bars indicate the means ± SD from 3 individual experiments. ***, P < 0.001. (C) Huh7-Vector or Huh7-NEURL3-Flag cells were infected with HCVcc (strain JFH1-E1A4) at an MOI of 1 for 48 h and subjected to immunofluorescence staining of NEURL3 (blue), E1 (green), and E2 (red). The colocalization of E1 and E2 was analyzed in pixel dot plots on the right, with the x axis representing red signal intensity (E2) and the y axis representing green signal (E1). (D) Pearson's coefficient, the indicator of colocalization between E1 and E2, was determined by regression of the pixel dot plots. Ten individual images were analyzed for each group. Error bars indicate the means ± SD. ***, P < 0.001.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Expressing, Immunoprecipitation, Western Blot, Plasmid Preparation, Infection, Immunofluorescence, Staining

    NEURL3 inhibits HCV infection of different genotypes. (A to F) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc of strain Con1 (genotype 1b), strain S52 (genotype 3a), and strain HK6a (genotype 6a) at an MOI of 0.01. The extracellular infectivity titers (A to C) and intracellular HCV RNA levels (D to F) at different time points were determined by RT-qPCR and titration assay, respectively. Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G to I) HEK293T cells were cotransfected with plasmids expressing NEURL3 together with Flag-tagged E1 of Con1 (G), S52 (H), or HK6a (I), and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 inhibits HCV infection of different genotypes. (A to F) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc of strain Con1 (genotype 1b), strain S52 (genotype 3a), and strain HK6a (genotype 6a) at an MOI of 0.01. The extracellular infectivity titers (A to C) and intracellular HCV RNA levels (D to F) at different time points were determined by RT-qPCR and titration assay, respectively. Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G to I) HEK293T cells were cotransfected with plasmids expressing NEURL3 together with Flag-tagged E1 of Con1 (G), S52 (H), or HK6a (I), and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Plasmid Preparation, Quantitative RT-PCR, Titration, Expressing, Immunoprecipitation, Western Blot

    The NHR domain but not the RING domain of NEURL3 is required for its anti-HCV activity. (A) Schematic presentation of functional domains of NEURL3 and the mutants used in the study. (B) HEK293T cells were cotransfected with plasmids expressing Flag-tagged E1 and NEURL3, NEURL3-ΔRING, or NEURL3-ΔNHR, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies. The asterisk indicates a smaller protein associated with NEURL3-ΔRING. (C) Western blotting of Huh7 cells that were transduced with lentivirus expressing NEURL3, NEURL3-ΔRING, NEURL3-ΔNHR, or empty vector control. The asterisk indicates a smaller protein associated with NEURL3-ΔRING. (D, E) Huh7-Vector, Huh7-NEURL3, Hnu7-NEURL3-ΔRING, or Huh7-NEURL3-ΔNHR cells were infected with HCVcc (strain JFH1) at an MOI of 0.01. The intracellular HCV RNA levels (D) and supernatant infectivity titers (E) at 24, 48, 72, and 96 h postinfection were determined by RT-qPCR and titration assay, respectively. Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: The NHR domain but not the RING domain of NEURL3 is required for its anti-HCV activity. (A) Schematic presentation of functional domains of NEURL3 and the mutants used in the study. (B) HEK293T cells were cotransfected with plasmids expressing Flag-tagged E1 and NEURL3, NEURL3-ΔRING, or NEURL3-ΔNHR, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies. The asterisk indicates a smaller protein associated with NEURL3-ΔRING. (C) Western blotting of Huh7 cells that were transduced with lentivirus expressing NEURL3, NEURL3-ΔRING, NEURL3-ΔNHR, or empty vector control. The asterisk indicates a smaller protein associated with NEURL3-ΔRING. (D, E) Huh7-Vector, Huh7-NEURL3, Hnu7-NEURL3-ΔRING, or Huh7-NEURL3-ΔNHR cells were infected with HCVcc (strain JFH1) at an MOI of 0.01. The intracellular HCV RNA levels (D) and supernatant infectivity titers (E) at 24, 48, 72, and 96 h postinfection were determined by RT-qPCR and titration assay, respectively. Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Activity Assay, Functional Assay, Expressing, Immunoprecipitation, Western Blot, Transduction, Plasmid Preparation, Infection, Quantitative RT-PCR, Titration

