Journal: The FEBS journal

Article Title: Defects of the spliceosomal gene SNRPB affect osteo- and chondro-differentiation.

doi: 10.1111/febs.16934

Figure Lengend Snippet: Fig. 2. Suppressed expression of osteodifferentiation genes in SNRPB reduced cells. (A) RT-qPCR analysis of osteodifferentiation genes in Saos-2 cells, following transfection with either control or SNRPB siRNA and induced for osteodifferentiation for 3, 5, 8 and 11 days as indi- cated. y-Axis shows fold expression (DDCt) in SNRPB siRNA-transfected cells normalised to control siRNA-transfected cells. y-Axis value 1 means that the SNRPB siRNA transfected cells show the same level of expression as control cells. (B) Western blot of control siRNA (Csi) or SNRPB siRNA (Ssi) transfected Saos-2 cells on days 3 and 8. GAPDH is a loading control. A representative of three biological repeats is shown. The molecular size marker (kDa) is shown on the right. (C) RT-qPCR analysis similar to (A), using CRISPR-mediated SNRPB-mutated Saos-2 cells. For the time-course changes in the expression level (normalised all data to day 3 control samples), see Fig. S3. (D) Western blot of control (C) or SNRPB-mutated (S) Saos-2 cells on days 3 and 8. (E–G) Rescue experiment of SNRPB siRNA by SNRPB (E, F) or Dlx5 (G) DNA transfection and subsequent 8 (E, F) or 5 (G) days of incubation. Grey bars show control siRNA transfected cells, whereas orange bars show SNRPB-siRNA transfected cells. SNRPB DNA transfection fully or partly rescues the expression of full-length encoding Dlx5 (full) (E) and Runx2 (F). Dlx5 transfection rescues the expression of Runx2 in SNRPB-knock-down cells (G). In (A, C, E–G), the graphs show the mean SD of three biological samples (n = 3), with each of them being the average of technical triplicates; *P ≤0.05, **P ≤0.01, ***P ≤0.001 in t-test. In (A, C), the comparison was made between control and SNRPB-deficient groups on each day. In (E–G), data were compared with control siRNA + control DNA, except that SNRPB siRNA + SNRPB DNA was compared with SNRPB siRNA + control DNA. (H) Western blot of Saos-2 cells transfected with control siRNA (Csi) or SNRPB siRNA (Ssi) together with control (C), SNRPB (S) or Dlx5 (D) DNA.

Article Snippet: Transfection of control siRNA (sc-44232; Santa Cruz Biotechnology, Dallas, TX, USA) and SNRPB siRNA (sc-36503; Santa Cruz, provided as a mix of siRNA targeting three sites on exon 2 of SNRPB) was performed using siRNA transfection reagent (sc-29528; Santa Cruz).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Western Blot, Marker, CRISPR, Incubation, Knockdown, Comparison

Journal: The FEBS journal

Article Title: Defects of the spliceosomal gene SNRPB affect osteo- and chondro-differentiation.

doi: 10.1111/febs.16934

Figure Lengend Snippet: Fig. 3. Enhanced expression of chondrodifferentiation genes in SNRPB reduced cells. (A–E) RT-qPCR analysis for indicated chondrodifferen- tiation genes, transfected with control siRNA (Ci), SNRPB siRNA (Si) or SNRPB sgRNA and Cas9 (Sg). The graph is presented as the fold change compared to the day 2 control siRNA transfected samples. Dlx5 primers used here detect the transcript encoding functional full- length Dlx5. The graphs show the mean SD of three biological triplicates (n = 3), with each of them being the average of technical tripli- cates; *P ≤0.05, **P ≤0.01, ***P ≤0.001 in t-test, by comparison with the control siRNA transfected samples collected on day 2. (F, G) Immunostaining of high-density 3D culture aggregates of HEPM cells incubated for 21 days without (no induction) or with cartilage induction supplements, transfected with either control siRNA, SNRPB siRNA or SNRPB sgRNA as indicated, stained with anti-Collagen II antibodies (pink) and DAPI (blue). The scale bar is 200 lm. The bar chart (G) is the quantification of Collagen II staining intensity in control siRNA (Ci), SNRPB siRNA (Si) or SNRPB sgRNA (Sg) aggregates, shown as the mean SD of four biological replicates (n = 4); *P ≤0.05, ***P ≤0.001 in t-test.

