Inhibition of SARS-CoV-2 Infection by Human Defensin HNP1 and Retrocyclin RC-101

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1. Version published to 10.1016/j.jmb.2021.167225

   Mar 1, 2022
2. 

   ScreenIT

   May 29, 2021

   SciScore for 10.1101/2021.05.27.445985: (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	Authentication: The identity and purity of the U2OS cells were verified by STR profiling (Amelogenin + 9 loci) at the Genomic Shared Resource (OSU, Comprehensive Cancer Center) with 100% match using The Cellosaurus cell line database 63. Contamination: All cell lines were mycoplasma-negative as determined by a PCR-based approach 64.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After 24 h, cells were trypsinized, fixed with 4% paraformaldehyde for 1 h at room temperature, permeabilized with 0.1% Triton X100 in PBS, and stained with …

   SciScore for 10.1101/2021.05.27.445985: (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	Authentication: The identity and purity of the U2OS cells were verified by STR profiling (Amelogenin + 9 loci) at the Genomic Shared Resource (OSU, Comprehensive Cancer Center) with 100% match using The Cellosaurus cell line database 63. Contamination: All cell lines were mycoplasma-negative as determined by a PCR-based approach 64.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After 24 h, cells were trypsinized, fixed with 4% paraformaldehyde for 1 h at room temperature, permeabilized with 0.1% Triton X100 in PBS, and stained with anti-SARS-CoV-2 N protein antibody (Sino Biological, Wayne, PA; #40143-MM08).	anti-SARS-CoV-2 N protein suggested: None
   Primary antibody labeling was followed by goat anti-mouse Alexa Fluor 647 secondary antibody (Life Technologies, Carlsbad, CA)	anti-mouse suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   H1299, Calu-3, Vero E6, and HEK293T cells were purchased from ATCC.	HEK293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
   For a negative control, U2OS cells were transfected with pmCherry alone.	U2OS suggested: CLS Cat# 300364/p489_U-2_OS, RRID:CVCL_0042)
   24-hours post-transfection, U2OS cells were trypsinized, mixed with Calu-3 cells on 96-well µ-plate (Ibidi, Germany), and incubated for 24 h in the presence and absence of HNP1 or RC-101 (40 µM; final concentration) with and without FBS (10%; final concentration).	Calu-3 suggested: None
   H1299 cells were incubated with the pseudovirus for 24 h followed by luciferase assays performed using NanoGlo Luciferase Assay System (Promega, Madison, WI).	H1299 suggested: None
   The virus stock was titered on Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
   Recombinant DNA
   Sentences	Resources
   Lentiviral NanoLuc expression vector (a gift from Erich Wanker; Addgene plasmid #113450; RRID:Addgene_113450) 62 was modified to introduce IL6 secretion signal (IL6ss) upstream of Myc-Nluc using PCR-based approach with NEBuilder DNA assembly (New England Biolabs, Ipswich, MA)	detected: RRID:Addgene_113450)
   , U2OS cells were co-transfected with pmCherry and pCAGGS vector encoding SARS-CoV-2 S protein.	pCAGGS suggested: RRID:Addgene_18926)
   Pseudotyped virus production, cell infection, and luciferase assays: Virus-like particles pseudotyped with SARS-CoV-2 Spike protein were produced by transfecting HEK293T cells with pLenti-IL6ss-Myc-Nluc, pCAGGS-SARS-CoV-2 Spike plasmid, HIV Gag-Pol plasmid, HIV Tat plasmid, and HIV Rev plasmid using Fugene 6 transfection reagent (Promega, Madison, WI)	pLenti-IL6ss-Myc-Nluc suggested: None pCAGGS-SARS-CoV-2 suggested: None
   Software and Algorithms
   Sentences	Resources
   Data was analyzed using FlowJo software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)

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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 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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 8 and 9. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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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 10.1101/2021.05.27.445985v1 on bioRxiv

   May 27, 2021