Journal: Nature Communications

Article Title: A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

doi: 10.1038/s41467-024-52803-7

Figure Lengend Snippet: a WT and STING KO SCC25, H596, OE21, and Detroit 562 cells were subjected to RNA-seq analysis. Heatmap shows differentially expressed genes (absolute log 2 fold change of > 2 and an adjusted q value of < 0.05 from n = 4 independent replicates for each cell type) in the IFN and inflammatory pathways in any given cell line between the WT vs. STING KO variant. b–g RT-qPCR analysis of IFIT2, MX1 and IFIT3 in uninfected SCC25, H596, OE21 and Detroit 562 cells treated with increasing concentrations of SN-011 ( b – d ) and H-151 ( e – g ) for 48 h. Data show the mean from n = 2 independent biological replicates, error bars show SEM ( h , i ) WT and STING KO SCC25 and STING KO cells were pre-treated with 20 μM SN-011 ( h ) or H-151 ( i ) for 24 h were infected with WT SARS-CoV-2 at MOI: 1 i.u./cell for 72 h. Data show the copies of cell-associated SARS-CoV-2 N RNA in compound-treated cells normalized relative to mock-treated samples. Data show the mean from n = 3 independent biological replicates, error bars show SEM. Significance was assessed by multiple unpaired two-tailed t tests and p- values provided in the figure. Source data are provided as a Source Data file.

Article Snippet: After washing with PBST, samples were incubated in a goat anti-mouse secondary antibody conjugated to Alexa Fluor Plus 488 (Invitrogen, Cat# A-11029, 1:1000) at room temperature for 1 h. IFIT3, IFIT2, ISG15 and STING were detected by incubation with a primary rabbit polyclonal IFIT3 antibody (Novus Biologicals NBP2-32500, 1:500), rabbit polyclonal IFIT2 antibody (Novus Biologicals NBP2-15180SS, 1:500), rabbit polyclonal ISG15 antibody (Proteintech 15981-1-AP, 1:250) and rabbit polyclonal STING antibody (Proteintech 19851-1-AP, 1:200) respectively as described above followed by incubation with a goat anti-rabbit fluorescent secondary antibody (Invitrogen Goat anti-Rabbit Alexa Fluor Plus 568TM, Cat# A-11036, 1:1000 or Invitrogen Goat anti-Rabbit Alexa FluorTM Plus 647, Catalog # A32733TR, 1:1000).

Techniques: RNA Sequencing, Variant Assay, Quantitative RT-PCR, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

doi: 10.1038/s41467-024-52803-7

Figure Lengend Snippet: a Relative expression of ISGs including ISG15, IFIT1, IFIT2, IFIT3, and IRF7 in SCC25 cGAS KO, STING KO or NT control cells infected with SARS-CoV-2 at a MOI of 2 i.u./cell. Cells were collected at 72 hpi, and ISG expression was analyzed by RT-qPCR. Data show the fold induction of ISGs in infected over uninfected cells from n = 3 biological replicates, errors bars show the SEM. b , c SCC25 STING KO or NT control cells were infected with SARS-CoV-2 at an MOI of 2 i.u./cell. Immunofluorescence detection for IFIT3 expression (in red) and SARS-CoV-2 nucleocapsid (N) viral protein (in green), and cellular nuclei (DAPI, in blue) at 72 hpi from a representative experiment ( n = 2) are shown. Images were collected with an epifluorescence microscope, 4X objective ( b ), or Zeiss LSM 880 Airyscan confocal microscope equipped with a × 63/1.4 objective ( c ) as detailed in Methods. Scale bars = 250 μm ( b ) or 10 μm ( c ). d , e SCC25 STING KO cells were infected with SARS-CoV-2-mNG at MOI:1 i.u./cell, and cells were single-cell sorted at 72 hpi to isolate infected (mNG positive) and bystander cells as detailed in Methods. Expression of the indicated ISGs ( d ) and inflammatory genes ( e ) were analyzed by RT-qPCR in these sorted cell populations. Data show the mean from two independent replicates, error bars show the SEM. Source data are provided as a Source Data file.

Article Snippet: After washing with PBST, samples were incubated in a goat anti-mouse secondary antibody conjugated to Alexa Fluor Plus 488 (Invitrogen, Cat# A-11029, 1:1000) at room temperature for 1 h. IFIT3, IFIT2, ISG15 and STING were detected by incubation with a primary rabbit polyclonal IFIT3 antibody (Novus Biologicals NBP2-32500, 1:500), rabbit polyclonal IFIT2 antibody (Novus Biologicals NBP2-15180SS, 1:500), rabbit polyclonal ISG15 antibody (Proteintech 15981-1-AP, 1:250) and rabbit polyclonal STING antibody (Proteintech 19851-1-AP, 1:200) respectively as described above followed by incubation with a goat anti-rabbit fluorescent secondary antibody (Invitrogen Goat anti-Rabbit Alexa Fluor Plus 568TM, Cat# A-11036, 1:1000 or Invitrogen Goat anti-Rabbit Alexa FluorTM Plus 647, Catalog # A32733TR, 1:1000).