Article Snippet: Transfection of control siRNA (sc-44232; Santa Cruz Biotechnology, Dallas, TX, USA) and SNRPB siRNA (sc-36503; Santa Cruz, provided as a mix of siRNA targeting three sites on exon 2 of SNRPB) was performed using siRNA transfection reagent (sc-29528; Santa Cruz).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Functional Assay, Comparison, Immunostaining, Incubation, Staining

Journal: The FEBS journal

Article Title: Defects of the spliceosomal gene SNRPB affect osteo- and chondro-differentiation.

doi: 10.1111/febs.16934

Figure Lengend Snippet: Fig. 5. SNRPB-knockdown results in low response to Wnt signals. (A–F) Reporter assays detecting activities of the Wnt/b-catenin (A, C, E) and BMP (B, D, F) pathways, in Saos-2 cells with siRNA-mediated knockdown (A, B) or CRISPR-mediated SNRPB mutation (C, D) and HEPM cells with SNRPB-knockdown (E, F). Each pathway was activated by transfection of cDNA plasmids [Wnt3a, constitutively active LRP6 (LRP6DN), b-catenin, BMP4, BMP7, constitutively active BMP receptor IA (c.a.BMPR)] or application of chemicals (BIO, CHIR, BMP4 protein) as indicated on the x-axis. The y-axis shows the relative luciferase unit (RLU) normalised to control cells without activation. White bars show control cells; grey bars show cells with affected SNRPB expression. The graphs show the mean SD of biological triplicates; *P ≤0.05, **P ≤0.01, ***P ≤0.001 in t-test.

Article Snippet: Transfection of control siRNA (sc-44232; Santa Cruz Biotechnology, Dallas, TX, USA) and SNRPB siRNA (sc-36503; Santa Cruz, provided as a mix of siRNA targeting three sites on exon 2 of SNRPB) was performed using siRNA transfection reagent (sc-29528; Santa Cruz).

Techniques: Knockdown, CRISPR, Mutagenesis, Transfection, Luciferase, Control, Activation Assay, Expressing

Journal: Journal of Virology

Article Title: Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain

doi: 10.1128/JVI.00406-21

Figure Lengend Snippet: Interferon regulatory factor 8 (IRF8) expression increases upon JEV infection in mouse brains. Three-weeks-old (C57BL/6) mice ( n = 3 mice/group) were administered with JEV, p20778 strain (10 7 pfu), or minimal Eagle’s medium (mock) through the intraperitoneal route. Brains were harvested 24 h postonset of encephalitic symptoms. The viral and IRF8 proteins and RNA were detected by immunofluorescence, Western blotting, and qRT-PCR method. (A) Brain sections were stained for NS1 (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). (B, C) Relative JEV RNA level and Irf8 transcript level were quantified by qRT-PCR (normalized to Gapdh mRNA; ****, P < 0.0001). (D) Total protein from brain homogenate was Western blotted for NS1, IRF8, and GAPDH detection. (E) Viral RNA and IRF8 transcript expressions were measured at six different time points (0, 2, 3, 4, 5, 6 days postinfection [dpi]) in the brains of mock and infected mice (normalized to Gapdh mRNA; ****, P < 0.0001). BALB/c mice (p-10, n = 3 mice/group) were infected either with JEV, GP78 strain (10 5 PFU), or with 1× PBS (mock), and 24 h postonset of encephalitic signs, brains were harvested to prepare RNA and protein. (F) Relative JEV RNA level and Irf8 transcript expression in the mock- and JEV-infected mice analyzed by quantitative RT-PCR normalized to Gapdh mRNA). **, P < 0.01; ****, P < 0.0001. (G) Total protein from brain homogenate was Western blotted for NS1, IRF8, and GAPDH detection. (H) Mouse neuronal cells (N2a) and microglia cell lines (BV2 and N9) were mock or JEV infected at an MOI of 5 and harvested 24 hours postinfection (h p.i.). Cell lysates were immunoblotted for IRF8 and GAPDH expression. All qRT-PCR data are represented as mean ± SD.

