Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The APC function of B cells is closely associated with their FA metabolism in metastatic OvCa. A GSVA pathway enrichment analysis of APC function low and APC function high B cells in patients with metastatic OvCa ( GSE235951 , GSE147082 and GSE154600 dataset in the GEO database, n = 13). B Representative images of the IHC staining of TLS structure (composed of CD3 + T cells, CD19 + B cells, and CD21 + FDC), CD80, and CD86, respectively, in the area adjacent to or away from tumor or adipose tissues in clinical HGSOC specimens (n = 20 for TLS and n = 5 for CD80 or CD86). Red dashed line area: TLS structure. Magnification × 200. C Representative image of lymphoid aggregates (white dashed line area) by immunofluorescent staining in ascites of OvCa mice with 3 w and 6 w in tumor-bearing mice. D Mean fluorescence intensity of CD80, CD86, CD83, and MHC class II molecules in ascitic CD19 + B cells of OvCa mice were detected by flow cytometry. E Comparison of mRNA levels of FA metabolic genes in ascitic B cells in OvCa mice. The relative expression of each gene was calculated using β -actin as the internal reference. F Protein expression of β-actin and FA metabolic proteins in ascitic B cells in OvCa mice was assessed by WB. β -Actin was used as the internal control to calculate the relative expression level of FA metabolic proteins. G Mean fluorescence intensity of Bodipy C16 in ascitic B cells of OvCa mice detected by flow cytometry. H Expression of A-CoA in ascitic B cells of OvCa mice detected by ELISA. I Expression of ATP in ascitic B cells of OvCa mice detected by ELISA. J Mean fluorescence intensity of intracellular oxidized lipid in ascitic B cells of OvCa mice detected by flow cytometry. I Comparison of mRNA levels of FA metabolic genes in ascitic B cells in OvCa mice. The relative expression of each gene was calculated using β -actin as the internal reference. Data are presented as the mean ± SD of three independent experiments. FA, fatty acid; TLS, tertiary lymphoid structure. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: Immunohistochemistry, Staining, Fluorescence, Flow Cytometry, Comparison, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The APC function and resulting anticancer immunity of B cells can be enhanced by oleic acid (OA) via reprogramming FA metabolism in vitro. A Mean fluorescence intensity of CD80 and MHC Class II molecules in CD19 + B cells from peripheral blood of healthy volunteers (n = 3) treated with OA and PA (both 150 μM), respectively. B Mean fluorescence intensity of CD80, CD86, CD83, MHC Class II molecules, and Ki67 in splenic CD19 + B cells of WT mice treated with 150 μM OA. C Mean fluorescence intensity of CD80, CD86, CD83, and MHC Class II molecules in ascitic CD19 + B cells from 3 w OvCa-bearing mice when treated with 150 μM OA. D Analysis of FA metabolism-related signaling pathways based on RNA-seq results. GSEA was used to analysised the FA metabolic pathways. E Comparison of mRNA levels of main FA metabolic genes in ascitic B cells from 3 w tumor-bearing mice when treated with 150 μM OA. The relative expression of each gene was calculated using β -actin as the internal reference. F Experimental scheme to detect the influence of inhibiting OA uptake on ascitic B cells. G Comparison of protein expressions of main FA metabolic molecules in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS and treated with OA. β -Actin was used as the internal control to calculate the relative expression level of the main FA metabolic molecules. H Mean fluorescence intensity of Bodipy C16 in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS and treated with OA was detected by flow cytometry. I The expression of A-CoA, ATP and the FAO activity in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS treated with OA was detected by ELISA. J Mean fluorescence intensity of intracellular oxidized lipid in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS and treated with OA was detected by flow cytometry. K Mean fluorescence intensity of CD80, CD86, and CD83 in 3 w OvCa-bearing mouse ascitic CD19 + B cells pretreated with BMS and treated with OA was detected by flow cytometry. L Mean fluorescence intensity of Bodipy C16, CD80, CD86 and CD83 in FABP4-knockdown CD19 + B cells, which are from the ascites of 3 w OvCa-bearing mice, treated with OA, was detected by flow cytometry. PBMC, peripheral blood mononuclear cell; SP, spleen; AS, Ascites; OA, oleic acid; PA, palmitic acid; BMS, BMS309403. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: In Vitro, Fluorescence, Protein-Protein interactions, RNA Sequencing, Comparison, Expressing, Control, Flow Cytometry, Activity Assay, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: Reprogramming of the FA metabolism of B cells by OA can improve anticancer immunity in vitro. A Experimental scheme to detect the in vitro effects of ascitic B cells treated with OA on anticancer immunity. B Mean fluorescence intensity of CD80, CD86, and CD83 in 3 w OvCa-bearing mouse ascitic CD19 + B cells after pulsed with ID8-Luc-cell-prepared antigenic peptides and pretreated with BMS and treated with OA, T cells and ID8-Luc. C Levels of IFN- γ , GZMB, and TNF-α in the supernatant of the coculture system constructed by ID8-Luc cells and 3 w OvCa-bearing mouse ascitic B and splenic T cells were detected by ELISA. D Cytotoxicity of T cells in the coculture system mentioned above was detected by luciferase assay. AS, Ascites; Ag, Antigen; OA, oleic acid; BMS, BMS309403. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: In Vitro, Fluorescence, Construct, Enzyme-linked Immunosorbent Assay, Luciferase

