Journal: Nature Communications

Article Title: Functional rare and low frequency variants in BLK and BANK1 contribute to human lupus

doi: 10.1038/s41467-019-10242-9

Figure Lengend Snippet: BLK variants fail to repress type 1 IFN expression. a IFNβ luciferase activity 24 h after co-transfection of HEK293T cells with IRF5, MyD88, and indicated BLK variants. Each data point represents the relative luminometer unit (RLU) relative to a cotransfected control plasmid expressing renilla luciferase, and is the average of three technical replicates. (* p < 0.05, ** p < 0.01, *** p < 0.001 Student t test). b IFNβ luciferase expression in CRISPR-Cas9 edited Ramos cells homozygous for the R131W variant, 24 h after stimulation with resiquimod (R848). Each data point represents the relative luminometer unit (RLU) relative to a cotransfected control plasmid expressing renilla luciferase, and is the average of five technical replicates. (* p < 0.05, ** p < 0.01, Mann–Whitney U ). c IFNβ luciferase activity relative to WT BLK 24 h after co-transfection of HEK293T cells with IRF5, MyD88, and indicated BLK variants found in healthy controls (HC) or SLE patients (SLE). Data points for each variant were normalized to the repression levels of WT BLK. The dotted line indicates 50% of repression by WT BLK (mean and SD). a – c representative of at least three experimental replicates ( p < 0.0001, two-tailed ANOVA). d . Heat map of 34 hierarchically clustered genes within IFN module M1.2. The data are normalized to healthy controls by subtracting out the mean of the healthy controls across all samples. The samples are ordered by condition, illustrating IFN upregulation in both BANK/BLK and non-BLK patients relative to the healthy controls. e For BLK samples, the map shows the percentage of genes within each module (minimum 10%) that are significant (False discovery rate (FDR) ≤ 0.05) and overexpressed (red) or underexpressed (blue) relative to healthy controls. As an example, a module in which 50% of the genes are significantly upregulated and 25% are significantly downregulated would appear on the map as 50–25% = 25% upregulated and would have a light red color

Article Snippet: IFNβ luciferase (Addgene) pRL-CMV (Promega). pCMV-HA-MyD88 was a gift from Bruce Beutler (Addgene plasmid # 12287), pX330-U6-Chimeric_BB-CBh-hSpCas9 was a gift from Feng Zhang (Addgene plasmid # 42230).

Techniques: Expressing, Luciferase, Activity Assay, Cotransfection, Control, Plasmid Preparation, CRISPR, Variant Assay, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Functional rare and low frequency variants in BLK and BANK1 contribute to human lupus

doi: 10.1038/s41467-019-10242-9

Figure Lengend Snippet: Impaired sequestration of TRAF6 by BANK1 increases nuclear IRF5. a Schematic of the role of BANK1 and BLK on regulation of T1 IFN production. Red lines indicate negative regulation. b Indirect immunofluorescence staining of HEK293T cells expressing either wild type (top panels) or W40C (bottom panels) BANK1-V5 cDNA constructs. BANK1 protein was detected using an antibody specific for the V5 tag (red). DNA was stained with DAPI (blue). Scale bar 20 μm. c Quantification of the percentage of HEK293T cells expressing WT or W40C BANK1-V5 with cytoplasmic inclusion bodies. Cells were transfected with 3 μg of the BANK1-V5 cDNA constructs and stained 72 h post transfection. Greyscale inserts show enlargements of the indicated cell sections. Double blind cell counting was carried out on at least 100 cells per experiment with three experimental replicates. d Representative images of indirect immunofluorescence staining of HEK293T cells expressing IRF5, FLAG-TRAF6 and either WT BANK1-V5 (top panel) or W40C BANK1-V5 (bottom panel). IRF5 protein was detected using an antibody specific for IRF5 (green) and BANK1 was detected using an antibody recognizing the V5 tag (red). Greyscale inserts show enlargements of the indicated cell. Scale bar 50 μm. Double blind cell counting was carried out on at least 100 cells per experiment with n = 3 experimental replicates. Bar graph represents quantification of the percentage of nuclear IRF5 as a proportion of the total IRF5 per cell based on indirect immunofluorescence staining of HEK293T cells expressing IRF5, FLAG-TRAF6 and either WT BANK1-V5 or W40C BANK1-V5 (as in ( d )). Quantification was performed using the ImageJ software . (Centre line = median, box = 25th to 75th percentile, whiskers = range; * p < 0.05, ** p < 0.01, Mann–Whitney U , representative of two experimental replicates)

Article Snippet: IFNβ luciferase (Addgene) pRL-CMV (Promega). pCMV-HA-MyD88 was a gift from Bruce Beutler (Addgene plasmid # 12287), pX330-U6-Chimeric_BB-CBh-hSpCas9 was a gift from Feng Zhang (Addgene plasmid # 42230).

