The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB signaling

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1. Version published to 10.1016/j.jbc.2022.101695

   Mar 1, 2022
2. 

   ScreenIT

   Aug 4, 2021

   SciScore for 10.1101/2021.08.01.454605: (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	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Purified human integrin α5β1 and anti-α5 (MAB1956Z) and anti-α5β1 (MAB1999) antibodies were from Merck (Kenilworth, NJ, USA).	anti-α5β1 suggested: None
   HUVEC monolayers were treated for 16 hours with TNFα, spike, the spike receptor-binding domain, and the RGD tripeptide alone or in combination with anti-α5β1 antibodies (5 μg mL−1), anti-α5 antibodies (5 μg mL−1), volociximab (5 μg mL−1), or ATN-161 (500 nM) in 20% FBS F12K without heparin or ECGS.	anti-α5 suggested: None
   Microplates were …

   SciScore for 10.1101/2021.08.01.454605: (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	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Purified human integrin α5β1 and anti-α5 (MAB1956Z) and anti-α5β1 (MAB1999) antibodies were from Merck (Kenilworth, NJ, USA).	anti-α5β1 suggested: None
   HUVEC monolayers were treated for 16 hours with TNFα, spike, the spike receptor-binding domain, and the RGD tripeptide alone or in combination with anti-α5β1 antibodies (5 μg mL−1), anti-α5 antibodies (5 μg mL−1), volociximab (5 μg mL−1), or ATN-161 (500 nM) in 20% FBS F12K without heparin or ECGS.	anti-α5 suggested: None
   Microplates were then incubated for 1 hour at RT, washed three times with PBST, incubated for 1 hour at RT with 1:1,000 of anti-spike antibody (Rabbit MAb, Sino Biological, Beijing, CN) diluted in blocking buffer, washed (three times in PBST), and incubated (1 hour at RT) with horseradish peroxidase-labeled goat anti-rabbit secondary antibodies (Thermo Fisher Scientific) diluted 1:2,500 in 50% blocking buffer.	anti-spike suggested: None anti-rabbit suggested: None
   The NF-κB activation inhibitor, BAY 11-7085 (5 μM), or anti-α5 antibodies (5 μg mL−1) were added 30 minutes prior to a 30-minutes incubation with spike (100 nM) or TNFα (1 nM).	TNFα suggested: None
   Cells were washed, fixed in 4% PFA for 30 minutes at RT, permeabilized for 30 minutes with 0.5% Tx-100-PBS, blocked with 0.05% Tx-100, 1% BSA, 5% normal goat serum-PBS at RT for 1 hour, and incubated overnight at 4°C in a humid chamber with 1:200 mouse monoclonal anti-NF-κB p65 antibody (Santa Cruz, CA, USA) in 0.1% Tx-100, 1% BSA-PBS.	anti-NF-κB p65 suggested: None
   Nuclear DNA was counterstained with 5 μg mL−1 Hoechst 33342 (Sigma-Aldrich) and the coverslips washed and mounted with Vectashield mounting medium (Vector laboratories, Burlingame, CA) and observed under fluorescence microscopy (Olympus IX51). qPCR: HUVEC grown to 80% confluency on 6-well plates were incubated under starving conditions (0.5% FBS, F12K medium) for 30 minutes with the NF-κB activation inhibitor, BAY 11-7085 (5 μM) or anti-α5 antibodies (5 μg mL−1) followed by a 4-hour incubation with 100 nM spike.	Vectashield mounting medium suggested: (Vector Laboratories Cat# H-1000, RRID:AB_2336789)
   Cells were washed, fixed with 4% PFA for 30 minutes, permeabilized with 0.5% Tx-100-PBS for 30 minutes, blocked with 0.1% Tx-100, 1% BSA, 5% normal goat serum-PBS for 1 hour at RT, and incubated overnight at 4°C in a humid chamber with 1: 100 anti-CD31 antibody (Abcam) in 0.1% Tx-100, 1% BSA-PBS.	anti-CD31 suggested: None
   Cells were washed and incubated in darkness with goat anti-mouse secondary antibodies (1:500, Alexa fluor 488, Abcam, Cambridge, UK) in 0.1% Tx-100, 1% BSA-PBS for 2 hours.	anti-mouse suggested: (Abcam Cat# ab19639, RRID:AB_2040847)
   Both, Rhotekin and PAK1 binding domains are coupled to agarose beads, and the isolation and detection of the active forms of RhoA, Rac1, and Cdc42 were done under conventional bead-precipitation and western blot protocols using goat anti-mouse alkaline phosphatase secondary antibodies (Jackson ImmunoResearch, Philadelphia, PA, USA, 1:5000) and a colorimetric detection kit (BioRad, Hercules, CA, USA).	PAK1 suggested: None Rac1 suggested: None Cdc42 suggested: None
   Forty-five μg of protein were resolved in SDS-PAGE and blotted with anti-phospho-eNOS (Ser1177) antibodies (Cell Signaling, Danvers, MA, USA, 1:250), followed by incubation with goat anti-rabbit horseradish peroxidase secondary antibodies (Jackson ImmunoResearch, 1:5000).	anti-phospho-eNOS suggested: None Ser1177) antibodies (Cell Signaling, Danvers, MA suggested: None
   Membranes were reblotted using antibodies against total eNOS (Cell Signaling, 1:500), goat anti-rabbit alkaline phosphatase secondary antibodies (Jackson ImmunoResearch, 1:5000), and a colorimetric detection kit (BioRad).	antibodies against total eNOS (Cell Signaling, 1:500) suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Fluorescence cytochemistry for CD31 and F-Actin: HUVEC were seeded on 18 mm coverslips coated with fibronectin (1 μg cm−1) and placed in a 12-well plate.	HUVEC suggested: None
   Software and Algorithms
   Sentences	Resources
   HUVEC monolayers were washed three times with warm PBS, and images were obtained in an inverted fluorescent microscope (Olympus IX51, Japan) and quantified using the CellProfiler software47.	CellProfiler suggested: None

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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: Please consider improving the rainbow (“jet”) colormap(s) used on page 17. 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 10.1101/2021.08.01.454605 on bioRxiv

   Aug 2, 2021