Identification of NPC1 as a novel SARS-CoV-2 intracellular target

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   ScreenIT

   Dec 22, 2020

   SciScore for 10.1101/2020.12.19.423584: (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
   Then, endogenous human NPC1 and HSP90 were purified using immobilized Recombinant Protein G Resin (Generon) and 4 µg of specific antibodies against NPC1 (Abcam, ab108921) or HSP90 (Enzo Life Sciences, ADI-SPA-835) respectively.	HSP90 suggested: None
   After that, plates were washed with PBST (PBS 0.1%Tween20) and the binding of NPC1 to SARS-CoV-2 N protein was detected with a rabbit anti-NPC1 antibody (1:2000), revealed with an anti-rabbit-horseradish …

   SciScore for 10.1101/2020.12.19.423584: (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
   Then, endogenous human NPC1 and HSP90 were purified using immobilized Recombinant Protein G Resin (Generon) and 4 µg of specific antibodies against NPC1 (Abcam, ab108921) or HSP90 (Enzo Life Sciences, ADI-SPA-835) respectively.	HSP90 suggested: None
   After that, plates were washed with PBST (PBS 0.1%Tween20) and the binding of NPC1 to SARS-CoV-2 N protein was detected with a rabbit anti-NPC1 antibody (1:2000), revealed with an anti-rabbit-horseradish peroxidase (HRP) (1:2000) using a colorimetric substrate (OPD) and finally, quantified by absorbance at 492 nm in the EnSight multimode plate reader of PerkinElmer.	anti-NPC1 suggested: None anti-rabbit-horseradish peroxidase (HRP suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture and viruses: Human embryonic kidney cells 293T/17 (HEK 293T; ATCC-CRL-11268) were cultured in Dulbecco modified Eagle medium (DMEM) at 37 °C and 5% CO2 atmosphere, supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 1X GlutaMAX (Thermo Fisher) and 10% heat-inactivated fetal bovine serum (FBS).	HEK suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
   Huh-7 Lunet C3 cells, a gift from T. Pietschman (Twincore, Germany), were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 10mM HEPES, 1X NEAA and 10% of heat-inactivated fetal bovine serum (FBS).	Lunet C3 suggested: None
   Expression of tagged-N protein and EGFP in HEK 293T cells: To transfect HEK 293T cells, four 60mm dishes were seeded with 2.5 ×106 cells each 24 hours prior to transfection in DMEM complete medium described above.	HEK 293T suggested: None
   Huh-7 cells were pre-treated with compounds at the indicated concentrations in growth medium for 1 h at 33 °C, followed by infection with 229E-GFP at a multiplicity of infection (MOI) of 1 pfu/cell for 24 h.	Huh-7 suggested: None
   Software and Algorithms
   Sentences	Resources
   To generate the SARS-CoV-2 N with N-terminal EGFP tag (EGFP-N), a codon optimized cDNA sequence for the ORF of SARS-CoV-2 N (NCBI reference sequence number: NC_045512) was cloned into the pEGFP-C1 (by GeneArt-Thermo Fisher Scientific).	GeneArt-Thermo Fisher Scientific suggested: None
   Production of SARS-CoV-2 N protein in the baculovirus system: The sequence of the N protein published in the NCBI database was selected (GenBank accession number: 43740575 / NCBI reference sequence number: NC_045512).	NCBI suggested: (NCBI, RRID:SCR_006472)
   Recombinant SARS-CoV-2 N protein produced in pupae was measured by band densitometry with the ChemiDoc™ XRS Gel Imaging System using Image Lab™ software (Bio-Rad).	Image Lab™ suggested: (Image Lab Software, RRID:SCR_014210)
   The IC50s values and dose-response curves were estimated using GraphPad Prism v6.0 with a 99% confidence interval.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   In order to determine the percentage of infected cells per condition, 8,000 cells/time point were scored using FACS Canto II flow cytometer (BD Sciences) and analyzed using the 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: 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.
   • No funding statement was detected.
   • No protocol registration statement was detected.

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2. 

   Version published to 10.1101/2020.12.19.423584v1 on bioRxiv

   Dec 20, 2020