t7 mscript standard mrna production kit  (Cellscript Inc)

Journal: Cell Death and Differentiation

Article Title: Spatio-temporal characterization of the antiviral activity of the XRN1-DCP1/2 aggregation against cytoplasmic RNA viruses to prevent cell death

doi: 10.1038/s41418-020-0509-0

Figure Lengend Snippet: a XRN1, DCP1, or DCP2 was ectopically expressed in various cell types for 48 h, followed by infection with indicated MOI of EMCV for 18 h. Crystal violet staining was conducted to determine cell viability. b L-929 cells were ectopically expressed with 1.0 µg of indicated plasmids, followed by infection with EMCV at MOI of 0.1 or 1 for 18 h. The released virions were measured by plaque assay. c Wild-type (WT) iMEFs were transfected with 1.0 μM of siRNAs for 48 h, followed by infection with NDV or EMCV (MOI = 1) for indicated hours, NDV N mRNA was measured by RT-qPCR (left). Supernatant was collected from EMCV-infected cells and viral titers were measured by plaque assay (right); n.s = not significant. d Irf3 −/− iMEFs were transfected with indicated siRNAs for 48 h, followed by infection with NDV (MOI = 1) for 9 h. (i) NDV N mRNA was measured by RT-qPCR. (ii) NDV F (fusion) RNA in each condition was analyzed by northern blot. e After EMCV infection (MOI = 1.0) for 18 h, supernatant from siRNA-transfected Irf3 −/− iMEFs and U138 cells was collected. Viral titers were measured by plaque assay. P -value in a was calculated by two-way analysis of variance, and P- value in b – e was calculated by Student’s unpaired t -test.

Article Snippet: The NDV mature mRNA was in vitro synthesized as previously described [ ] using T7 mScript Standard mRNA kit (CellScript).

Techniques: Infection, Staining, Plaque Assay, Transfection, Quantitative RT-PCR, Northern Blot

Journal: Cell Death and Differentiation

Article Title: Spatio-temporal characterization of the antiviral activity of the XRN1-DCP1/2 aggregation against cytoplasmic RNA viruses to prevent cell death

doi: 10.1038/s41418-020-0509-0

Figure Lengend Snippet: a (i–ii) Confocal micrographs of XRN1 distribution in NDV-infected cells. HeLa cells were either mock treated or infected with NDV (MOI = 1.0) for 9 h. Immunostaining for endogenous XRN1, viral dsRNA, N proteins (NP), and polymerase (Pol) was conducted. (iii) Percentage of cells with XRN1 foci was quantified. b Huh-7 cells were either mock or SeV (MOI = 1.0) infected for 12 h, followed by immunostaining with indicated antibodies. Percentage of cells with foci was quantified. c HeLa cells were either mock treated or infected with PolioV or EMCV (MOI = 1.0) for 5 h, followed by immunostaining with indicated antibodies. White box for a specific region was enlarged. d U-2 OS cells stably expressing mRFP-DCP1a and EGFP-AGO1 were infected with NDV for 9 h. Immunostaining for viral NP was performed and percentage of cells with Dcp1a foci was quantified. e (i) U-2 OS stable cells were either mock treated or NDV infected for indicated time points. Cells were lysed for RNA Co-IP analysis and NDV N mRNA was evaluated by RT-qPCR. (ii) Similar IP experiments were performed using HeLa cells expressing mRFP-DCPa, which were infected with SeV for 6 h and immunoblotting was conducted with indicated antibodies. All the white scale bars correspond to 10 μm. n.d., not detected.

Article Snippet: The NDV mature mRNA was in vitro synthesized as previously described [ ] using T7 mScript Standard mRNA kit (CellScript).

Techniques: Infection, Immunostaining, Stable Transfection, Expressing, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Western Blot

Journal: Cell Death and Differentiation

Article Title: Spatio-temporal characterization of the antiviral activity of the XRN1-DCP1/2 aggregation against cytoplasmic RNA viruses to prevent cell death

doi: 10.1038/s41418-020-0509-0

Figure Lengend Snippet: a Schematic diagram of human DCP1 deletion and point mutants. b U138 cells were ectopically expressed with indicated plasmids at increased dosages of 0.1, 0.5, and 1.0 μg for 48 h, followed by either NDV or EMCV (MOI = 1.0) infection for 12 h and 24 h, respectively. NDV N and L mRNA was measured by RT-qPCR. Supernatant from EMCV-infected cells was collected for plaque assay. c U138 cells were overexpressed with indicated plasmids at the dosages of 0.1, 0.5, and 1.0 μg for 48 h, followed by NDV (MOI = 1.0) infection for 12 h. NDV N mRNA was assessed by RT-qPCR. Supernatant from U138 cells infected with EMCV (MOI = 0.1) was subjected to plaque assay. P -value was calculated by two-way analysis of variance analysis.

Article Snippet: The NDV mature mRNA was in vitro synthesized as previously described [ ] using T7 mScript Standard mRNA kit (CellScript).

