The SARS-CoV-2 spike protein binds and modulates estrogen receptors

Article activity feed

1. 

   ScreenIT

   May 25, 2022

   SciScore for 10.1101/2022.05.21.492920: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: , Radiation Safety, and Animal Care and Use Committees.
   Sex as a biological variable	Male golden Syrian hamsters (7 to 8 weeks of age) were purchased from Envigo (Haslett, MI).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The wells were washed and then incubated with rabbit anti-ERα (1:2000, 1 h, RT) and horseradish-conjugated secondary antibody (1:2000, 1 h, RT) that were provided with the kit.	anti-ERα suggested: (Santa Cruz Biotechnology Cat# sc-542, RRID:AB_631470)
   The cells were then incubated at 4°C overnight with 2 μg/ml each of anti-ERα(H222) rat IgG1 monoclonal antibody …

   SciScore for 10.1101/2022.05.21.492920: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: , Radiation Safety, and Animal Care and Use Committees.
   Sex as a biological variable	Male golden Syrian hamsters (7 to 8 weeks of age) were purchased from Envigo (Haslett, MI).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The wells were washed and then incubated with rabbit anti-ERα (1:2000, 1 h, RT) and horseradish-conjugated secondary antibody (1:2000, 1 h, RT) that were provided with the kit.	anti-ERα suggested: (Santa Cruz Biotechnology Cat# sc-542, RRID:AB_631470)
   The cells were then incubated at 4°C overnight with 2 μg/ml each of anti-ERα(H222) rat IgG1 monoclonal antibody (mAb) (Santa Cruz Biotech, sc-5349, 1:100) and HA-probe (	anti-ERα(H222 suggested: None rat IgG1 suggested: None
   Afterwards, the cells were washed 4 times with PBS + 0.1% Tween-20 (PBS-T) for 5 minutes and incubated at room temperature for 1 hour in the dark with a fluorescent secondary antibody mixture contaning mouse IgGk BP-CFL594 (Santa Cruz Biotech, sc-516178, 1:100) and anti-rat IgG AF488 (ThermoFisher Scientific, cat no. A-11006, 1:500).	anti-rat IgG suggested: None
   Then, cells were washed and were incubated with detector anti-BrdU antibody for 1 hour at RT.	anti-BrdU suggested: None
   After the incubation cells were washed and incubated with the horseradish peroxidase conjugated goat anti-mouse antibody for 30 minutes at RT.	anti-mouse suggested: None
   Briefly, 50 μl of standard were added to standard wells and 40 μl of sample-to-sample wells and then added 10 μl of anti-TRAP antibody to sample wells and 50 μl of streptavidin-HRP to sample wells and standard wells.	anti-TRAP suggested: None
   After 72 hours, cells were washed, fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100 in PBS and stained overnight at 4°C with ACE2 protein-specific antibody (Abcam Ab15348).	ACE2 suggested: None
   Cells were then incubated with anti-rabbit secondary antibody (Alexa Fluor 536 anti-rabbit, Invitrogen Life Technologies) for 1 hour at 37°C.	anti-rabbit suggested: None
   Sections were then incubated with cocktails of primary antibodies: rabbit anti-SARS-CoV-2 Spike Protein (1:100, Invitrogen, #MA5-36087) + rat anti-ERα H222 (1:100, Santa Cruz Biotechnology, #sc53492) overnight at 4°C.	anti-SARS-CoV-2 Spike Protein ( 1:100 , Invitrogen , #MA5-36087 ) suggested: None
   Sections were then incubated with the primary antibodies rat anti-ERα H222(1:100, Santa Cruz Biotechnology, #sc53492), diluted in 1% normal goat serum (NGS), 4% BSA, 0.02% saponin in PB at 4°C overnight.	anti-ERα H222 suggested: None
   Sections were rinsed and incubated overnight at 4°C in the secondary antibody Nanogold-Fab’ goat anti-rat-IgG (1:100	anti-rat-IgG suggested: None