Article Snippet: After blocking with 5% (wt/vol) skimmed milk, the membrane was probed with rabbit GAPDH antibody (at dilution 1:10,000, GTX100118; GeneTex, Irvine, CA, USA), in-house rabbit polyclonal antibody to JEV NS1 protein (at dilution 1:10,000), mouse JE1 antibody (at dilution 1:1,000, ab71671; Abcam, Cambridge, UK), rabbit Mannose receptor (at dilution 1:1,000, ab64693; Abcam, Cambridge, UK), mouse CD68 (at dilution 1:1,000, ab955; Abcam, Cambridge, UK), mouse IBA1 (at dilution 1:1,000, MABN92; Merck Millipore, Darmstadt, Germany), rabbit STAT1 (at dilution 1:1,000, sc346; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit Phospho-STAT1 (at dilution 1:1,000, Tyr701-58D6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT2 (at dilution 1:1,000, Y690-D3P2P; Cell Signaling Technology, MA, USA), rabbit STAT2 (at dilution 1:1,000, D9J7L; Cell Signaling Technology, MA, USA), mouse STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), and mouse IRF8 antibody (at dilution 1:1,000, sc365042; Santa Cruz Biotechnology, Dallas, TX, USA) to check their expression in infected and uninfected mouse brain lysate.

Techniques: Expressing, Infection, Immunofluorescence, Western Blot, Quantitative RT-PCR, Staining

Journal: Journal of Virology

Article Title: Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain

doi: 10.1128/JVI.00406-21

Figure Lengend Snippet: Effect of Irf8 deficiency on microglia and infiltrating macrophage phenotype. Total RNA was isolated from the WT and Irf8 −/− mouse brains of either mock- or JEV-infected groups at 24 h postonset of symptoms to evaluate the gene expression. (A) The heat map represents different microglia marker expression levels between WT and Irf8 −/− infected mice obtained from the qPCR array. (B) Representative Western blotting data show the status of different activation markers for microglia and infiltrated macrophages. (C) qRT-PCR was performed for quantitation of relative levels of indicated genes. Gapdh mRNA level was used for normalization. All qRT-PCR data are represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (D) Cytokine expression was quantified in the protein lysates of WT and Irf8 −/− mouse brains of either mock- or JEV-infected groups using cytokine bead array kits analyzed by FCAP and Qognit software. Representative data are shown; ****, P < 0.0001.

Article Snippet: After blocking with 5% (wt/vol) skimmed milk, the membrane was probed with rabbit GAPDH antibody (at dilution 1:10,000, GTX100118; GeneTex, Irvine, CA, USA), in-house rabbit polyclonal antibody to JEV NS1 protein (at dilution 1:10,000), mouse JE1 antibody (at dilution 1:1,000, ab71671; Abcam, Cambridge, UK), rabbit Mannose receptor (at dilution 1:1,000, ab64693; Abcam, Cambridge, UK), mouse CD68 (at dilution 1:1,000, ab955; Abcam, Cambridge, UK), mouse IBA1 (at dilution 1:1,000, MABN92; Merck Millipore, Darmstadt, Germany), rabbit STAT1 (at dilution 1:1,000, sc346; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit Phospho-STAT1 (at dilution 1:1,000, Tyr701-58D6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT2 (at dilution 1:1,000, Y690-D3P2P; Cell Signaling Technology, MA, USA), rabbit STAT2 (at dilution 1:1,000, D9J7L; Cell Signaling Technology, MA, USA), mouse STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), and mouse IRF8 antibody (at dilution 1:1,000, sc365042; Santa Cruz Biotechnology, Dallas, TX, USA) to check their expression in infected and uninfected mouse brain lysate.

Techniques: Isolation, Infection, Gene Expression, Marker, Expressing, Western Blot, Activation Assay, Quantitative RT-PCR, Quantitation Assay, Software

