Increased mTOR signaling, impaired autophagic flux and cell-to-cell viral transmission are hallmarks of SARS-CoV-2 infection

Article activity feed

1. 

   ScreenIT

   Oct 22, 2021

   SciScore for 10.1101/2021.10.13.464225: (What is this?)

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

   Table 1: Rigor

   Ethics	Euthanasia Agents: The plates were incubated at 37 °C, 5 % of CO2 atmosphere for 48 h.
   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
   Cells were then incubated overnight at 4 °C with primary antibodies at 1:100 dilution in PBST and 1 % BSA [SARS-COV-2 Spike S1 antibody (#HC2001 GenScript - #A02038), p62 antibody (#BD 610832), Lamp1 antibody (#BD 555798) and CD63 antibody (#BD 556019), according to the desired double IF.	p62 suggested: (BD Biosciences Cat# 610832, RRID:AB_398151) Lamp1 suggested: (BD Biosciences Cat# 555798, RRID:AB_396132) CD63 sugges…

   SciScore for 10.1101/2021.10.13.464225: (What is this?)

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

   Table 1: Rigor

   Ethics	Euthanasia Agents: The plates were incubated at 37 °C, 5 % of CO2 atmosphere for 48 h.
   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
   Cells were then incubated overnight at 4 °C with primary antibodies at 1:100 dilution in PBST and 1 % BSA [SARS-COV-2 Spike S1 antibody (#HC2001 GenScript - #A02038), p62 antibody (#BD 610832), Lamp1 antibody (#BD 555798) and CD63 antibody (#BD 556019), according to the desired double IF.	p62 suggested: (BD Biosciences Cat# 610832, RRID:AB_398151) Lamp1 suggested: (BD Biosciences Cat# 555798, RRID:AB_396132) CD63 suggested: (BD Biosciences Cat# 556019, RRID:AB_396297)
   The slides were washed and incubated for 2 h with secondary antibodies (Alexa 488 anti-Human IgG Thermo Fisher - #A11013 and Alexa Fluor 555 Anti-Mouse IgG #A21422), diluted 1:500 in PBST+ 1 % BSA.	anti-Human IgG suggested: (Molecular Probes Cat# A-11013, RRID:AB_141360) Anti-Mouse IgG suggested: (Molecular Probes Cat# A-21422, RRID:AB_141822)
   After blocking, the membranes were incubated with anti-mTOR (Cell Signaling, #2972), anti p-mTOR (Cell Signaling, #2971), p-S6K1 (Cell Signaling, #9234), anti-S6K1 (Cell Signaling, #2708), anti-pS6 (Cell Signaling, #2215), anti-S6 (Cell Signaling, #2317), anti-pULK1 (Cell Signaling, #5869), anti-ATG7 (Cell Signaling, #8558), anti-LC3 (Cell Signaling, #4108), anti-p62 (Cell Signaling, #88588), and anti-β-actin (Cell Signaling, #4967), antibodies overnight at 4 °C.	anti-mTOR (Cell Signaling, suggested: (Cell Signaling Technology Cat# 2972, RRID:AB_330978) anti-mTOR (Cell Signaling, #2972), anti p-mTOR (Cell Signaling, #2971), p-S6K1 (Cell Signaling, #9234), anti-S6K1 (Cell Signaling, #2708) suggested: None anti p-mTOR (Cell Signaling, #2971), p-S6K1 (Cell Signaling, #9234), anti-S6K1 (Cell Signaling, suggested: None anti-S6 suggested: (Cell Signaling Technology Cat# 2317, RRID:AB_2238583) anti-pULK1 (Cell Signaling, suggested: None anti-ATG7 suggested: (Cell Signaling Technology Cat# 8558, RRID:AB_10831194) anti-LC3 (Cell Signaling, #4108), anti-p62 (Cell Signaling, #88588), suggested: None anti-β-actin (Cell Signaling, suggested: (Cell Signaling Technology Cat# 4967, RRID:AB_330288)
   Membranes were washed with TBS-T (3 times for 10 min) and incubated with horseradish peroxidase-conjugated secondary antibodies anti-mouse (Millipore, #AP308P), or anti-rabbit (Thermo Scientific, #31460) according to the primary antibody for 1 h at room temperature with constant agitation.	horseradish peroxidase-conjugated secondary antibodies anti-mouse (Millipore, suggested: None anti-rabbit suggested: (Thermo Fisher Scientific Cat# 31460, RRID:AB_228341)
   Experimental Models: Cell Lines
   Sentences	Resources
   Vero E6: Vero E6 (African green monkey, Cercopithicus aethiops, kidney) cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, USA) and 1% Penicillin-Streptomycin 100 U/mL and 100 μg/mL (Sigma-Aldrich, USA), and incubated in 5 % carbon dioxide atmosphere at 37 °C.	Vero E6 suggested: RRID:CVCL_XD71)
   To access the production of viable viral particles inside Vero cells in the experiments, the supernatants were removed from the wells, cells were washed once with PBS 1X, and the monolayer was collected and frozen with liquid nitrogen, followed by three series of freeze-and-thaw in liquid nitrogen and 37 °C water bath.	Vero suggested: None
   Software and Algorithms
   Sentences	Resources
   Before image analysis, raw.czi files were automatically processed into deconvoluted Airyscan images using Zen Black 2.3 software.	Zen suggested: None
   Gene level quantification was performed using Salmon [25] with the Chlorocebus sabaeus reference transcriptome obtained from NCBI as the index.	Salmon suggested: (Salmon, RRID:SCR_017036)
   The R package tximport [26] was used to load quant.sf files to R and to create a DESeq2 object for differential expression analysis.	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   Geneset enrichment analysis (GSEA) of differentially expressed genes was performed using the fgsea R package [28] against a custom geneset containing all pathways and terms from Reactome, Gene Ontology (GO) biological process, GO cellular compartment, GO molecular function, Biocarta, KEGG, Hallmark pathways, and Wikipathways.	KEGG suggested: (KEGG, RRID:SCR_012773) Wikipathways suggested: (WikiPathways, RRID:SCR_002134)
   Figures were elaborated using the ggplot2 and pheatmap R packages.	ggplot2 suggested: (ggplot2, RRID:SCR_014601) pheatmap suggested: (pheatmap, RRID:SCR_016418)
   Band densitometry was measured using ImageJ software.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Quantification and Statistical Analysis: The software packages GraphPad Prism 8.0 and 9.0 were used for statistical analyses of Western blotting and PFU, respectively.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

   ---

   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 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).

   ---

   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 10.1101/2021.10.13.464225 on bioRxiv

   Oct 14, 2021