Journal: Communications Biology

Article Title: Bmp8a is an essential positive regulator of antiviral immunity in zebrafish

doi: 10.1038/s42003-021-01811-0

Figure Lengend Snippet: a , b , e , f Expression of irf3 , irf7 , and tbk1 mRNA after transfected with 2 μg bmp8a or empty vector in ZFL ( a , b ) or EPC ( e , f ) cells. The cells were collected at 36 h ( a , e ) or 48 h ( b , f ) post-transfection. c , d , g , h Expression of irf3 , irf7 , and tbk1 mRNA after transfected with 2 μg bmp8a or empty vector in ZFL ( c , d ) or EPC ( g , h ) cells for 24 h, followed by infection with GCRV for another 24 h ( c , g ) or 36 h ( d , h ). i – l Expression of irf3 , irf7 , and tbk1 mRNA after bmp8a knockdown in ZFL cells. The cells were collected at 36 h ( i ) and 48 h ( j ) post-knockdown or at 24 h ( k ) and 36 h ( l ) post-infected with GCRV. m , o Immunoblot analysis of phosphorylated (p-) Tbk1 and Irf3 after transfected with 2 μg bmp8a or empty vector in ZFL ( m ) or EPC ( o ) cells. The cells were collected at 36 or 48 h post-transfection for Immunoblot analysis. n , p Immunoblot analysis of phosphorylated (p-) Tbk1 and Irf3 after transfected with 2 μg bmp8a or empty vector in ZFL ( n ) or EPC ( p ) cells for 24 h, followed by infection with GCRV for another 24 or 36 h. q , r Immunoblot analysis of phosphorylated (p-) TBK1 and IRF3 after bmp8a knockdown in ZFL cells. The cells were collected at 36 and 48 h post-knockdown or at 24 and 36 h post-infected with GCRV. s – u EPC cells were cotransfected with IFN-φ1pro-luc (200 ng, s ), IFN-φ3pro-luc (200 ng, t ) or EPC IFNpro-luc (200 ng, u ), and bmp8a (100 ng) together with each of the dominant negative plasmids including tbk1–K38M (100 ng), irf3DN (100 ng) and irf7DN (100 ng). At 48 h post-transfection, the cells were collected for luciferase assays. Renilla luciferase was used as the internal control. v – y Expression of irf3 , irf7 , and tbk1 mRNA in the liver, kidney, intestine, and spleen from WT or bmp8a −/− zebrafish injected i.p. with 50 µl of GCRV (10 8 TCID 50 per ml). The expression of zebrafish actb1 or EPC actin was used as an internal control for the qRT-PCR. Data were from three independent experiments and were analyzed by Student’s t -test (two-tailed) for comparison of two groups or one-way ANOVA followed by Games–Howell post hoc tests for comparison of multiple groups. All data were presented as mean ± SD (** p < 0.01, *** p < 0.001).

Article Snippet: Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R).

Techniques: Expressing, Transfection, Plasmid Preparation, Infection, Western Blot, Dominant Negative Mutation, Luciferase, Injection, Quantitative RT-PCR, Two Tailed Test

Journal: Communications Biology

Article Title: Bmp8a is an essential positive regulator of antiviral immunity in zebrafish

doi: 10.1038/s42003-021-01811-0

Figure Lengend Snippet: a , b Expression of alk2 , alk3 , alk6a , bmpr2a , bmpr2b , actr2a , and actr2b mRNA in ZFL cells stimulated with poly(I:C) (2 μg/ml, a ) or GCRV (5 × 10 4 TCID 50 per ml, b ) for 48 h. c – e Expression of ifnφ1 ( c ) and ifnφ3 ( d ) mRNA in ZFL cells or EPC ifn ( e ) in EPC cells which were transfected with 2 μg of alk2, alk3, alk6a, bmpr2a, bmpr2b, actr2a, actr2b or empty vector for 48 h. f , h Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg of pcDNA3.1-alk6a or empty vector in ZFL ( f ) or EPC ( h ) cells for 48 h. g , i Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg of of pcDNA3.1-alk6a or empty vector in ZFL ( g ) or EPC ( i ) cells for 24 h, followed by infection with GCRV for another 36 h. j Schematic drawing of the alk6a-ΔGS mutation that the GS domain of Alk6a was deleted. k , m Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg pcDNA3.1-alk6a-ΔGS or empty vector in ZFL ( k ) or EPC ( m ) cells for 48 h. l , n Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg pcDNA3.1-alk6a-ΔGS or empty vector in ZFL ( l ) or EPC ( n ) cells for 24 h, followed by infection with GCRV for another 36 h. The expression of zebrafish actb1 or EPC actin was used as an internal control for the qRT-PCR. Data were from three independent experiments and were analyzed by Student’s t -test (two-tailed) for comparison of two groups or one-way ANOVA followed by Games–Howell posthoc tests for comparison of multiple groups. All data were presented as mean ± SD (* p < 0.05, ** p < 0.01, and *** p < 0.001, ns means no significant difference).

