Journal: bioRxiv

Article Title: The most common human ADAR1p150 Zα domain mutation P193A is well tolerated in mice

doi: 10.1101/2022.06.24.497437

Figure Lengend Snippet: A) Schematic outline of how the HOXA9 immortalised myeloid cell lines are derived. B) Experimental outline. When tamoxifen is added to the cells this activates deletion of the floxed Adar1 allele leaving the cells either heterozygous (Controls; 1′/+) or P195A only expressing (1′/P195A). C) Genomic DNA genotyping demonstrating efficient recombination of the floxed Adar1 allele following tamoxifen treatment. D) Proliferation of cell lines of the indicated genotypes with and without tamoxifen treatment (isogenic pairs). Cells were counted on a Countess II cell counter. E) Cell viability of cell lines of the indicated genotypes with and without tamoxifen treatment (isogenic pairs) assessed by trypan blue staining and on a Countess II cell counter. F) qPCR (SYBR green) based analysis of Ifit1 and Irf7 expression in the cell lines collected at day 14 of analysis. Data expressed as mean +/- SEM gene expression relative to Ppia expression. G) qPCR (SYBR green) based analysis of Ifit1 , Irf7 and Ifnb expression in the cell lines transfected with the indicated dose of high molecular weight Polyinosinic-polycytidylic acid (polyI:C). Data expressed as mean +/- SEM gene expression relative to Ppia expression. Statistical analysis by t-test using individually calculated AUC per sample. Cell lines had been treated for 14 days with tamoxifen, tamoxifen withdrawn, genotyped, then used for polyI:C dose response. Data from three independently derived cell lines (different donor bone marrow) for each genotype.

Article Snippet: The cells were treated with a dose range of high molecular weight poly(I:C) (1.5-8kb; 1mg/mL stock) by nucleofection following manufacturer’s instructions (Lonza 4D-Nucleofector TM Kits).

Techniques: Derivative Assay, Expressing, Staining, SYBR Green Assay, Transfection, Molecular Weight

Journal: Frontiers in Immunology

Article Title: Function conservation and disparities of zebrafish and human LGP2 genes in fish and mammalian cells responsive to poly(I:C)

doi: 10.3389/fimmu.2022.985792

Figure Lengend Snippet: Zebrafish and human LGP2s play antithetic roles in regulating IFN response by poly(I:C) in fish cells (A–C) DrLGP2 and HsLGP2 downregulated fish IFN and human IFN promoter activation by poly(I:C) at a high concentration of 1 μg/ml in EPC cells. EPC cells seeded in 24-wells plates were co-transfected with DrIFNφ1pro-luc or CaIFNpro-luc (A, B) , or HsIFNβpro-luc (200 ng each) (C) , together with DrLGP2 or HsLGP2 at increasing doses (0, 10, 50, 100, 200 ng). 24 h later, cells were transfected with 1 μg/ml poly(I:C) for another 24 h, followed by luciferase assays. P values were calculated using ANOVA. ** P < 0.01, *P < 0.05. (D–G) DrLGP2 and HsLGP2 switched a first positive role to a following negative one in regulating IFN response by increasing concentrations of poly(I:C) in EPC cells. EPC cells seeded overnight in 24-wells plates were co-transfected with DrIFNφ1pro-luc or CaIFNpro-luc (D, E) , or HsIFNβpro-luc (F, G) , together with DrLGP2 (D, F) or HsLGP2 (E, G) (200 ng each). 24 h later, cells were transfected with poly(I:C) at increasing doses for another 24 h, followed by luciferase assays. P values were calculated using Student’s t-test. ** P <0.01. (H) Agarose electrophoresis showed the molecular weight spanning of MMW poly(I:C) and HMW poly(I:C). (I) RNA pull-down assays verified the binding of poly(I:C) to DrLGP2 and HsLGP2 in fish cells. CO cells seeded in 10 cm dishes were transfected with DrLGP2-HA, HsLGP2-HA or GFP-HA as control. 24 h later, cells were lysed. One-tenth of cell lysates were taken as input, the remaining was incubated with 100 ng biotinylated poly(I:C), followed by western blots with anti-HA antibody.

Article Snippet: The medium molecular weight (MMW) poly(I:C) was purchased from SIGMA (Catalog no. I3036), and the high molecular weight (HMW) poly(I:C) from Enzo Life Sciences (Catalog no. ALX-746-021).

Techniques: Activation Assay, Concentration Assay, Transfection, Luciferase, Electrophoresis, Molecular Weight, Binding Assay, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: Function conservation and disparities of zebrafish and human LGP2 genes in fish and mammalian cells responsive to poly(I:C)

doi: 10.3389/fimmu.2022.985792

Figure Lengend Snippet: Zebrafish and human LGP2s play antithetic roles under low concentrations of poly(I:C) in mammalian cells and do so alone in fish cells. (A–E) Zebrafish and human LGP2s played antithetic roles under low concentrations of poly(I:C) in mammalian cells. HEK293T cells seeded in 24-wells plates were co-transfected with HsIFNβpro-luc (200ng), together with HsLGP2 (A–D) or DrLGP2 (E) (200 ng each). 24h later, cells were transfected again with MMW poly(I:C) [indicated as poly(I:C) in the text or all Figures] at 2 μg/ml (B, E) or at 4 ng/ml (C) , or with HMW poly(I:C) at 4 ng/ml (D) . Renilla vector (pRL-TK, 0.2 ng) was transfected as internal control. Another 24 h later, cells were collected for luciferase assays. P values were calculated using ANOVA. ** P < 0.01. (F) Overexpression of zebrafish or human LGP2s alone revealed antithetic roles in mammalian cells. EPC cells seeded in 24-well plates were transfected with DrLGP2 at the indicated increasing doses for 48 h. Or at 24 h post transfection, cells were transfected again with poly(I:C) at a high concentration of 1 μg/ml for another 24 h, followed by luciferase assays. P values were calculated using ANOVA. ** P < 0.01.

Article Snippet: The medium molecular weight (MMW) poly(I:C) was purchased from SIGMA (Catalog no. I3036), and the high molecular weight (HMW) poly(I:C) from Enzo Life Sciences (Catalog no. ALX-746-021).

Techniques: Transfection, Plasmid Preparation, Luciferase, Over Expression, Concentration Assay