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The enhanced APC function of B cells by OA in vitro is achieved through H3K27ac-mediated upregulation of PPAR γ expression. A Protein expression of β -actin and H3K27ac in 3 w OvCa-bearing mouse ascitic CD19 + B cells pretreated with BMS and treated with OA was assessed by WB. β -Actin was used as the internal control to calculate the relative expression level of H3K27ac. B The enrichment percentage of H3K27ac at the PPAR γ , CD80, CD86, and CD83 promoter regions was quantified using ChIP-seq analysis. C Correlation analysis of PPAR γ and APC function-related genes (CD80, CD86, CD83, MHC II) in B cells in OvCa patients in the TCGA database (n = 426). D Analysis of protein interaction among FABP4, PPAR γ , CD80, CD86, and CD83. E The enrichment percentage of PPAR γ at the CD80, CD86, and CD83 promoter regions was quantified using ChIP-seq analysis. F Mean fluorescence intensity of CD80, CD86, and CD83 in 3 w OvCa-bearing mouse ascitic CD19 + B cells pretreated with BMS, GW9662/Trog, and treated with OA was detected by flow cytometry. AS, Ascites; Ag, Antigen; OA, oleic acid; BMS, BMS309403. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: In Vitro, Expressing, Control, ChIP-sequencing, Fluorescence, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The combination of adoptive immunotherapy with APC function-enhanced B cells and LDC improved anticancer immunity in metastatic OvCa mice. A Experimental scheme to evaluate the effects of combining adoptive immunotherapy with APC function-enhanced B cells and LDC in metastatic OvCa mice. B Representative BLI images and comparison of OvCa progression in each group (n = 3). C Representative images ( a ) and the number of tumor nodules ( b ) in the abdominal wall of mice in each group (n = 3). Areas marked by yellow dashed lines: Representative tumor nodules in the abdominal wall. D (a) Representative images of lymphoid aggregates (composed of CD3 + T cells, CD19 + B cells, and CD21 + FDC) in the ascitic cells of mice in each group. (b) Comparison of the amount of CD3 + T, CD8 + T, Ki67 + CD3 + T, Ki67 + CD8 + T, CD19 + B, CD80 + B, CD86 + B, and lymphoid aggregates respectively in the ascitic cells of mice in each group (n = 3) in frozen sections detected by immunofluorescence. E Comparison of the proportion of CD3 + T, CD8 + T, CD19 + B, CD19 − CD138 + B cells, mean fluorescence intensity of Ki67 in CD3 + T and CD8 + T, and mean fluorescence intensity of CD80, CD86, CD83, MHC II in CD19 + B, respectively, in ascitic cells of each group of mice (n = 3) detected by flow cytometry. F Mean fluorescence intensity values of IL-2, IFN- γ , GZMB, and CTLA-4 in CD8 + T cells, respectively. G Kaplan Meier analysis of survival time of each group of mice (n = 7). LDC, low-dose chemotherapy; B OA , B cells that have been treated with OA for 24h; L-DDP, low-dose DDP (1 mg/kg); H-DDP, high-dose DDP (2 mg/kg); BLI, bioluminescence imaging. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: Comparison, Immunofluorescence, Fluorescence, Flow Cytometry, Imaging