Journal: Journal of Virology

Article Title: Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain

doi: 10.1128/JVI.00406-21

Figure Lengend Snippet: Transcriptome profiling to elucidate genes significantly affected in WT and Irf8 −/− mouse brains following JEV infection analyzed by RNA-Seq. To reveal the global transcriptional changes in the whole brain, RNA sequencing of RNA samples isolated from WT and Irf8 −/− mouse brains of either mock- or JEV-infected groups was performed. (A) Venn diagram showing differentially expressed genes generated by a comparative analysis of WT JEV versus WT, Irf8 −/− JEV versus Irf8 −/− , and Irf8 −/− versus WT groups. (B) Venn diagram and heat map plot of genes associated with the interferon signaling pathway identified using Metascape pathway enrichment analysis of infected versus uninfected WT and Irf8 −/− transcript profiles. Blue represented higher expression values. (C) Clustered heat map analysis of interferon regulatory factors extracted from infected versus uninfected WT and Irf8 −/− transcript profiles. (D) Representative Western blotting data showing the status of different STATs. (E) mRNA expression of interferon-stimulated genes identified during RNA sequencing analysis was validated in the mock/infected WT and Irf8 −/− mouse brains, analyzed by quantitative RT-PCR (normalized to Gapdh mRNA). All qRT-PCR data are represented as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: After blocking with 5% (wt/vol) skimmed milk, the membrane was probed with rabbit GAPDH antibody (at dilution 1:10,000, GTX100118; GeneTex, Irvine, CA, USA), in-house rabbit polyclonal antibody to JEV NS1 protein (at dilution 1:10,000), mouse JE1 antibody (at dilution 1:1,000, ab71671; Abcam, Cambridge, UK), rabbit Mannose receptor (at dilution 1:1,000, ab64693; Abcam, Cambridge, UK), mouse CD68 (at dilution 1:1,000, ab955; Abcam, Cambridge, UK), mouse IBA1 (at dilution 1:1,000, MABN92; Merck Millipore, Darmstadt, Germany), rabbit STAT1 (at dilution 1:1,000, sc346; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit Phospho-STAT1 (at dilution 1:1,000, Tyr701-58D6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT2 (at dilution 1:1,000, Y690-D3P2P; Cell Signaling Technology, MA, USA), rabbit STAT2 (at dilution 1:1,000, D9J7L; Cell Signaling Technology, MA, USA), mouse STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), and mouse IRF8 antibody (at dilution 1:1,000, sc365042; Santa Cruz Biotechnology, Dallas, TX, USA) to check their expression in infected and uninfected mouse brain lysate.

Techniques: Infection, RNA Sequencing, Isolation, Generated, Expressing, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain

doi: 10.1128/JVI.00406-21

Figure Lengend Snippet: Effect of Ifnγ deficiency on viral load and activation genes of microglia and infiltrated macrophage. Total RNA was isolated from the WT and Ifnγ −/− mouse brains of mock- or JEV-infected groups at 24 h postonset of clinical signs. (A, B) The relative RNA level of Irf8 and JEV transcripts were quantified by qRT- PCR in WT (C57BL/6) and Ifnγ −/− mouse brain RNA (normalized to Gapdh mRNA). ***, P < 0.001; **, P < 0.01. (C) Virus titers in the brain homogenates of infected WT and Ifnγ −/− mouse brains were determined through plaque assay on the PS cell line. Virus titer is expressed as the number of virion particles per gram; **, P < 0.01. (D) Total protein in the indicated groups was Western blotted for E protein, NS1, and GAPDH detection. (E) qRT-PCR was performed for quantitation of relative levels of indicated genes (normalized to Gapdh mRNA). Representative data are shown. All qRT-PCR data are represented as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F) A survival study was performed to assess the survival of Ifnγ −/− mice to JEV infection. WT (C57BL/6) and Ifnγ −/− mice ( n = 12 in each group) were mock and JEV (10 7 PFU) infected, followed by the appearance of encephalitic symptoms until death; ****, P < 0.0001. The data shown are the combined results of three experiments.

Article Snippet: After blocking with 5% (wt/vol) skimmed milk, the membrane was probed with rabbit GAPDH antibody (at dilution 1:10,000, GTX100118; GeneTex, Irvine, CA, USA), in-house rabbit polyclonal antibody to JEV NS1 protein (at dilution 1:10,000), mouse JE1 antibody (at dilution 1:1,000, ab71671; Abcam, Cambridge, UK), rabbit Mannose receptor (at dilution 1:1,000, ab64693; Abcam, Cambridge, UK), mouse CD68 (at dilution 1:1,000, ab955; Abcam, Cambridge, UK), mouse IBA1 (at dilution 1:1,000, MABN92; Merck Millipore, Darmstadt, Germany), rabbit STAT1 (at dilution 1:1,000, sc346; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit Phospho-STAT1 (at dilution 1:1,000, Tyr701-58D6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT2 (at dilution 1:1,000, Y690-D3P2P; Cell Signaling Technology, MA, USA), rabbit STAT2 (at dilution 1:1,000, D9J7L; Cell Signaling Technology, MA, USA), mouse STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), and mouse IRF8 antibody (at dilution 1:1,000, sc365042; Santa Cruz Biotechnology, Dallas, TX, USA) to check their expression in infected and uninfected mouse brain lysate.

