Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo

doi: 10.1128/JVI.01423-20

Figure Lengend Snippet: SLA-DR-presented NSP immunopeptides evoke antibody response in vivo. (A) cDNAs of PRRSV NSP immunopeptides from PRRSV strain JXA1 were cloned into the pCAGEN vector and fused with NanoLuc luciferase, followed by transfection of HEK-293T cells for transient expression. Cell lysates from 293T cells with transfection of indicated plasmids were harvested and subjected to Western blotting using rabbit anti-NanoLuc polyclonal antibodies to confirm the expression of NanoLuc-fused NSP immunopeptides. (B) Serum samples collected at 21 days postinoculation (dpi) from PRRSV JXA1-infected pigs (n = 6) and noninfected piglets (n = 6) were subjected to luciferase-linked antibody capture assay (LACA) using NanoLuc luciferase fused with indicated immunopeptides from different NSPs to identify corresponding antibody levels as determined by sample-to-negative (S/N) ratio. (C) Amounts of 1 μg of indicated artificial immunopeptides were used for coating ELISA plates. After blocking with 5% skim milk, indirect ELISA was conducted using 21-dpi serum from PRRSV JXA1-infected pigs (n = 6) or noninfected piglets (n = 6) (1-to-200 dilution in PBS), along with visualization by HRP-conjugated goat anti-swine IgG and TMB substrate. (D) Amounts of 400 ng of indicated KLH-conjugated artificial immunopeptides were used for coating ELISA plates. After blocking with 5% skim milk, indirect ELISA was conducted using 21-dpi serum from PRRSV JXA1-infected pigs (n = 6) or noninfected piglets (n = 6) (1 to 200 dilution in PBS), along with visualization by HRP-conjugated goat anti-swine IgG and TMB substrate. (E) Serum samples collected at 21 dpi from PRRSV SD16- and PRRSV NADC30-like strain-infected pigs (n = 5 for each) and noninfected piglets (n = 5) were subjected to LACA using NanoLuc luciferase fused with indicated immunopeptides from different NSPs (JXA1 strain) and evaluated by S/N ratio. OD 450, optical density at 450 nm. All data are expressed as mean values ± standard deviations and were subjected to Student’s t test. Error bars show standard deviations. Significant differences between indicated groups are marked as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

Article Snippet: Serum samples of immunized mice were evaluated by using the Idexx PRRS X3 enzyme-linked immunosorbent assay (ELISA) kit by replacing the original horseradish peroxidase (HRP)-labeled anti-swine antibodies with HRP-labeled anti-mouse IgG (Thermo Fisher Scientific) in order to determine the serum conversion as previously described ( 68 ).

Techniques: In Vivo, Clone Assay, Plasmid Preparation, Luciferase, Transfection, Expressing, Western Blot, Infection, Enzyme-linked Immunosorbent Assay, Blocking Assay, Indirect ELISA

Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo

doi: 10.1128/JVI.01423-20

Figure Lengend Snippet: SLA-DR-mediated antigen presentation of PRRSV NSPs evokes a nonneutralizing antibody response in vivo. (A) Swine serum (28 dpi) from PRRSV JXA1-infected swine or noninfected swine was used to incubate Sepharose 4B resin conjugated with mixed NSP immunopeptides listed in Table 1, except NSP7α, for 2 h at RT, followed by washing resin with PBS. Next, immunopeptide-specific antibodies were eluted and subjected to SDS-PAGE, and the existence of immunopeptide-specific antibodies was visualized using HRP-labeled goat anti-swine IgG. (B) MARC-145 cells were infected by PRRSV JXA1 at an MOI of 1 for 24 h. After cells were fixed and permeabilized, PRRSV-infected cells were costained by anti-PRRSV N protein MAb PP7EF11 and purified PRRSV NSP immunopeptide (IM-Pep) antibodies, followed by visualization via Alexa Fluor 555-labeled goat anti-mouse IgG(H+L) (red channel) and FITC-labeled goat anti-swine IgG (green channel). (C) MARC-145 cells were infected by PRRSV JXA1 at an MOI of 1 for 24 h and harvested for SDS-PAGE. PVDF membrane was blotted using purified PRRSV NSP immunopeptide swine antibodies or anti-PRRSV N MAb 6D10, followed by visualization via HRP-labeled goat anti-mouse IgG(H+L) or goat anti-swine IgG. (D) PRRSV JXA1 (MOI of 0.1) was preincubated with indicated doses of purified PRRSV NSP immunopeptide swine antibodies at 37°C for 1 h and then used to inoculate MARC-145 cells for 4 h. After removing antibodies and unbound virions by washing cells with PBS, virus-infected cells were incubated at 37°C for another 14 h. Replication of PRRSV was visualized by IFA using anti-PRRSV N MAb PP7EF11 for counting of fluorescent spots. All data are expressed as mean values ± standard deviations from at least three independent experiments.

Article Snippet: Serum samples of immunized mice were evaluated by using the Idexx PRRS X3 enzyme-linked immunosorbent assay (ELISA) kit by replacing the original horseradish peroxidase (HRP)-labeled anti-swine antibodies with HRP-labeled anti-mouse IgG (Thermo Fisher Scientific) in order to determine the serum conversion as previously described ( 68 ).