Techniques: Expressing, Control, Infection, Quantitative RT-PCR, Immunofluorescence, Microscopy

Journal: Nature Communications

Article Title: A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

doi: 10.1038/s41467-024-52803-7

Figure Lengend Snippet: a WT and STING KO SCC25, H596, OE21, and Detroit 562 cells were subjected to RNA-seq analysis. Heatmap shows differentially expressed genes (absolute log 2 fold change of > 2 and an adjusted q value of < 0.05 from n = 4 independent replicates for each cell type) in the IFN and inflammatory pathways in any given cell line between the WT vs. STING KO variant. b–g RT-qPCR analysis of IFIT2, MX1 and IFIT3 in uninfected SCC25, H596, OE21 and Detroit 562 cells treated with increasing concentrations of SN-011 ( b – d ) and H-151 ( e – g ) for 48 h. Data show the mean from n = 2 independent biological replicates, error bars show SEM ( h , i ) WT and STING KO SCC25 and STING KO cells were pre-treated with 20 μM SN-011 ( h ) or H-151 ( i ) for 24 h were infected with WT SARS-CoV-2 at MOI: 1 i.u./cell for 72 h. Data show the copies of cell-associated SARS-CoV-2 N RNA in compound-treated cells normalized relative to mock-treated samples. Data show the mean from n = 3 independent biological replicates, error bars show SEM. Significance was assessed by multiple unpaired two-tailed t tests and p- values provided in the figure. Source data are provided as a Source Data file.

Article Snippet: After washing with PBST, samples were incubated in a goat anti-mouse secondary antibody conjugated to Alexa Fluor Plus 488 (Invitrogen, Cat# A-11029, 1:1000) at room temperature for 1 h. IFIT3, IFIT2, ISG15 and STING were detected by incubation with a primary rabbit polyclonal IFIT3 antibody (Novus Biologicals NBP2-32500, 1:500), rabbit polyclonal IFIT2 antibody (Novus Biologicals NBP2-15180SS, 1:500), rabbit polyclonal ISG15 antibody (Proteintech 15981-1-AP, 1:250) and rabbit polyclonal STING antibody (Proteintech 19851-1-AP, 1:200) respectively as described above followed by incubation with a goat anti-rabbit fluorescent secondary antibody (Invitrogen Goat anti-Rabbit Alexa Fluor Plus 568TM, Cat# A-11036, 1:1000 or Invitrogen Goat anti-Rabbit Alexa FluorTM Plus 647, Catalog # A32733TR, 1:1000).

Techniques: RNA Sequencing, Variant Assay, Quantitative RT-PCR, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

doi: 10.1038/s41467-024-52803-7

Figure Lengend Snippet: a Relative expression of ISGs including ISG15, IFIT1, IFIT2, IFIT3, and IRF7 in SCC25 cGAS KO, STING KO or NT control cells infected with SARS-CoV-2 at a MOI of 2 i.u./cell. Cells were collected at 72 hpi, and ISG expression was analyzed by RT-qPCR. Data show the fold induction of ISGs in infected over uninfected cells from n = 3 biological replicates, errors bars show the SEM. b , c SCC25 STING KO or NT control cells were infected with SARS-CoV-2 at an MOI of 2 i.u./cell. Immunofluorescence detection for IFIT3 expression (in red) and SARS-CoV-2 nucleocapsid (N) viral protein (in green), and cellular nuclei (DAPI, in blue) at 72 hpi from a representative experiment ( n = 2) are shown. Images were collected with an epifluorescence microscope, 4X objective ( b ), or Zeiss LSM 880 Airyscan confocal microscope equipped with a × 63/1.4 objective ( c ) as detailed in Methods. Scale bars = 250 μm ( b ) or 10 μm ( c ). d , e SCC25 STING KO cells were infected with SARS-CoV-2-mNG at MOI:1 i.u./cell, and cells were single-cell sorted at 72 hpi to isolate infected (mNG positive) and bystander cells as detailed in Methods. Expression of the indicated ISGs ( d ) and inflammatory genes ( e ) were analyzed by RT-qPCR in these sorted cell populations. Data show the mean from two independent replicates, error bars show the SEM. Source data are provided as a Source Data file.