   On day one, slides were blocked with a peroxidase blocker (Bio SB Catalog No. BSB 0054), washed with an immunoDNA washer buffer (Bio SB, Catalog No. BSB 0150); then, incubated with 0.2 μg/mL of anti-SARS-CoV-2 spike glycoprotein antibody (abcam, Catalog No. ab272504) for 1 hour.	anti-SARS-CoV-2 spike glycoprotein suggested: (Abcam Cat# ab272504, RRID:AB_2847845)
   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, MCF-7 nuclear extracts (5 μg; ab14860, Abcam) were treated with either S (0.01-300 nM; Acro Biosystems)	MCF-7 suggested: None
   Proliferation assays: MCF-7 and MDA-MB-23 cells were obtained from ATCC and growth in DMEM without phenol red, supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin at 37 °C in a 5% CO2 and 95% humidified atmosphere.	MDA-MB-23 suggested: None
   TRAP activity by ELISA assay in RAW-OCs: RAW264.7 (murine macrophages ATCC, USA) were cultured as manufacturer’s protocol.	RAW264.7 suggested: CLS Cat# 400319/p462_RAW-2647, RRID:CVCL_0493)
   ACE2 expression in Calu-3 cells: Calu-3 cell line was obtained from ATCC and maintained in Eagle’s Minimum Essential Medium(EMEM; Lonza) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin/streptomycin solution at 37°C in a humidified atmosphere of 5% CO2.	Calu-3 suggested: None
   Recombinant DNA
   Sentences	Resources
   The next day, cells in each well were transfected with 1.5 μl of ViaFect reagent (Promega, cat no. E498A) and 0.5 μg of empty pcDNA3.1 vector, or an expression vector for the wild-type (WT) SARS-CoV2 S with a C-terminal hemagglutinin (HA) epitope tag (pBOB-CAG-SARS-CoV2-S-HA) or the double mutant (R682S,R685S) SARS-CoV2 S with a C-terminal flag epitope tag (pCAGGS-SARS2-S-FKO).	pcDNA3.1 suggested: RRID:Addgene_79663) pBOB-CAG-SARS-CoV2-S-HA suggested: None
   pBOB-CAG-SARS-CoV2-S-HA was a gift from Gerald Pao (Addgene plasmid # 141347; http://n2t.net/addgene:141347; RRID:Addgene_141347).	detected: RRID:Addgene_141347)
   pCAGGS-SARS2-S-FKO (C-flag) was a gift from Hyeryun Choe & Michael Farzan (Addgene plasmid # 159364; http://n2t.net/addgene:159364; RRID:Addgene_159364).	detected: RRID:Addgene_159364)
   Software and Algorithms
   Sentences	Resources
   Data were fitted using the non-linear curve fitting routines in Prism® (Graphpad Software Inc).	Graphpad suggested: (GraphPad, RRID:SCR_000306)
   The digitized images were also analyzed using ProtoArray Prospector v5.2 and potential hits were identified using the software’s algorithm.	ProtoArray Prospector suggested: None
   Protein binding responses were analyzed using BiaEval software.	BiaEval suggested: None
   Interactome analysis: The STRING database52, that integrates all known and predicted associations between proteins, including both physical interactions as well as functional associations has been used to analyses functional associations between biomolecules.	STRING suggested: (STRING, RRID:SCR_005223)
   Images were prepared for presentation using ImageJ v.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   After three washes, the Mouse/Rabbit PolyDetector Plus link &HRP label (Bio SB, Catalog No. BSB 0270) were applied.	Mouse/Rabbit PolyDetector Plus suggested: None PolyDetector suggested: None

   ---

   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).

   ---

   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.

   ---

   Results from TrialIdentifier: No clinical trial numbers were referenced.

   ---

   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

   ---

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

   ---

   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.

   ---

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

   ---

   Read the original source
2. 

   Version published to 10.1101/2022.05.21.492920v1 on bioRxiv

   May 23, 2022