Journal: The Journal of Neuroscience

Article Title: β1-C121W Is Down But Not Out: Epilepsy-AssociatedScn1b-C121WResults in a Deleterious Gain-of-Function

doi: 10.1523/jneurosci.0405-16.2016

Figure Lengend Snippet: Figure2. Comparisonof1-C121WandWT1proteinexpression.A,RepresentativeWesternblotscomparinganti-1 immunoreactivity in Scn1b / and Scn1b W/W mouse brain membrane preparations. 1-C121W polypeptides migrate at a lower apparent molecular weight than WT 1 separated on a 10% SDS-PAGE gel. Immunoreactive bands were quantified using densitometry. Each band density was first normalized to its corresponding -tubulin signal and 1 levels in Scn1b W/Wmice were expressed as a percentage of 1 in Scn1b /mice. Scn1b W/Wmice had 45 10% of Scn1b /1 expression (2-way ANOVA; p 0.0001; 3 replicate experiments with 4 animals of each genotype). B, Representative Western blots showing mock-digested, PNGaseF-digested, and control brain membrane inputs from Scn1b / and Scn1b W/W brains separated on a 12% SDS-PAGE gel. WT 1 and 1-C121W polypeptides migrate at similar molecular weights after removal of N-linked glycosylation by PNGaseF (n 3). C, Representative Western blots, separated on a 15% SDS-PAGE gel, comparing levels of WT 1 and 1-C121W polypeptides in P14–P15, P17, and 20-week-old Scn1b /, Scn1b /W, and Scn1b W/W mice. -tubulin is shown as a loading control. Every Scn1b W/W mouse tested showed the lower molecular band only (P14–P15, n 5; P17, n 5). Every Scn1b /W sample tested showed both bands (P14–P15, n 11; P17, n 6; 20 weeks, n 4).

Article Snippet: Primary antibodies were as follows: rabbit anti-Scn1b (directed against an intracellular 1 epitope; Cell Signaling Technology, preproduction serum of D4Z2N, catalog #14684, 1:3000 used for Western blotting); rabbit anti-Scn1b (directed against an extracellular 1 epitope; Cell Signaling Technology, preproduction serum of D9T5B, catalog #13950, 1:25 used for immunofluorescence in optic nerves); rabbit anti-Scn1b (directed against an extracellular 1 epitope; Cell Signaling Technology, production version of D9T5B, catalog #13950, 1:250 used for immunofluorescence in brains); guinea pig anti-Caspr (gift from Dr. James Salzer, New York University School of Medicine, 1:1000 used for immunofluorescence in optic nerves); mouse anti-PAN VGSC -subunit (Sigma-Aldrich, catalog #S8809, 1:200 used for immunofluorescence in optic nerves and coimmunoprecipitation); goat anti-ankyrinG (recognizing total ankyrinG, gift from Dr. Vann Bennett, He et al., 2014; Jenkins et al., 2015, 1:500 used for immunofluorescence in brains); mouse anti-calbindin (Sigma-Aldrich, catalog #C9848, 1:400 used for immunofluorescence in brains); rat anti-Ctip2 (Abcam, catalog #ab18465, 1:400 used for immunofluorescence in brains); rabbit anti-PAN VGSC -subunit (Cell Signaling Technology, D2I9C, catalog #14380, 1:1000 used for Western blotting); mouse anti-human -tubulin (Cedarlane Laboratories, catalog #CLT9002; 1:5000, used for Western blotting); mouse anti-HSP90 (Enzo Life Sciences, catalog #AC88, 1:1000, used for Western blotting).

Techniques: Membrane, Molecular Weight, SDS Page, Expressing, Western Blot, Control, Glycoproteomics

Journal: Blood Cancer Journal

Article Title: Discovery and characterization of LY2784544, a small-molecule tyrosine kinase inhibitor of JAK2V617F

doi: 10.1038/bcj.2013.6

Figure Lengend Snippet: LY2784544 effectively inhibits JAK2V617F-STAT5 signaling in ascitic tumor cells from Ba/F3-JAK2V617F-GFP tumor-bearing mice. ( a ) Dose-dependent inhibition of JAK2V617F STAT5 signaling. Oral LY2784544 was administered at the indicated doses, with ascitic cells harvested 30 min later and STAT5 phosphorylation measured by FACS. Left: representative histograms showing decreased pSTAT5 Y694 -positive ascitic tumor cells collected from animals treated with LY2784544 in dose-dependent manner. Right: quantification of decreased pSTAT5 Y694 -positive ascitic tumor cells shown in the histograms. The horizontal line indicates the threshold effective dose (TED 50 ). Standard error is indicated by the error bars with statistically significant inhibition, defined as P <0.05 using Dunnett's test, indicated by an asterisk (*). ( b ) Plasma concentration–dependent inhibition of JAK2V617F activation. Blood was collected 30 min after oral LY2784544 exposure, and the concentration of LY2784544 was measured in the plasma and plotted versus percentage inhibition of STAT5 phosphorylation. Ba/F3, murine pro-B-cells; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; JAK, janus kinase; STAT5, signal transducer and activator of transcription 5. TED50, threshold effective dose required to cause 50% inhibition.