Techniques: Immunofluorescence, Staining, Expressing, Construct, Transfection, Cell Counting, Software, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Entirely plasmid-based reverse genetics system for rotaviruses

doi: 10.1073/pnas.1618424114

Figure Lengend Snippet: IFN-β promoter activation by the NSP1 mutant. (A) Expression of NSP1 protein from a recombinant virus. MA104 cells were infected with rsSA11 or rsSA11-dC103 at an MOI of 0.1 pfu per cell. As a control, cells were transfected with NSP1 expression plasmid (pCAG-NSP1). Viral protein expression in cells was assessed by immunoblotting using anti-NSP1 antibody after 48 h of incubation. An actin-specific antibody was used as a loading control. Molecular masses were determined by coelectrophoresis of prestained protein markers. (B) IFN-β promoter activation assay. The 293T cells were transfected with a firefly luciferase (Fluc) reporter plasmid under the control of the IFN-β promoter (pIFN-β–Fluc) and a pSV40-Rluc reporter plasmid that expresses Renilla luciferase (Rluc) under the control of the SV40 promoter (Promega). Twelve hours after transfection, cells were infected with rsSA11 or rsSA11-dC103 at an MOI of 10 pfu per cell. Cells were harvested 10 h postinfection, and luciferase activities were determined. Relative luminescence units (Fluc/Rluc) are shown. *P < 0.05, **P < 0.01 (Tukey’s multiple comparison test).

Article Snippet: The 293T cells were transfected with a firefly luciferase (Fluc) reporter plasmid under the control of the IFN-β promoter (pIFN-β–Fluc) and a pSV40-Rluc reporter plasmid that expresses Renilla luciferase (Rluc) under the control of the SV40 promoter (Promega).

Techniques: Activation Assay, Mutagenesis, Expressing, Recombinant, Infection, Transfection, Plasmid Preparation, Western Blot, Incubation, Luciferase

Journal: Virus Research

Article Title: Porcine reproductive and respiratory syndrome virus (PRRSV) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in MARC-145 cells

doi: 10.1016/j.virusres.2010.07.028

Figure Lengend Snippet: PRRSV phosphorylated IRF-3 and weakly activated the IFN-β promoter in MARC-145 at 24 h p.i., but their activities were rapidly inhibited in the following infection. (A) MARC-145 cells were infected with PRRSV at an MOI of 0.05. After 6 h, 18 h, 30 h respectively, the cells were co-transfected with p-284 Luc and phRL-TK, and 18 h later, cells were harvested for dual luciferase reporter assay system in MicroBeta-1450. Cells were transfected with Poly(I:C) for 9 h as positive controls. (B) MARC-145 cells were infected with PRRSV at an MOI of 0.05, and harvested at 24, 36, 48 h p.i. for the immunoblots of IRF-3 and pIRF-3. The results shown were from one of three independent experiments with similar observations, and each including three or two replicates. Control (Con): Sham virus mentioned in part of Section were added into MARC-145 cells. *: p < 0.05 compared to the control.

Article Snippet: The firefly- luciferase plasmid for monitoring IFN-β promoter activation (p-284 Luc) was cloned from genetic DNA of MARC-145 cells and was ligated into pGL-417 (Prpmega). p55C1B Luc ( , , ), a firefly- luciferase reporter gene plasmid containing repetitive pIRF-3 binding sites, was kindly provided by Dr. Takashi Fujita. phRL-TK (Promega), contained a Renilla -luciferase reporter gene, was used as an internal control in dual luciferase reporter assay system. pcDNA3.1-His was constructed by using His primers.