    Knockout of NEURL3 promotes HCV infection. (A) Schematic illustration of the NEURL3-sgRNA targeting sites on the genome. (B) Alignment of the subcloned NEURL3 mutations in NEURL3 knockout Huh7-MAVSR cells. The sequences spanning the NEURL3-sgRNA targeting sites in the knockout cells were amplified by PCR and subcloned. The sequences of 8 subclones are presented. The designed sgRNA targeting sequences are marked with a box. (C to E) NEURL3 knockout or control Huh7-MAVSR cells were infected with HCVcc (strain JFH1) at an MOI of 1, and the intracellular HCV RNA levels (C), infectivity titers (D), and NEURL3 mRNA levels (E) at 24, 48, 72, and 96 h postinfection were determined. Assays were performed in duplicates, and error bars indicate the means ± SD. (F) Sequences of the sgRNA-resistant NEURL3. The introduced mutations change only nucleotide sequences but not amino acid sequences. (G) Western blot analysis of Huh7-MAVSR cells in which NEURL3 knockout was rescued by exogenous expression of sgRNA-resistant NEURL3 using an anti-NEURL3 antibody. (H, I) The NEURL3 knockout Huh7-MAVSR cells were transduced with lentivirus expressing sgRNA-resistant NEURL3 to rescue the NEURL3 expression. These cells were infected with HCVcc (strain JFH1) at an MOI of 1 and then collected at 24, 48, 72, and 96 h postinfection to determine intracellular HCV RNA by RT-qPCR (H) or supernatant infectivity titers by titration assay (I). Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: Knockout of NEURL3 promotes HCV infection. (A) Schematic illustration of the NEURL3-sgRNA targeting sites on the genome. (B) Alignment of the subcloned NEURL3 mutations in NEURL3 knockout Huh7-MAVSR cells. The sequences spanning the NEURL3-sgRNA targeting sites in the knockout cells were amplified by PCR and subcloned. The sequences of 8 subclones are presented. The designed sgRNA targeting sequences are marked with a box. (C to E) NEURL3 knockout or control Huh7-MAVSR cells were infected with HCVcc (strain JFH1) at an MOI of 1, and the intracellular HCV RNA levels (C), infectivity titers (D), and NEURL3 mRNA levels (E) at 24, 48, 72, and 96 h postinfection were determined. Assays were performed in duplicates, and error bars indicate the means ± SD. (F) Sequences of the sgRNA-resistant NEURL3. The introduced mutations change only nucleotide sequences but not amino acid sequences. (G) Western blot analysis of Huh7-MAVSR cells in which NEURL3 knockout was rescued by exogenous expression of sgRNA-resistant NEURL3 using an anti-NEURL3 antibody. (H, I) The NEURL3 knockout Huh7-MAVSR cells were transduced with lentivirus expressing sgRNA-resistant NEURL3 to rescue the NEURL3 expression. These cells were infected with HCVcc (strain JFH1) at an MOI of 1 and then collected at 24, 48, 72, and 96 h postinfection to determine intracellular HCV RNA by RT-qPCR (H) or supernatant infectivity titers by titration assay (I). Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Knock-Out, Infection, Amplification, Western Blot, Expressing, Transduction, Quantitative RT-PCR, Titration

    Induction of NEURL3 in liver biopsy specimens of HCV-infected patients. The data of NEURL3 mRNA levels in liver biopsy specimens of HCV-infected patients and control patients were obtained from three published studies and analyzed. GEO2R was used to obtain the data, and the t test was used for statistical analysis. (A) NEURL3 expression was analyzed in an mRNA microarray analysis of 87 HCV-associated HCC liver samples and 8 control samples (HCV- and hepatitis B virus [HBV]-free metastatic liver tumors). Raw microarray data were obtained from Gene Expression Omnibus (GSE17856). (B) NEURL3 expression was analyzed in an mRNA microarray analysis of 24 HCV-positive liver biopsy samples from HCV-induced cirrhosis patients and 6 HCV-negative liver biopsy samples from patients with non-HCV-associated liver diseases (autoimmune hepatitis, alcohol-induced cirrhosis, or primary biliary cirrhosis). Raw microarray data were obtained from GEO (GSE15331). (C) NEURL3 expression was analyzed in an RNA-Seq analysis of 28 liver biopsy specimens from chronic hepatitis C and 6 control patients with non-HCV-associated liver diseases. The normalized raw data were obtained from GEO (GSE84346).

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: Induction of NEURL3 in liver biopsy specimens of HCV-infected patients. The data of NEURL3 mRNA levels in liver biopsy specimens of HCV-infected patients and control patients were obtained from three published studies and analyzed. GEO2R was used to obtain the data, and the t test was used for statistical analysis. (A) NEURL3 expression was analyzed in an mRNA microarray analysis of 87 HCV-associated HCC liver samples and 8 control samples (HCV- and hepatitis B virus [HBV]-free metastatic liver tumors). Raw microarray data were obtained from Gene Expression Omnibus (GSE17856). (B) NEURL3 expression was analyzed in an mRNA microarray analysis of 24 HCV-positive liver biopsy samples from HCV-induced cirrhosis patients and 6 HCV-negative liver biopsy samples from patients with non-HCV-associated liver diseases (autoimmune hepatitis, alcohol-induced cirrhosis, or primary biliary cirrhosis). Raw microarray data were obtained from GEO (GSE15331). (C) NEURL3 expression was analyzed in an RNA-Seq analysis of 28 liver biopsy specimens from chronic hepatitis C and 6 control patients with non-HCV-associated liver diseases. The normalized raw data were obtained from GEO (GSE84346).

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Expressing, Microarray, RNA Sequencing Assay