Techniques: Infection, Quantitative RT-PCR, Plaque Assay

Journal: Cell Death and Differentiation

Article Title: Spatio-temporal characterization of the antiviral activity of the XRN1-DCP1/2 aggregation against cytoplasmic RNA viruses to prevent cell death

doi: 10.1038/s41418-020-0509-0

Figure Lengend Snippet: a U-2 OS EGFP-AGO1 and mRFP-DCP1a cells were either mock treated or NDV infected (MOI = 1.0) for 1 h. Cells were then exposed to UV (500 millisevert) or CHX (100 μg/mL). Cells were harvested for either (i) RT-qPCR to quantify NDV N mRNA, or (ii) immunostaining to visualize DCP1a foci. (iii) Percentage of cells with DCP1a foci was quantified. b gRNA from respective viruses was purified, followed by transfection into (i) WT and Rig-i − /− Mda5 −/− MEFs for Ifnb1 mRNA measurement by RT-qPCR, or (ii–iii) HeLa cells for immunostaining of endogenous XRN1 and quantitation of percentage of cells with XRN1 foci. c CEFs were either mock treated or infected with NDV and CVB4 (MOI = 1.0) for indicated time point. ss- and dsRNA were fractionated and analyzed on (i) agarose gel containing ethidium bromide; (ii) 7.5% PAGE by immunoblotting with anti-dsRNA antibody. d Fractionated viral ss- and dsRNA were transfected into HeLa cells and immunostained with indicated antibodies. e Percentage of cells with XRN1 aggregates was quantified. f (i) I.V.T viral mRNA was transfected into HeLa cells, followed by immunostaining for endogenous XRN1 and DCP1. (ii) Percentage of cells with XRN1-DCP1a foci was quantified. Nuclei were stained with DAPI (blue). All the white scale bars correspond to 10 μm. n.d., not detected.

Article Snippet: The NDV mature mRNA was in vitro synthesized as previously described [ ] using T7 mScript Standard mRNA kit (CellScript).

Techniques: Infection, Quantitative RT-PCR, Immunostaining, Purification, Transfection, Quantitation Assay, Agarose Gel Electrophoresis, Western Blot, Staining

Journal: Cell Death and Differentiation

Article Title: Spatio-temporal characterization of the antiviral activity of the XRN1-DCP1/2 aggregation against cytoplasmic RNA viruses to prevent cell death

doi: 10.1038/s41418-020-0509-0

Figure Lengend Snippet: a Total RNA extracted from mock- or NDV-infected HeLa cells was treated with or without XRN1 recombinant enzyme. cDNA was then synthesized. NDV N mRNA was measured by PCR and analyzed on 2% agarose gel. b In vitro synthesized RNA treated under indicated conditions for 12 h was analyzed on agarose gel containing PFA. c (i) In vitro synthesized RNA as indicated was transfected into HeLa cells for 12 h. Immunostaining was carried out with XRN1 antibody. (ii) Percentage of cells with XRN1 foci for indicated experimental conditions was quantified. d HeLa cells were transfected with (i) intact, or (ii) denatured HCV 5′-UTR RNA, followed by immunostaining with XRN1 antibody. (iii) Percentage of cells with XRN1 foci was quantified. All the white scale bars correspond to 10 μm. n.d., not detected. P -value was calculated by Student’s unpaired t-test by comparing to 5′-OH control.

Article Snippet: The NDV mature mRNA was in vitro synthesized as previously described [ ] using T7 mScript Standard mRNA kit (CellScript).

Techniques: Infection, Recombinant, Synthesized, Agarose Gel Electrophoresis, In Vitro, Transfection, Immunostaining, Control

Journal: Cell Death and Differentiation

Article Title: Spatio-temporal characterization of the antiviral activity of the XRN1-DCP1/2 aggregation against cytoplasmic RNA viruses to prevent cell death

doi: 10.1038/s41418-020-0509-0

Figure Lengend Snippet: a , b HeLa cells were transfected with indicated siRNAs for 48 h, followed by either arsenite or TNFα treatment at the indicated concentration. Cell viability was measured by MTS assay. c Knockdown efficiency of Bax and Bcl-2 gene was measured by qRT-PCR. d U138 cells were transfected with indicated siRNAs for 48 h. Cells were then infected with EMCV (MOI = 0, 0.01, 0.1, 2, 10, or 20) for 5 h, followed by MTS assay. e MDA5-deficient HeLa cells were generated using Crispr/Cas9 system. Knockout of MDA5 was confirmed by immunoblotting (left). These cells were transfected with indicated siRNAs for 48 h, followed by treatment with vehicle as negative controls or BIP-V5 peptide (100 μM). Cells were then infected with CVB3 (MOI = 0, 0.01, 0.1, 1, 5, 10, or 20) for 5 h and subjected to MTS assay. f MDA5-deficient HeLa cells were transfected with indicated siRNAs for 48 h. Cells were then either treated with dimethyl sulfoxide (DMSO, vehicle), caspase-1 inhibitor (50 μ M), or glycine (5 mM), followed by mock or CVB3 infection (MOI = 1) for 5 h. Supernatant was collected to measure LDH release. g Schematic diagram indicating that, during quiescent state, decapping enzyme DCP1/2 and host 5′–3′ exonuclease XRN1 are mainly localized in PB. Upon viral infection, viral gRNA released from the virion initiates viral replication process, generating copies of different vRNA species (e.g., ssRNA, dsRNA, and mRNA) in various length or with 5′- or 3′-end modifications. The presence of both ss-vRNA and ds-vRNA with the minimum length of at least 100 nt, or multistem loop structure might trigger the specific relocation (blue arrow) of XRN1 and DCP1/2 decapping enzymes into viral replication complexes for vRNA degradation. Consequently, virus-induced, caspase-1-dependent pyroptosis, and Bax-dependent apoptosis are inhibited. (Statistical analyses in c , and f are unpaired Student’s t -test).

Article Snippet: The NDV mature mRNA was in vitro synthesized as previously described [ ] using T7 mScript Standard mRNA kit (CellScript).

Techniques: Transfection, Concentration Assay, MTS Assay, Knockdown, Quantitative RT-PCR, Infection, Generated, CRISPR, Knock-Out, Western Blot, Virus