Techniques: Activation Assay, Isolation, Infection, Quantitative RT-PCR, Virus, Plaque Assay, Western Blot, Quantitation Assay

Journal: Journal of Virology

Article Title: Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain

doi: 10.1128/JVI.00406-21

Figure Lengend Snippet: Immune cells infiltration into the brain 24 h postonset of clinical signs in WT, Ifnγ −/− , and Irf8 −/− mice. Myeloid and lymphoid cells were enriched from the brains of WT ( n = 3), Ifnγ −/− ( n = 3), and Irf8 −/− ( n = 5) mice either of mock- or JEV-infected groups, by Percoll gradient for the flow cytometric analysis. Representative dot plots (left) show percentage of cells in each quadrant; positive for the indicated markers on the axis and bar graph (right panel) indicates the quantification of CD45 + , CD11b + , CD68 + , CD86 + , Ly6C + , Ly6G + , CD3 + , CD4 + , CD8 + , and NK1.1 + expression on leukocytes (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Each dot represents cells from an individual mouse. Error bars are represented as mean ± SD.

Article Snippet: After blocking with 5% (wt/vol) skimmed milk, the membrane was probed with rabbit GAPDH antibody (at dilution 1:10,000, GTX100118; GeneTex, Irvine, CA, USA), in-house rabbit polyclonal antibody to JEV NS1 protein (at dilution 1:10,000), mouse JE1 antibody (at dilution 1:1,000, ab71671; Abcam, Cambridge, UK), rabbit Mannose receptor (at dilution 1:1,000, ab64693; Abcam, Cambridge, UK), mouse CD68 (at dilution 1:1,000, ab955; Abcam, Cambridge, UK), mouse IBA1 (at dilution 1:1,000, MABN92; Merck Millipore, Darmstadt, Germany), rabbit STAT1 (at dilution 1:1,000, sc346; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit Phospho-STAT1 (at dilution 1:1,000, Tyr701-58D6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT2 (at dilution 1:1,000, Y690-D3P2P; Cell Signaling Technology, MA, USA), rabbit STAT2 (at dilution 1:1,000, D9J7L; Cell Signaling Technology, MA, USA), mouse STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), and mouse IRF8 antibody (at dilution 1:1,000, sc365042; Santa Cruz Biotechnology, Dallas, TX, USA) to check their expression in infected and uninfected mouse brain lysate.

Techniques: Infection, Expressing

Journal: Journal of Virology

Article Title: Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain

doi: 10.1128/JVI.00406-21

Figure Lengend Snippet: Immune cell abundance in blood 24 h postonset of clinical signs in WT, Ifnγ −/− , and Irf8 −/− mice. Leukocytes from the blood of WT ( n = 3), Ifnγ −/− ( n = 3), and Irf8 −/− ( n = 5) mice, either of mock- or JEV-infected groups, were subjected to flow cytometric analysis. Representative dot plot (left) shows the percentage of cells in each quadrant, positive for the indicated markers on the axis. Bar graph (right) indicates the quantification of CD45 + , CD11b + , Ly6C + , Ly6G + , CD3 + , CD4 + , CD8 + , and NK1.1 + expression on leukocytes (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Each dot in the bar graph represents cells from an individual mouse. Error bar are represented as mean ± SD.

Article Snippet: After blocking with 5% (wt/vol) skimmed milk, the membrane was probed with rabbit GAPDH antibody (at dilution 1:10,000, GTX100118; GeneTex, Irvine, CA, USA), in-house rabbit polyclonal antibody to JEV NS1 protein (at dilution 1:10,000), mouse JE1 antibody (at dilution 1:1,000, ab71671; Abcam, Cambridge, UK), rabbit Mannose receptor (at dilution 1:1,000, ab64693; Abcam, Cambridge, UK), mouse CD68 (at dilution 1:1,000, ab955; Abcam, Cambridge, UK), mouse IBA1 (at dilution 1:1,000, MABN92; Merck Millipore, Darmstadt, Germany), rabbit STAT1 (at dilution 1:1,000, sc346; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit Phospho-STAT1 (at dilution 1:1,000, Tyr701-58D6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT2 (at dilution 1:1,000, Y690-D3P2P; Cell Signaling Technology, MA, USA), rabbit STAT2 (at dilution 1:1,000, D9J7L; Cell Signaling Technology, MA, USA), mouse STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), and mouse IRF8 antibody (at dilution 1:1,000, sc365042; Santa Cruz Biotechnology, Dallas, TX, USA) to check their expression in infected and uninfected mouse brain lysate.