Techniques: In Vivo, Infection, SDS Page, Labeling, Purification, Incubation

Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo

doi: 10.1128/JVI.01423-20

Figure Lengend Snippet: PRRSV structural protein-derived immunopeptides could be recognized by PRRSV hyperimmune serum but not early serum samples from PRRSV-infected pigs. (A) cDNAs of PRRSV structural protein (SP) immunopeptides from PRRSV strain JXA1 were synthesized and ligated into the pET-28a-His-NanoLuc expression vector to fuse with NanoLuc luciferase, followed by expression in E. coli cells. After dialysis and purification, 10 μg of each recombinant protein was subjected to SDS-PAGE and Western blot analysis using rabbit anti-NanoLuc polyclonal antibodies to confirm the expression. (B) Amounts of 200 ng of indicated NanoLuc-fused immunopeptides were used for coating ELISA plates. After blocking with 5% skim milk, indirect ELISA was conducted using 14-dpi or 21-dpi serum from PRRSV JXA1-infected pigs (n = 4) or PRRSV hyperimmune serum samples (n = 4) (20-fold dilution in PBS), along with visualization by HRP-conjugated goat anti-swine IgG and TMB substrate. OD 450, optical density at 450 nm. All data are expressed as mean values ± standard deviations and were subjected to Student’s t test. Significant differences between indicated groups are marked as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

Article Snippet: Serum samples of immunized mice were evaluated by using the Idexx PRRS X3 enzyme-linked immunosorbent assay (ELISA) kit by replacing the original horseradish peroxidase (HRP)-labeled anti-swine antibodies with HRP-labeled anti-mouse IgG (Thermo Fisher Scientific) in order to determine the serum conversion as previously described ( 68 ).

Techniques: Derivative Assay, Infection, Synthesized, Expressing, Plasmid Preparation, Luciferase, Purification, Recombinant, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Blocking Assay, Indirect ELISA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: SNAI1 promotes epithelial-mesenchymal transition and maintains cancer stem cell-like properties in thymic epithelial tumors through the PIK3R2/p-EphA2 Axis

doi: 10.1186/s13046-024-03243-0

Figure Lengend Snippet: Validation of the oncogenic effect of SNAI1 both in vitro and in vivo. a . qRT-PCR reveals the relative mRNA expression levels in Ty82 cells under various conditions, including those overexpressing SNAI1 (LV-SNAI1), suppressing SNAI1 (LV-shSNAI1), treated with the SNAI1 inhibitor (Rosiglitazone), and the control group. b - c . Western blot analysis depicting the protein expression levels of SNAI1 in TET cells under different experimental conditions. d - e . The impact of SNAI1 overexpression on the proliferation of TET cells was assessed using the CCK-8 assay. Absorbance at 450 nm was measured at various time points. f - i . Transwell assays were employed to evaluate the impact of SNAI1 overexpression or suppression on the in vitro migration and invasion ability of TET cells. Representative micrographs and quantification of cells attached to the lower surface of the chamber are presented (Scale bars: 200 μm). j - m . The clone assay was utilized to assess the influence of SNAI1 on the in vitro proliferation of TET cells. Representative micrographs are presented (Scale bars: 2 mm). The quantifications of cell clone spheres are presented. n . The apoptosis assay was conducted using an Annexin V-APC/PI Apoptosis Kit in Ty82 cells treated with Rosiglitazone or the control group. o - p . Western blot analysis was performed to assess the protein expression levels of EMT markers, including N-cadherin, E-cadherin, and Vimentin, in TET cells transfected with LV-SNAI1 or LV-Ctrl. q . Representative images depict Ty82 cells transfected with LV-SNAI1, LV-shSNAI1, or LV-Ctrl grown as spheres (Scale bars: 200 μm). r . Ty82 cells transfected with LV-SNAI1 or LV-Ctrl were injected into the left ventricle of 5- to 6-week-old male wild-type BALB/c nude mice. Representative bioluminescent images depict TET cell metastases. * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001, and ns denotes not statistically significant

Article Snippet: For the cell apoptosis assay, the Annexin V-APC/PI Apoptosis Kit (Elabscience) was employed.

Techniques: Biomarker Discovery, In Vitro, In Vivo, Quantitative RT-PCR, Expressing, Control, Western Blot, Over Expression, CCK-8 Assay, Migration, Clone Assay, Apoptosis Assay, Transfection, Injection

Journal: eLife

Article Title: Binary outcomes of enhancer activity underlie stable random monoallelic expression

doi: 10.7554/eLife.74204

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , NEBNext Ultra II DNA Library Prep Kit for Illumina , New England Biolabs , Cat # E7645S , .

Techniques: Generated, Microinjection, Electroporation, CRISPR, Purification, cDNA Synthesis, Cell Isolation, Multiplex Assay, TA Cloning, Subcloning, Staining, Flow Cytometry, Recombinant, Cell Culture, Software