Article Snippet: Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R).

Techniques: Expressing, Transfection, Plasmid Preparation, Infection, Mutagenesis, Quantitative RT-PCR, Two Tailed Test

Journal: Communications Biology

Article Title: Bmp8a is an essential positive regulator of antiviral immunity in zebrafish

doi: 10.1038/s42003-021-01811-0

Figure Lengend Snippet: Upon virus infection, the transcriptions of bmp8a are activated through the Jak-Stat1 pathway. The Bmp8a binds to BMP type I receptor Alk6a, promoting phosphorylation of Tbk1 and Irf3 to induce the expression of Ifn through p38 MAPK pathway.

Article Snippet: Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R).

Techniques: Infection, Expressing

Journal: PLoS ONE

Article Title: Chronic Oral Infection with Porphyromonas gingivalis Accelerates Atheroma Formation by Shifting the Lipid Profile

doi: 10.1371/journal.pone.0020240

Figure Lengend Snippet: Cells were stimulated with 1.0 µg/ml of P. gingivalis LPS, 0.1 µg/ml of E. coli LPS or 0.1 µg/ml of Pam3CSK4 for 12 hrs. Phosphorylation of IRF3 cellular extracts was analyzed by western blotting. Blot is a representative of three independent experiments.

Article Snippet: Rabbit anti-mouse IRF3 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse phospho-IRF3 (Cell Signaling Technology, Danvers, MA), rabbit anti-mouse GAPDH (Santa Cruz Biotechnology), peroxidase labeled anti-rabbit antibody (GE Healthcare, Munich, Germany) and ECL Plus Western Blotting Reagent Pack (Amersham Biosciences, Buckinghamshire, UK) were used for western blotting experiments.

Techniques: Phospho-proteomics, Western Blot

Journal: Molecular cell

Article Title: A Quantitative Genetic Interaction Map of HIV Infection

doi: 10.1016/j.molcel.2020.02.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-IRF7 , Cell Signaling , 13014S, RRID:AB_2737060.

Techniques: Virus, Luciferase, Mutagenesis, Recombinant, Purification, esiRNA, Cloning, Synthesized, Software

Journal: EBioMedicine

Article Title: A STING antagonist modulating the interaction with STIM1 blocks ER-to-Golgi trafficking and inhibits lupus pathology

doi: 10.1016/j.ebiom.2021.103314

Figure Lengend Snippet: ISD017 associates with STING and STIM1 and prevents ER-to-Golgi Trafficking . (a) PMA-differentiated THP1 cells were treated with ISD017 (200 µg/ml). One h later, the cells were transfected with dsDNA (2 µg/ml). Cells were lysed at the indicated time points post-stimulation and immunoblotted by antibodies against pTBKs (S172), pSTING (S366), pIRF3 (S396), and Vinculin. ( n = 3). (b) PMA-differentiated THP1 cells were pretreated with ISD017 (200 µg/ml) for 1 h. The cells were transfected with dsDNA (6 µg/ml) and fixed 2 h later for imaging analysis after staining with anti-STING, anti-Calreticulin (ER), and anti-GM130 (Golgi) ( n = 3). Scale bar, 10 µm. (c) Lysates from PMA-differentiated THP1 cells were incubated with biotinylated ISD017, washed, and precipitated with streptavidin beads. Precipitates were immunoblotted with antibodies binding the indicated proteins ( n = 3). The light blue color of the bands for STEEP and vinculin in the input lane illustrates saturation of the signal. (d) Lysates from PMA-differentiated Wt, STING KO, and STIM1 KO THP1 cells were incubated with biotinylated ISD017, washed, and precipitated with streptavidin beads. Precipitates were immunoblotted with antibodies against STIM1, STING, and vinculin ( n = 3). (e) PMA-differentiated THP1 cells were treated with ISD017 (200 µg/ml) for 1 h, followed by stimulation with dsDNA for 2 hs. Cell lysates were subjected to immunoprecipitation with anti-STING and immunoblotting with anti-STIM1, anti-STING, and anti-vinculin ( n = 3). (f,g) Wt and STIM1 KO THP1 macrophages were treated with dsDNA in the presence or absence of ISD017 (200 µg/ml, pre-treatment). Supernatants were harvested 16 h post-stimulation, and IFN-I activity and TNFα levels were measured ( n = 3). The data in panel f and g are presented as means of biological triplicates +/- st.dev. [Statistical analysis of the data in f, g were performed using two-tailed one-way ANOVA test].

