TMPRSS2 expression dictates the entry route used by SARS‐CoV‐2 to infect host cells

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1. Version published to 10.15252/embj.2021107821

   Jul 13, 2021
2. 

   ScreenIT

   Dec 27, 2020

   SciScore for 10.1101/2020.12.22.423906: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibodies, reagents, and plasmids: The mouse mAb against the SARS-CoV nucleoprotein NP (40143-MM05) was purchased from Sino biologicals and used at dilutions of 1:500 for flow cytometry analysis and 1:1,000 for titration in TCID50 assays.	SARS-CoV nucleoprotein NP suggested: (Sino Biological Cat# 40143-MM05, RRID:AB_2827977)
   The rabbit antibodies against TMPRSS2 (ab92323) and actin (A2066) were obtained from Abcam and Sigma, respectively.	TMPRSS2 suggested: …

   SciScore for 10.1101/2020.12.22.423906: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibodies, reagents, and plasmids: The mouse mAb against the SARS-CoV nucleoprotein NP (40143-MM05) was purchased from Sino biologicals and used at dilutions of 1:500 for flow cytometry analysis and 1:1,000 for titration in TCID50 assays.	SARS-CoV nucleoprotein NP suggested: (Sino Biological Cat# 40143-MM05, RRID:AB_2827977)
   The rabbit antibodies against TMPRSS2 (ab92323) and actin (A2066) were obtained from Abcam and Sigma, respectively.	TMPRSS2 suggested: (Abcam Cat# ab92323, RRID:AB_10585592) actin suggested: (Sigma-Aldrich Cat# A2066, RRID:AB_476693)
   Anti-rabbit secondary antibodies conjugated to IRDye 800CW were purchased from LI-COR.	Anti-rabbit suggested: None
   The membranes were first blocked with Intercept blocking buffer (LI-COR) and then incubated with primary antibodies against TMPRSS2, cathepsin L, actin, and α-tubulin, all diluted in Tris-buffered saline containing 0.1% Tween and Intercept blocking buffer (1:1,000, 1:400, 1:5,000, and 1:2,000, respectively).	cathepsin L , actin , suggested: None α-tubulin suggested: None
   Cells were then exposed to primary antibody at RT for 1 h, washed, and subsequently incubated with secondary anti-mouse antibodies at RT for another 1 h.	anti-mouse suggested: None
   Infected cells were fixed 24 hpi and subjected to immunostaining using the primary mouse mAb anti-SARS-CoV-2 NP and then the secondary anti-mouse antibody 800CW (1:10,000).	anti-SARS-CoV-2 suggested: None NP suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cells: The African green monkey Vero kidney epithelial cells (ATCC CRL 1586), the human Caco-2 colorectal adenocarcinoma (ATCC HTB-37), the human Calu-3 lung adenocarcinoma (ATCC HTB-55), and the human epithelial lung cells A549 stably expressing ACE2 (A549*; a kind gift from Prof. Ralf Bartenschlager) were all maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 units.mL−1 penicillin, and 100 μg.mL−1 streptomycin.	Calu-3 suggested: ATCC Cat# HTB-55, RRID:CVCL_0609) A549 suggested: None
   Baby hamster kidney cells (BHK-21) were grown in Glasgow’s minimal essential medium containing 10% tryptose phosphate broth, 5% FBS, 100 units.mL−1 penicillin, and 100 μg.mL−1 streptomycin.	BHK-21 suggested: None
   DNA transfection: As previously described (56), Vero cells were transfected with 750 ng of plasmids using Lipofectamine 2000 (Invitrogen) in 24-well-plates according to the manufacturer’s recommendations and washed 5 h later.	Vero suggested: None
   Virus titration by TCID50 assay: Confluent monolayers of Vero and Caco-2 cells in 96-well plates were infected with 10-fold serial dilutions of SARS-CoV-2.	Caco-2 suggested: None
   Software and Algorithms
   Sentences	Resources
   , Concanamycin B (BioViotica), SB412515 (Cayman Chemical), colcemid (Cayman Chemical), and MG-132 (Selleck Chemicals) were all dissolved in DMSO.	BioViotica suggested: None
   Infected cells were quantified with a FACSCelesta cytometer (Becton Dickinson) and FlowJo software (TreeStar)	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Graph plotting of numerical values, as well as the statistics, were achieved with GraphPad Prism v5.00 (GraphPad Software).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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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 did not find any issues relating to the usage of bar graphs.

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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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3. 

   Version published to 10.1101/2020.12.22.423906v1 on bioRxiv

   Dec 23, 2020