Techniques: Infection, Expressing

Journal: Journal of Virology

Article Title: Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain

doi: 10.1128/JVI.00406-21

Figure Lengend Snippet: Intracellular staining of Ifnγ in infiltrating NK1.1 + and CD3 + cells in infected brains. Lymphocytes were enriched from the brains of WT ( n = 3) and Irf8 −/− ( n = 3) mice from either the mock- or JEV-infected group by Percoll gradient for flow cytometric analysis. Representative dot plot (left) shows the percentage of cells in each quadrant, positive for the indicated markers on the axis. Bar graph (right) indicates the quantification of NK1.1 + , NK1.1 + IFN-γ + , CD3 + , and CD3 + IFN-γ + expression on lymphocytes (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Each dot in the bar graph represents cells from an individual mouse. Error bar represents mean ± SD.

Article Snippet: After blocking with 5% (wt/vol) skimmed milk, the membrane was probed with rabbit GAPDH antibody (at dilution 1:10,000, GTX100118; GeneTex, Irvine, CA, USA), in-house rabbit polyclonal antibody to JEV NS1 protein (at dilution 1:10,000), mouse JE1 antibody (at dilution 1:1,000, ab71671; Abcam, Cambridge, UK), rabbit Mannose receptor (at dilution 1:1,000, ab64693; Abcam, Cambridge, UK), mouse CD68 (at dilution 1:1,000, ab955; Abcam, Cambridge, UK), mouse IBA1 (at dilution 1:1,000, MABN92; Merck Millipore, Darmstadt, Germany), rabbit STAT1 (at dilution 1:1,000, sc346; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit Phospho-STAT1 (at dilution 1:1,000, Tyr701-58D6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT2 (at dilution 1:1,000, Y690-D3P2P; Cell Signaling Technology, MA, USA), rabbit STAT2 (at dilution 1:1,000, D9J7L; Cell Signaling Technology, MA, USA), mouse STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), and mouse IRF8 antibody (at dilution 1:1,000, sc365042; Santa Cruz Biotechnology, Dallas, TX, USA) to check their expression in infected and uninfected mouse brain lysate.

Techniques: Staining, Infection, Expressing

Journal: Journal of Virology

Article Title: Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain

doi: 10.1128/JVI.00406-21

Figure Lengend Snippet: Effect of loss of IRF8 on JEV replication. (A) A survival study was performed to assess the survival of Irf8 −/− mice to JEV infection. WT (C57BL/6) and Irf8 −/− mice ( n = 18 in each group) were mock or JEV (10 7 PFU) infected and followed from the appearance of encephalitic signs until death; ****, P < 0.0001. Data shown are the combined results of three experiments. (B) The relative RNA level of Irf8 transcripts was quantified by qRT-PCR in WT (C57BL/6) and Irf8 −/− mice of mock- and JEV-infected RNA samples (normalized to Gapdh mRNA). (C, D) The relative level of viral and virus titer in the brain homogenates of infected WT and Irf8 −/− mouse brains was determined through qRT-PCR and plaque assay. Virus titer is expressed as the number of virion particles per gram; **, P < 0.01. (D, C) ***, P < 0.001; ****, P < 0.0001. (E) The total protein of indicated groups was Western blotted for NS1 and GAPDH detection.

