The circadian clock component BMAL1 regulates SARS-CoV-2 entry and replication in lung epithelial cells

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   ScreenIT

   Mar 24, 2021

   SciScore for 10.1101/2021.03.20.436163: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: Participants provided written informed consent. IRB: The study was reviewed by the Oxford Research Ethics Committee B (18/SC/0361).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	C57BL/6J male mice were purchased from Charles River and housed in individually ventilated cages under a 12/12 dark/light cycle with a ZT0 corresponding to 7am.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After 1h, the cells were washed with PBS before medium containing a VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700, ATCC) and supernatants harvested after 24h.	VSV-G suggeste…

   SciScore for 10.1101/2021.03.20.436163: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: Participants provided written informed consent. IRB: The study was reviewed by the Oxford Research Ethics Committee B (18/SC/0361).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	C57BL/6J male mice were purchased from Charles River and housed in individually ventilated cages under a 12/12 dark/light cycle with a ZT0 corresponding to 7am.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After 1h, the cells were washed with PBS before medium containing a VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700, ATCC) and supernatants harvested after 24h.	VSV-G suggested: None I1 suggested: None
   Membranes were blocked in 5% milk in PBS/0.1% Tween-20, then incubated with anti-ACE2 (Abcam ab108252), anti-TMPRSS2 (SCBT sc-515727), anti-BMAL1 (Abcam Ab93806) or Anti-β-actin (Sigma A5441) primary antibodies and appropriate HRP-conjugated secondary antibodies (DAKO).	anti-ACE2 suggested: (Abcam Cat# ab108252, RRID:AB_10864415) anti-TMPRSS2 suggested: None anti-BMAL1 suggested: (Abcam Cat# ab93806, RRID:AB_10675117) Anti-β-actin suggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture: Calu-3, Huh-7, HEK293T and VERO E6 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 U/mL penicillin and 10μg/mL streptomycin (all reagents from Life Technologies/Thermo Fisher).	Huh-7 suggested: None HEK293T suggested: None
   SARS-CoV-2 pseudoparticle genesis and infection: SARS-CoV-2 lentiviral pp were generated by transfecting HEK-293T cells with p8.91 (Gag-pol), pCSFW (luciferase reporter) and a codon optimised expression construct pcDNA3.1-SARS-CoV-2-Spike, as previously reported53.	HEK-293T suggested: None
   The VSV*ΔG used for generating the pps was propagated in BHK-21 G43 cells stably expressing VSV-G.	BHK-21 G43 suggested: None
   Viral titres were determined by infecting Calu-3 cells and measuring cellular luciferase after 48h.	Calu-3 suggested: None
   Briefly, Vero E6 cells were inoculated with serial dilutions of SARS-CoV-2 stocks for 2h followed by addition of a semi-solid overlay consisting of 3% carboxymethyl cellulose (SIGMA).	Vero E6 suggested: RRID:CVCL_XD71)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   C57BL/6J male mice were purchased from Charles River and housed in individually ventilated cages under a 12/12 dark/light cycle with a ZT0 corresponding to 7am.	C57BL/6J suggested: None
   Software and Algorithms
   Sentences	Resources
   Airway epithelial cells were taken using 2mm diameter cytology brushes from 3rd to 5th order bronchi and cultured in Airway Epithelial Cell medium (PromoCell, Heidelberg, Germany) in submerged culture.	PromoCell suggested: None
   Anti-β-actin-HRP conjugate (Abcam ab49900) and/or Coomassie brilliant blue staining was used to verify equal protein loading and densitometric analysis performed using ImageJ software (NIH).	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Promoters (−1kb from TSS) of genes encoding SARS-CoV-2 host factors were analyzed with the HOMER (Hypergeometric Optimization of Motif EnRichment) tool for motif discovery (E-box motif CANNTG; RORE motif RGGTCA).	HOMER suggested: (HOMER, RRID:SCR_010881)
   P values were determined using Mann-Whitney testing (two group comparisons) or with a Kruskal–Wallis ANOVA (multi group comparisons) using PRISM version 9.	PRISM suggested: (PRISM, RRID:SCR_005375)

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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: 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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   Version published to 10.1101/2021.03.20.436163 on bioRxiv

   Mar 21, 2021