Article Snippet: After washing with PBST, samples were incubated in a goat anti-mouse secondary antibody conjugated to Alexa Fluor Plus 488 (Invitrogen, Cat# A-11029, 1:1000) at room temperature for 1 h. IFIT3, IFIT2, ISG15 and STING were detected by incubation with a primary rabbit polyclonal IFIT3 antibody (Novus Biologicals NBP2-32500, 1:500), rabbit polyclonal IFIT2 antibody (Novus Biologicals NBP2-15180SS, 1:500), rabbit polyclonal ISG15 antibody (Proteintech 15981-1-AP, 1:250) and rabbit polyclonal STING antibody (Proteintech 19851-1-AP, 1:200) respectively as described above followed by incubation with a goat anti-rabbit fluorescent secondary antibody (Invitrogen Goat anti-Rabbit Alexa Fluor Plus 568TM, Cat# A-11036, 1:1000 or Invitrogen Goat anti-Rabbit Alexa FluorTM Plus 647, Catalog # A32733TR, 1:1000).

Techniques: Expressing, Control, Infection, Quantitative RT-PCR, Immunofluorescence, Microscopy

Journal: Nature Communications

Article Title: A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

doi: 10.1038/s41467-024-52803-7

Figure Lengend Snippet: a WT and STING KO SCC25, H596, OE21, and Detroit 562 cells were subjected to RNA-seq analysis. Heatmap shows differentially expressed genes (absolute log 2 fold change of > 2 and an adjusted q value of < 0.05 from n = 4 independent replicates for each cell type) in the IFN and inflammatory pathways in any given cell line between the WT vs. STING KO variant. b–g RT-qPCR analysis of IFIT2, MX1 and IFIT3 in uninfected SCC25, H596, OE21 and Detroit 562 cells treated with increasing concentrations of SN-011 ( b – d ) and H-151 ( e – g ) for 48 h. Data show the mean from n = 2 independent biological replicates, error bars show SEM ( h , i ) WT and STING KO SCC25 and STING KO cells were pre-treated with 20 μM SN-011 ( h ) or H-151 ( i ) for 24 h were infected with WT SARS-CoV-2 at MOI: 1 i.u./cell for 72 h. Data show the copies of cell-associated SARS-CoV-2 N RNA in compound-treated cells normalized relative to mock-treated samples. Data show the mean from n = 3 independent biological replicates, error bars show SEM. Significance was assessed by multiple unpaired two-tailed t tests and p- values provided in the figure. Source data are provided as a Source Data file.

Article Snippet: Samples were then incubated in a primary rabbit polyclonal antibody against IFIT3 (Novus Biologicals, NBP2-32500, 1:500) at 4 °C overnight, followed by staining with a goat anti-rabbit Alexa Fluor Plus 647 (Life Technologies, A32733TR, 1:1000) for 1 h at room temperature.

Techniques: RNA Sequencing, Variant Assay, Quantitative RT-PCR, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

doi: 10.1038/s41467-024-52803-7

Figure Lengend Snippet: a Relative expression of ISGs including ISG15, IFIT1, IFIT2, IFIT3, and IRF7 in SCC25 cGAS KO, STING KO or NT control cells infected with SARS-CoV-2 at a MOI of 2 i.u./cell. Cells were collected at 72 hpi, and ISG expression was analyzed by RT-qPCR. Data show the fold induction of ISGs in infected over uninfected cells from n = 3 biological replicates, errors bars show the SEM. b , c SCC25 STING KO or NT control cells were infected with SARS-CoV-2 at an MOI of 2 i.u./cell. Immunofluorescence detection for IFIT3 expression (in red) and SARS-CoV-2 nucleocapsid (N) viral protein (in green), and cellular nuclei (DAPI, in blue) at 72 hpi from a representative experiment ( n = 2) are shown. Images were collected with an epifluorescence microscope, 4X objective ( b ), or Zeiss LSM 880 Airyscan confocal microscope equipped with a × 63/1.4 objective ( c ) as detailed in Methods. Scale bars = 250 μm ( b ) or 10 μm ( c ). d , e SCC25 STING KO cells were infected with SARS-CoV-2-mNG at MOI:1 i.u./cell, and cells were single-cell sorted at 72 hpi to isolate infected (mNG positive) and bystander cells as detailed in Methods. Expression of the indicated ISGs ( d ) and inflammatory genes ( e ) were analyzed by RT-qPCR in these sorted cell populations. Data show the mean from two independent replicates, error bars show the SEM. Source data are provided as a Source Data file.

Article Snippet: Samples were then incubated in a primary rabbit polyclonal antibody against IFIT3 (Novus Biologicals, NBP2-32500, 1:500) at 4 °C overnight, followed by staining with a goat anti-rabbit Alexa Fluor Plus 647 (Life Technologies, A32733TR, 1:1000) for 1 h at room temperature.

Techniques: Expressing, Control, Infection, Quantitative RT-PCR, Immunofluorescence, Microscopy

Journal: Cell Reports

Article Title: CK2 phosphorylation of CMTR1 promotes RNA cap formation and influenza virus infection

doi: 10.1016/j.celrep.2024.114405

Figure Lengend Snippet:

Article Snippet: Anti-IFIT3 rabbit polyclonal , Proteintech , 15201-1-AP; RRID:AB_2248738.

Techniques: Recombinant, Transduction, RNA Sequencing, Software