Techniques: Infection, Transfection, Luciferase, Reporter Assay, Western Blot, Control, Virus

Journal: Virus Research

Article Title: Porcine reproductive and respiratory syndrome virus (PRRSV) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in MARC-145 cells

doi: 10.1016/j.virusres.2010.07.028

Figure Lengend Snippet: PRRSV nsp1 inhibited the activations of IFN-β promoter and pIRF-3-dependent synthetic promoter (p55C1B Luc) induced by Poly(I:C). (A) The mRNA of nsp1 and N in MARC-145 cells transfected with pcDNA3.1-nsp1 (nsp1), pcDNA3.1-N (N) or pcDNA3.1-His (Con) were analyzed by RT-PCR. Lanes 1–3 were amplified by PRRSV N primers, while lanes 4–6 were amplified by PRRSV nsp1 primers. (B) Western blot analyzed nsp1 and N protein by anti-His primary antibody in MARC-145 transfected with pcDNA3.1-nsp1 (nsp1) or pcDNA3.1-N (N). The control cells were transfected with pcDNA 3.1-His (Con). Since the nsp1 should be cleaved into nsp1α and nsp1β subunits, the arrow-pointed band was only the nsp1β subunits. (C) MARC-145 cells were co-transfected with p-284 Luc, phRL-TK and different expression plasmid, and 24 h later, cells were either mock-treated (Con) or transfected with Poly(I:C) for 6 h. Cells were analyzed by dual luciferase reporter assay. (D) MARC-145 cells were co-transfected with p55C1B Luc, phRL-TK and different expression plasmid. Twenty four hours later, cells were either mock-treated (Con) or transfected with Poly(I:C) for 6 h. Cells were analyzed by dual luciferase reporter assay. LCPS was the luminescence counts per second detected by luminescence counting on the Microbeta-1450. Bars showed relative luciferase activity (Relative LCPS) normalized to phRL-TK activity (relative LCPS = LCPS of firefly- luciferase/LCPS of Renilla- luciferase). Data represented means of three replicates, and experiments were repeated three times. Vector (pcDNA3.1-His) showed in figure was the control plasmid. *: p < 0.05 compared to the cells transfected with pcDNA3.1-His (Con).

Article Snippet: The firefly- luciferase plasmid for monitoring IFN-β promoter activation (p-284 Luc) was cloned from genetic DNA of MARC-145 cells and was ligated into pGL-417 (Prpmega). p55C1B Luc ( , , ), a firefly- luciferase reporter gene plasmid containing repetitive pIRF-3 binding sites, was kindly provided by Dr. Takashi Fujita. phRL-TK (Promega), contained a Renilla -luciferase reporter gene, was used as an internal control in dual luciferase reporter assay system. pcDNA3.1-His was constructed by using His primers.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Control, Expressing, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay

Journal: Virus Research

Article Title: Porcine reproductive and respiratory syndrome virus (PRRSV) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in MARC-145 cells

doi: 10.1016/j.virusres.2010.07.028

Figure Lengend Snippet: PRRSV nsp1 inhibited activities of IFN-β promoter and pIRF-3 dependent promoters (p55C1B Luc) induced by RIG-N, VISA, TRIF or TBK1, but did not inhibit the activities of IFN-β promoter and pIRF-3 dependent promoter induced by IRF-3(5D). MARC-145 cells were co-transfected with p-284 Luc (A) or p55C1B Luc (B), phRL-TK and different expression plasmids. Thirty hours later, cells were harvested for dual luciferase reporter assay. Con: cells transfected with pcDNA3.1-His. Data represented means of three replicates, and experiments were repeated three times. *: p < 0.05 compared to cells transfected with pcDNA3.1-His (Con).

Article Snippet: The firefly- luciferase plasmid for monitoring IFN-β promoter activation (p-284 Luc) was cloned from genetic DNA of MARC-145 cells and was ligated into pGL-417 (Prpmega). p55C1B Luc ( , , ), a firefly- luciferase reporter gene plasmid containing repetitive pIRF-3 binding sites, was kindly provided by Dr. Takashi Fujita. phRL-TK (Promega), contained a Renilla -luciferase reporter gene, was used as an internal control in dual luciferase reporter assay system. pcDNA3.1-His was constructed by using His primers.

Techniques: Transfection, Expressing, Luciferase, Reporter Assay