Article Snippet: Dose- and time-dependent in vivo inhibition of JAK2V617F signaling was assessed by measuring inhibition of STAT5 phosphorylation in a mouse ascitic tumor model. Ba/F3-JAK2V617F-GFP cells (1 × 10 7 ) were implanted in the intraperitoneal cavity of severe combined immunodeficiency mice (SCID, strain-CB17, Taconic, Hudson, NY, USA) and allowed to develop into ascitic tumors for 7 days.

Techniques: Inhibition, Phospho-proteomics, Clinical Proteomics, Concentration Assay, Activation Assay, Fluorescence, FACS

Journal: Blood Cancer Journal

Article Title: Discovery and characterization of LY2784544, a small-molecule tyrosine kinase inhibitor of JAK2V617F

doi: 10.1038/bcj.2013.6

Figure Lengend Snippet: Reduction of splenomegaly and Ba/F3-JAK2V617F-GFP-positive tumor cell burden in a JAK2V617-induced mouse hematologic disease model after treatment with LY2784544, twice daily, for 14 days. LY2784544 was administered orally twice daily for 14 days at the specified doses, 7 days after intravenous infusion of tumor cells, with six mice per dosage or time point group, and a six-mouse vehicle control group. Standard error is indicated by the error bars with statistically significant inhibition, defined as P <0.05 using Dunnett's test, indicated by an asterisk (*). ( a ) Dose-dependent inhibition of splenomegaly in LY2784544-treated groups. Percentage inhibition of splenomegaly was determined by comparing the mean spleen mass per dose group with the mean of the untreated control group. ( b ) Dose-dependent reduction of Ba/F3 JAK2V617F-GFP-positive tumor cell burden (total GFP-positive cells) in spleen as measured by flow cytometry. Splenocytes were isolated from the spleens of mice treated with LY2784544 at the indicated doses. Total GFP-positive Ba/F3-JAK2V617F cells were measured and determined by flow cytometry analysis. The horizontal line indicates the threshold effective dose (TED 50 ). ( c ) Actual versus predicted efficacy as a function of the area under the plasma concentration-time curve for the dosing interval (AUCtau). A mouse PK/efficacy model was developed using the JAK2V617-induced MPN mouse model, in which LY2784544 was administered every 12 h (AUC 0-12 ). The model-predicted data are represented with the actual data superimposed. ( d ) Immunostaining of spleen tissue from vehicle and LY2784544-treated Ba/F3-JAK2V617F-GFP mice with antibody against GFP. In the left column of the immunohistochemistry images, the brown staining indicates antibody binding. The right column of the fluorescent images reveals antibody staining as yellow, orange, and red. Brightfield microscopy images were acquired at room temperature using a whole-slide imaging system, Aperio's Scanscope XT (Aperio Technologies, Inc., Vista, CA, USA), at a × 20 magnification setting. The image analysis was performed using Aperio's Positive Pixel Count Algorithm (version 9.1) (Aperio Technologies, Inc.) using the default parameter settings. AUC, area under the curve; JAK, janus kinase; Ba/F3, murine pro-B-cell; GFP, green fluorescent protein; MPN, myeloproliferative neoplasm.

Article Snippet: Dose- and time-dependent in vivo inhibition of JAK2V617F signaling was assessed by measuring inhibition of STAT5 phosphorylation in a mouse ascitic tumor model. Ba/F3-JAK2V617F-GFP cells (1 × 10 7 ) were implanted in the intraperitoneal cavity of severe combined immunodeficiency mice (SCID, strain-CB17, Taconic, Hudson, NY, USA) and allowed to develop into ascitic tumors for 7 days.

Techniques: Control, Inhibition, Flow Cytometry, Isolation, Clinical Proteomics, Concentration Assay, Immunostaining, Immunohistochemistry, Staining, Binding Assay, Microscopy, Imaging

Journal: Vaccines

Article Title: A Bivalent Trans-Amplifying RNA Vaccine Candidate Induces Potent Chikungunya and Ross River Virus Specific Immune Responses

doi: 10.3390/vaccines10091374

Figure Lengend Snippet: TR-RNA amplification by the CHIKV replicase. HEK 293T cells were transfected with 2 μg of the replicase RNA or TR-luc-RNA as irrelevant RNA together with 0.5 μg of the indicated TR-RNAs. The total amount of TR-RNA was kept constant between the single and double transfections. For comparison, cells were infected with CHIKV or RRV (MOI 3). RNA was harvested after 6 h, 16 h, and 24 h, and ( A ): CHIKV E2 and ( B ): RRV E2 RNA levels were measured by RT-qPCR. Numbers indicate the fold change in TR-RNA amount 24 h after co-transfection with the replicase. Ct values, which were below the cutoff, were set to 0.1 for plotting. Data are mean values ± SEM of three independent experiments.