Article Snippet: The following antibodies and dilutions were used for immunoblotting anti‐STING (Cell Signaling, D2P2F/#13,647, 1:1000), sheep anti‐STING (R&D Systems, AF6516, 1:500), rabbit anti‐phosphoSTING (S366) (Cell Signaling Technology, #85,735, 1:1000), rabbit anti‐TBK1 (Cell Signaling, 3504, 1:1000), rabbit anti‐phospho-TBK1 (Ser172) (Cell Signaling, D52C2/#5483, 1:1000), IRF3 (Cell signaling, 11,904, 1:1000), phospho-IRF3 (S396) (Cell Signalling, 4947,1:1000, mouse anti‐p62 (Cell Signaling, # 88,588, 1:1000), anti-STEEP (Proteintech, 24,021–1-AP, 1:1000), anti-STIM1 (D88E10, Cell Signaling, #5668, 1:1000), rabbit anti‐LC3 (Cell Signaling, #3868, 1:1000), rabbit anti-cleaved caspase3 (Cell Signaling, #9664,1:1000), and mouse anti‐vinculin (Sigma, #V9131, 1:10,000).

Techniques: Transfection, Imaging, Staining, Incubation, Binding Assay, Immunoprecipitation, Western Blot, Activity Assay, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

doi: 10.4049/jimmunol.2101054

Figure Lengend Snippet: FIGURE 4. RelB deficiency triggers elevated basal expressions of T1-IFNs involving IRF7-mediated positive autoregulation. (A) GSEA comparing WT and Relb−/−

Article Snippet: Anti-IRF7 Ab (AHP-1180) was from Bio-Rad Laboratories.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

doi: 10.4049/jimmunol.2101054

Figure Lengend Snippet: FIGURE 5. T1-IFNs and IRF7 cooperatively restrict viral multiplication in Relb−/−cells. Plaque assay reveal- ing the titer of progeny virus particles produced by WT and Relb−/−cells, including primary MEFs (A) or BMDMs (C, left panel) or immortalized MEFs (C, right panel), upon infection for 12 h with the indicated MOI of CHPV. (B) RT-qPCR showing the relative abundance of viral genomic RNA and mRNAs encoding viral nucleo- capsid protein and phosphoprotein in WT and Relb−/−

Article Snippet: Anti-IRF7 Ab (AHP-1180) was from Bio-Rad Laboratories.

Techniques: Plaque Assay, Virus, Produced, Infection, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

doi: 10.4049/jimmunol.2101054

Figure Lengend Snippet: FIGURE 6. Vitamin D modulates the T1-IFNIRF7 pathway through RelB to exert antiviral effects. Vitamin D naive or vitamin Dconditioned WT or Relb−/−primary MEFs were examined for the relative abundance of IRF7 mRNA (A) and protein (B). Similarly, vitamin Dinduced expressions of IFN-b mRNA were scored in Relb−/−MEFs (C) and immortalized IRF7DCRISPR MEFs (E). Virus propaga- tion was scored in vitamin D naive and vitamin Dconditioned Relb−/−MEFs (D) or immortalized IRF7DCRISPR MEFs (F) subsequent to cell infection with 0.1 or 2 MOI of CHPV. The RT-qPCR data represent mean ± SEM of three indepen- dent experiments. The immunoblots represent three experi- mental replicates. The plaque assay data represent mean ± SEM of four biological replicates. *p# 0.05, **p# 0.01.

Article Snippet: Anti-IRF7 Ab (AHP-1180) was from Bio-Rad Laboratories.

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