Article Snippet: After blocking with 5% (wt/vol) skimmed milk, the membrane was probed with rabbit GAPDH antibody (at dilution 1:10,000, GTX100118; GeneTex, Irvine, CA, USA), in-house rabbit polyclonal antibody to JEV NS1 protein (at dilution 1:10,000), mouse JE1 antibody (at dilution 1:1,000, ab71671; Abcam, Cambridge, UK), rabbit Mannose receptor (at dilution 1:1,000, ab64693; Abcam, Cambridge, UK), mouse CD68 (at dilution 1:1,000, ab955; Abcam, Cambridge, UK), mouse IBA1 (at dilution 1:1,000, MABN92; Merck Millipore, Darmstadt, Germany), rabbit STAT1 (at dilution 1:1,000, sc346; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit Phospho-STAT1 (at dilution 1:1,000, Tyr701-58D6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT2 (at dilution 1:1,000, Y690-D3P2P; Cell Signaling Technology, MA, USA), rabbit STAT2 (at dilution 1:1,000, D9J7L; Cell Signaling Technology, MA, USA), mouse STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), and mouse IRF8 antibody (at dilution 1:1,000, sc365042; Santa Cruz Biotechnology, Dallas, TX, USA) to check their expression in infected and uninfected mouse brain lysate.

Techniques: Infection, Quantitative RT-PCR, Virus, Plaque Assay, Western Blot

Journal: Journal of Virology

Article Title: Lack of Interferon (IFN) Regulatory Factor 8 Associated with Restricted IFN-γ Response Augmented Japanese Encephalitis Virus Replication in the Mouse Brain

doi: 10.1128/JVI.00406-21

Figure Lengend Snippet: Effect of IRF8 overexpression in myeloid cell lines. (A) Haploid1 (HAP1) cell line was mock or JEV infected at an MOI of 5 for 24 h. Expression of IRF8 and viral envelope protein were detected by the immunofluorescence method. The relative level of human Irf8 transcript was quantified by qRT-PCR in a HAP1 cell that was mock or JEV infected at an MOI of 5 for 24 h (right). (B, C) The relative level of Irf8 and viral RNA were determined by qRT-PCR (**, P < 0.01). (D) Viral protein (NS1) was immunoblotted in the lysates of the mock and infected HAP1 and HAP1 Irf8 5bp del cells. GAPDH was used as an internal control. (E) HAP1 Irf8 5 bp del cells transduced with pMig-CD8t control or pMig-IRF8-cd8t plasmid were mock or JEV infected at an MOI of 5 for 24 h. Cell lysate was prepared from mock- and JEV-infected HAP1 Irf8 5 bp del transduced with pMig-CD8t control or pMig-IRF8-cd8t plasmid, and Western blotting was performed to determine JEV and mouse IRF8 level in overexpressed cells. All qRT-PCR data are represented as mean ± SD and were normalized to Gapdh . (F) hIFN-γ expression was quantified in the protein lysates (left) and supernatant (right) of mock- and JEV-infected HAP1 Irf8 5 bp del transduced with pMig-CD8t control or pMig-IRF8-cd8t plasmid groups using cytokine bead array kits analyzed by FCAP software. Representative data is shown; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: After blocking with 5% (wt/vol) skimmed milk, the membrane was probed with rabbit GAPDH antibody (at dilution 1:10,000, GTX100118; GeneTex, Irvine, CA, USA), in-house rabbit polyclonal antibody to JEV NS1 protein (at dilution 1:10,000), mouse JE1 antibody (at dilution 1:1,000, ab71671; Abcam, Cambridge, UK), rabbit Mannose receptor (at dilution 1:1,000, ab64693; Abcam, Cambridge, UK), mouse CD68 (at dilution 1:1,000, ab955; Abcam, Cambridge, UK), mouse IBA1 (at dilution 1:1,000, MABN92; Merck Millipore, Darmstadt, Germany), rabbit STAT1 (at dilution 1:1,000, sc346; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit Phospho-STAT1 (at dilution 1:1,000, Tyr701-58D6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT2 (at dilution 1:1,000, Y690-D3P2P; Cell Signaling Technology, MA, USA), rabbit STAT2 (at dilution 1:1,000, D9J7L; Cell Signaling Technology, MA, USA), mouse STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), rabbit Phospho-STAT3 (at dilution 1:1,000, 124H6; Cell Signaling Technology, MA, USA), and mouse IRF8 antibody (at dilution 1:1,000, sc365042; Santa Cruz Biotechnology, Dallas, TX, USA) to check their expression in infected and uninfected mouse brain lysate.

Techniques: Over Expression, Infection, Expressing, Immunofluorescence, Quantitative RT-PCR, Control, Transduction, Plasmid Preparation, Western Blot, Software