USP22 controls type III interferon signaling and SARS-CoV-2 infection through activation of STING

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1. Version published to 10.1038/s41419-022-05124-w

   Aug 6, 2022
2. 

   ScreenIT

   Feb 5, 2022

   SciScore for 10.1101/2022.02.01.478628: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: All cell lines were regularly negatively tested for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The following antibodies are used: rabbit anti-STING (13647S, Cell Signaling Beverly, MA, USA), rabbit anti-phospho-STAT1 (9167L, Cell Signaling), mouse anti-STAT1 (9176S, Cell signaling), rabbit anti-USP22 (#ab195298, Abcam), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (5G4cc, HyTest, Turku, Finland), mouse anti-Vinculin (#V9131-100UL, Merck), rabbit anti-TBK1 (ab40676, Abcam), rabbit anti-phospho-TBK1 …

   SciScore for 10.1101/2022.02.01.478628: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: All cell lines were regularly negatively tested for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The following antibodies are used: rabbit anti-STING (13647S, Cell Signaling Beverly, MA, USA), rabbit anti-phospho-STAT1 (9167L, Cell Signaling), mouse anti-STAT1 (9176S, Cell signaling), rabbit anti-USP22 (#ab195298, Abcam), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (5G4cc, HyTest, Turku, Finland), mouse anti-Vinculin (#V9131-100UL, Merck), rabbit anti-TBK1 (ab40676, Abcam), rabbit anti-phospho-TBK1 (ab109272, Abcam), rabbit anti-Histone H2B (#07-371, Merck), mouse anti-Ubiquityl-Histone H2B (#05-1312, Merck)	anti-STING suggested: (Cell Signaling Technology Cat# 13647, RRID:AB_2732796) anti-phospho-STAT1 suggested: None anti-STAT1 suggested: (Cell Signaling Technology Cat# 9176, RRID:AB_2240087) anti-USP22 suggested: None anti-glyceraldehyde 3-phosphate dehydrogenase suggested: None GAPDH suggested: (Millipore Cat# 17-650, RRID:AB_1977238) anti-Vinculin ( #V9131-100UL , Merck) suggested: None anti-TBK1 suggested: (Abcam Cat# ab40676, RRID:AB_776632) anti-phospho-TBK1 suggested: (Abcam Cat# ab109272, RRID:AB_10862438) anti-Histone H2B suggested: (Millipore Cat# 07-371, RRID:AB_310561) anti-Ubiquityl-Histone H2B suggested: (Millipore Cat# 05-1312, RRID:AB_1587119)
   Infected cells were stained with 1:1000 diluted anti-dsRNA (J2) for 1 h at room temperature, washed three times with 0.1 % PBT-T, followed by incubation with secondary antibody (anti-mouse CW800) and DNA dye Draq5 (Abcam, Cambridge, UK), diluted 1:10000 in blocking buffer and incubated for 1 h at room temperature.	anti-dsRNA (J2 suggested: (SCICONS Cat# 10010200, RRID:AB_2651015) anti-mouse CW800 suggested: None Draq5 suggested: (Biostatus Cat# DR50050, RRID:AB_2314341)
   Cells were washed in 1X PBS three times and incubated with secondary antibodies goat-anti mouse Alexa Fluor 568 and DAPI for 45 mins at RT.	mouse suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture, reagents and chemicals: The human colon carcinoma cell line HT-29 was obtained from DSMZ (Braunschweig, Germany) and cultivated in McCoy’s 5A Medium GlutaMAXTM-I (Life Technologies, Inc., Eggenstein, Germany), supplemented with 10 % fetal calf serum (FCS) (Biochrom, Ltd., Berlin, Germany) and 1 % penicillin-streptomycin (Invitrogen).	HT-29 suggested: None
   For the generation of viral particles, multiple gene-specific gRNAs were combined and co-transfected with pMD2.G (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260) in HEK293T cells using FuGENE HD Transfection Reagent (Promega), according to the manufacturer’s protocol.	HEK293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   TCID50 virus titration: Vero E6 cells were seeded (20000 per well) in 96-well plates 24 h prior to infection.	Vero E6 suggested: None
   A volume of 100 µl of viral supernatant from the indicated SARS-CoV-2-infected Caco-2 cells was added to the first well.	Caco-2 suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   SARS-CoV-2 infection: SARS-CoV-2 (strain BavPat1/2020) was obtained from the European Virology Archive and amplified in Vero E6 cells and used at passage 3.	SARS-CoV-2 suggested: None
   Recombinant DNA
   Sentences	Resources
   Addgene plasmid #51763, #51762 and #51760) were ligated into pLentiCRISPRv2 (Addgene plasmid # 52961) using restriction cloning.	pLentiCRISPRv2 suggested: RRID:Addgene_127644)
   For the generation of viral particles, multiple gene-specific gRNAs were combined and co-transfected with pMD2.G (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260) in HEK293T cells using FuGENE HD Transfection Reagent (Promega), according to the manufacturer’s protocol.	pMD2 . G suggested: None psPAX2 suggested: RRID:Addgene_12260)
   Software and Algorithms
   Sentences	Resources
   Genes with differential expression between NHT control and USP22 KO have been identified using the linear model-based approach limma R package 85.	limma suggested: (LIMMA, RRID:SCR_010943)
   Gene-set enrichment analysis was performed with gage R package 86 using the MSigDB 87 as gene set repository.	gage suggested: (GAGE, RRID:SCR_017067)
   Statistical analysis: Significance was assessed using Student’s t-test (two-tailed distribution, two-sample, equal variance) using Microsoft Excel, unless indicated otherwise.	Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137)
   Data and code availability: Microarray data are available on Gene Expression Omnibus under the accession number GSE190036.	Gene Expression Omnibus suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2022.02.01.478628 on bioRxiv

   Feb 2, 2022