Characterization of the interaction between SARS-CoV-2 Membrane Protein and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target

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   ScreenIT

   Dec 11, 2021

   SciScore for 10.1101/2021.12.06.471464: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: The use of this material for research purposes has been previously approved by the Institutional Ethical Board CAAE #30364720.0.0000.0068)
   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
   The samples were diluted with lysis buffer without inhibitors and incubated overnight at 4°C with anti-PCNA antibody (1:500, #2586, Cell signaling) under mild agitation.	anti-PCNA suggested: (Cell Signaling Technology Cat# 2586, RRID:AB_2160343)
   The secondary antibodies used were: Alexa-Fluor-594 Goat anti-Rabbit (Jackson ImmunoResearch #711-585-152),	anti-Rabbit

   SciScore for 10.1101/2021.12.06.471464: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: The use of this material for research purposes has been previously approved by the Institutional Ethical Board CAAE #30364720.0.0000.0068)
   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
   The samples were diluted with lysis buffer without inhibitors and incubated overnight at 4°C with anti-PCNA antibody (1:500, #2586, Cell signaling) under mild agitation.	anti-PCNA suggested: (Cell Signaling Technology Cat# 2586, RRID:AB_2160343)
   The secondary antibodies used were: Alexa-Fluor-594 Goat anti-Rabbit (Jackson ImmunoResearch #711-585-152),	anti-Rabbit suggested: (Jackson ImmunoResearch Labs Cat# 711-585-152, RRID:AB_2340621)
   Briefly, cells were blocked and stained with primary antibodies (anti-FLAG 1:400 - Sigma F7425 and anti-PCNA 1:400 – Sta Cruz sc-56) and PLA probes (anti-mouse MINUS - DUO92004 and anti-rabbit PLUS - DUO92002).	anti-FLAG suggested: None anti-mouse MINUS - DUO92004 suggested: None anti-rabbit PLUS - DUO92002 suggested: None
   Membranes were blocked for one hour at room temperature (RT) with 5% non-fat powdered milk dissolved in TBS-Tween-20 (TBS-T) (50 mM Tris-Cl, pH 7.5; 150 mM NaCl; 0.1% Tween-20), incubated overnight (4°C) with primary antibodies and with secondary antibodies against mouse or rabbit IgG conjugated with peroxidase (Amersham) for one hour at RT.	rabbit IgG suggested: None
   The following antibodies were used: anti-PCNA (#2586); anti-γH2AX (#9718); anti-β-actin (#4967) (Cell Signaling), anti-α-tubulin (CP06) (Calbiochem) Immunohistochemistry: Lung tissue sections, obtained via Minimally Invasive Autopsies,	anti-γH2AX suggested: None anti-β-actin ( #4967 ) ( Cell Signaling) , anti-α-tubulin ( CP06 suggested: None anti-α-tubulin suggested: (Millipore Cat# CP06, RRID:AB_2617116)
   Experimental Models: Cell Lines
   Sentences	Resources
   Viral infections were performed in Vero cells seeded in 24-well plates (5 × 105 cells/well) for the experiments with treatments and immunofluorescence assays, and six-well plates (1 × 106 cells/well) for Western blots.	Vero suggested: None
   Immunoprecipitation: For anti-FLAG immunoprecipitation, HEK293T cells cultivated in 100 mm diameter dishes expressing the FLAG-tagged GFP, E, M, and N proteins were washed twice with PBS and harvested by pipetting up and down in PBS after 48 hours.	HEK293T suggested: None
   Western blotting: The proteins were collected from Vero E6 and HEK293 cells using a cell lysis buffer (50 mM Tris-Cl pH 7.5; 150 mM NaCl, 1 mM EDTA, 1% Triton x-100, and protease and phosphatase inhibitor cocktail).	HEK293 suggested: None
   Briefly, after seeding, Vero E6 (8 × 105 cells per well, 6-well plates) were incubated overnight and infected with 200 PFU per well.	Vero E6 suggested: RRID:CVCL_XD71)
   Recombinant DNA
   Sentences	Resources
   Cloning: To do the FLAG-tagged protein expression, a modified pcDNA 3.1 (+) (Thermo Fisher Scientific) was generated by cloning the FLAG peptide coding sequence upstream of the multiple cloning sites, using the NheI and BamHI restriction sites and generating the pcDNA-FLAG vector 60.	pcDNA 3.1 suggested: RRID:Addgene_20407) pcDNA-FLAG suggested: RRID:Addgene_60940)
   The full-length of M, N, and E genes were codon-optimized, synthesized (Geneart-Thermo Fisher Scientific), and cloned into pcDNA-FLAG, generating plasmid pFLAG-M, pFLAG-N, and pFLAG-E.	pFLAG-N suggested: None pFLAG-E suggested: None
   Subcellular Fractionation: Vero E6 cells were seeded at 8×106 in a P100 plate and transfected with pFLAG-GFP or pFLAG-M.	pFLAG-GFP suggested: None
   The quantification was performed by BCA. Proximity Ligation Assay (PLA.): For PLA protocol, Vero E6 cells were grown on chambered slides at a confluency of 70% and transfected with pFLAG-M.	pFLAG-M suggested: None
   Software and Algorithms
   Sentences	Resources
   To quantify the signal on immunofluorescences we used the ImageJ software to transform the images in 8 bit (grayscale), and check-in Set Measurements the options: Area and Integrated density; select an ROI of nuclei area (Hoechst staining) to determinate the fluorescence signal on nuclei; the cytoplasmatic fluorescence signal was obtained by a total cell - nuclei area ROI.	ImageJ suggested: (ImageJ, RRID:SCR_003070)

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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.
   • 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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   Version published to 10.1101/2021.12.06.471464v1 on bioRxiv

   Dec 8, 2021