Article Snippet: Primary antibodies were directed against CHIKV E2 (Eurogentec, Cologne, Germany; custom made), RRV E2 (ATCC VR-1246AF), and β-actin (Sigma, Munich, Germany; no. A5441).

Techniques: RNA Amplification, Transfection, Comparison, Infection, Quantitative RT-PCR, Cotransfection

Journal: Vaccines

Article Title: A Bivalent Trans-Amplifying RNA Vaccine Candidate Induces Potent Chikungunya and Ross River Virus Specific Immune Responses

doi: 10.3390/vaccines10091374

Figure Lengend Snippet: Antigen expression from TR-RNAs. ( A ): CHIKV E2 and RRV E2 protein expression in cellular lysates 6 h and 24 h after transfection of 2 μg of replicase-RNA together with 0.5 μg of the indicated TR-RNAs. As control, cells were infected with CHIKV or RRV (MOI 3) or left untreated (ctrl). ( B ): Protein expression 48 h after transfection of 8 μg of the replicase-RNA with 2 μg of the indicated TR-RNAs in cellular lysates or concentrated supernatants. The depicted Western blots are representative of three independent experiments. Uncropped blots and densitometry readings are given in .

Article Snippet: Primary antibodies were directed against CHIKV E2 (Eurogentec, Cologne, Germany; custom made), RRV E2 (ATCC VR-1246AF), and β-actin (Sigma, Munich, Germany; no. A5441).

Techniques: Expressing, Transfection, Control, Infection, Western Blot

Journal: mBio

Article Title: Flavivirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control

doi: 10.1128/mBio.01255-17

Figure Lengend Snippet: Progression of POWV protein synthesis in infected organ cultures. Magnification, ×20 (POWV-infected MG, salivary gland [SG], and synganglion [SN]). md, midgut diverticulum; ld, lobular duct; a.c., acinus; pg/h, periganglionic sinus/sheath; n.p., neuropile; rca, retrocerebral area; mgt, midgut tissue. POWV proteins within tissue are denoted by the prominent purple color. Mock-infected organs at 192 hpi were processed and stained in the same manner, using POWV antibody to identify any potential nonspecific staining background (see Fig. S3 ).

Article Snippet: Anti-POWV and anti-LGTV virus immunoreactivities were detected using POWV ascites fluid (which detects multiple viral proteins; ATCC) ( ) at a 1:2,000 dilution and anti-LGTV-E mouse monoclonal antibody (detects viral E protein; 11H12) at a 1:50 dilution, respectively.

Techniques: Infection, Staining

Journal: mBio

Article Title: Flavivirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control

doi: 10.1128/mBio.01255-17

Figure Lengend Snippet: dsRNA transfection effect on infectious LGTV and POWV replication from infected midgut and salivary gland cultures. pGEM, pGEM plasmid (negative control); LGTV 3UTR, 3′ UTR of LGTV strain TP21. Transfection of LGTV-infected midgut (A), POWV-infected midgut (B), LGTV-infected salivary gland pair (C), and POWV-infected salivary gland pair (D) with 10 ng pGEM dsRNA and LGTV 3′ UTR dsRNA. Transfections of midguts and salivary glands were completed for 108 and 180 h, respectively, before supernatants were collected for immunofocus assays, which were normalized to the pGEM dsRNA negative-control response. An unpaired t test was completed to compare FFU per milliliter of the LGTV 3′ UTR dsRNA to that for the pGEM negative control. Error bars represent standard errors of the means, and data are representative of two technical replicates for each sample for the immunofocus assays of two biological replicates. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: Anti-POWV and anti-LGTV virus immunoreactivities were detected using POWV ascites fluid (which detects multiple viral proteins; ATCC) ( ) at a 1:2,000 dilution and anti-LGTV-E mouse monoclonal antibody (detects viral E protein; 11H12) at a 1:50 dilution, respectively.

Techniques: Transfection, Infection, Plasmid Preparation, Negative Control