Natural Killer cell activation, reduced ACE2, TMPRSS2, cytokines G-CSF, M-CSF and SARS-CoV-2-S pseudovirus infectivity by MEK inhibitor treatment of human cells

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   ScreenIT

   Aug 6, 2020

   SciScore for 10.1101/2020.08.02.230839: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	The number of red/green color cells in random fields was determined using thresholding and particle analysis in the Fiji modification of ImageJ and expressed as a dead/live cell ratio.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Authentication: Cell lines were authenticated and tested to ensure the cultures were free of mycoplasma infection. Contamination: Cell lines were authenticated and tested to ensure the cultures were free of mycoplasma infection.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Abnova ACE2 polyclonal antibody #PAB13444, Santa Cruz Biotechnology ACE2 …

   SciScore for 10.1101/2020.08.02.230839: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	The number of red/green color cells in random fields was determined using thresholding and particle analysis in the Fiji modification of ImageJ and expressed as a dead/live cell ratio.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Authentication: Cell lines were authenticated and tested to ensure the cultures were free of mycoplasma infection. Contamination: Cell lines were authenticated and tested to ensure the cultures were free of mycoplasma infection.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Abnova ACE2 polyclonal antibody #PAB13444, Santa Cruz Biotechnology ACE2 Antibody (E-11) #sc-390851, Sigma Anti-TMPRSS2 Antibody, clone P5H9-A3 #MABF2158, Sigma Anti-IL6 antibody produced in rabbit #SAB1408591, Santa Cruz Biotechnology TMPRSS2 Antibody (H-4) #sc-515727, TMPRSS2 (EMD Millipore #MABF2158), Cell Signaling Technology Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E)	Anti-IL6 suggested: (Sigma-Aldrich Cat# SAB1408591, RRID:AB_10742282) #SAB1408591 suggested: (Sigma-Aldrich Cat# SAB1408591, RRID:AB_10742282) TMPRSS2 suggested: None
   Rabbit mAb #4370, Cell Signaling Technology p44/42 MAPK (Erk1/2) Antibody #9102, Ran (BD Biosciences #610341)	Erk1/2 suggested: (Cell Signaling Technology Cat# 9102, RRID:AB_330744)
   , Caspase-8 (Cell Signaling #9746), Sigma Monoclonal Anti-β-Actin antibody produced in mouse #A5441.	Anti-β-Actin suggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744)
   Invitrogen Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP # 31460 and Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP # 31430 were diluted 1:5000 in 2.5% non-fat milk. qRT-PCR methods and primers: Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen)	anti-Rabbit IgG suggested: (Thermo Fisher Scientific Cat# 31460, RRID:AB_228341) anti-Mouse IgG suggested: (Thermo Fisher Scientific Cat# 31430, RRID:AB_228307)
   Western blots evaluating cleaved caspase 8 were performed using Cell Signaling antibody (#9746).	cleaved caspase 8 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines and culture conditions: Normal human primary small airway epithelial cells HSAEC, normal human bronchial epithelial cells BEAS-2B, normal human lung fibroblast MRC-5, human NSCLC cells H1975, H1299, Calu-3, Calu-6, human mesothelioma cells MSTO-211H, NSCLC patient-derived cell line, human natural killer cells NK-92, normal human colon epithelial cells CCD 841 CoN and human colorectal cancer cells HT-29, HCT116 were used in this study.	HSAEC suggested: None MRC-5 suggested: None H1299 suggested: None
   H1975, H1299, MSTO-211H and NSCLC patient-derived cell line were cultured in RPMI-1640 medium supplemented 10% FBS.	H1975 suggested: None
   NK-92 cells were cultured in Alpha Minimum Essential medium without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate supplemented with 0.2 mM inositol; 0.1 mM 2-mercaptoethanol; 0.02 mM folic acid; 100 U/ml recombinant IL-2, 12.5% horse serum and 12.5% FBS.	NK-92 suggested: None
   HCT116 and HT-29 were cultured in McCoy’s 5A (modified) medium supplemented 10% FBS.	HCT116 suggested: None HT-29 suggested: None
   Briefly, HEK293T cells at 75% confluency were co-transfected with the backbone vector pHAGE-fullEF1α-ZsGreen-IRES-Puro(R), plasmids expressing lentiviral proteins Tat, Rev and Gag/Pol, and plasmids expressing D614 or G614 S protein (a gift from Dr. Hyeryun Choe, The Scripps Research Institute, Jupiter, FL).	HEK293T suggested: None
   SARS-CoV-2 pseudoviruses (5 x 106) or VSV-G lentivirus (2 x 105) were used to spin-infect (931 g for 2 hr at 30°C) Calu-3 or BEAS-2B cells in a 6-well plate.	BEAS-2B suggested: RRID:CVCL_WZ49)
   To test the inhibitors, Calu-3 or BEAS-2B cells were pre-treated with the inhibitors for 48 hr, spun-infected with pseudovirus followed by another 48 hr incubation with the inhibitors.	Calu-3 suggested: None
   Software and Algorithms
   Sentences	Resources
   The number of red/green color cells in random fields was determined using thresholding and particle analysis in the Fiji modification of ImageJ and expressed as a dead/live cell ratio.	Fiji suggested: (Fiji, RRID:SCR_002285) ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Flow cytometry analysis of ZsGreen+ cells was carried out 48 hr after infection on a BD LSRII flow cytometer and with the FlowJo software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)

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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: We detected the following sentences addressing limitations in the study:

   This is a limitation of the work, as is lack of direct evidence that MEKi attenuate SARS-CoV-2 infection of human cells. Our evidence is indirect, and the effects are predicted based on current knowledge of SARS-CoV-2 infectivity factors, and consistent with recent results showing SARS-Cov-2 effects on the kinome including MAPK p38 activation [45]. Our results showing infection of SARS-CoV-2-S pseudovirus of human bronchial epithelial cells, human small airway epithelial cells, or lung cancer cells provide an experimental model system to discover or test therapeutics with potential to block coronavirus infection. The observed effect of MEKi to reduce SARS-CoV-2-S pseudovirus infection of human cells is consistent with our other evidence that MEKi may attenuate coronavirus infectivity factors to inhibit infection. In pursuit of a therapeutic agent that could attenuate cytokine storm while reducing viral infectivity and boosting NK cells activity, we found that VS-6766 decreases G-CSF and other cytokines. These cytokines of interest were increased in COVID-19-(+) patient plasma samples in our study. The combination of remdesivir and VS-6766 was not associated with increased cytokine expression at nontoxic doses of the drugs. The MEKi plus remdesivir drug combinations do not block NK-mediated cell killing and in fact the MEKi stimulate NK killing activity towards target cells. Moreover, the drug combinations do not inhibit TRAIL-mediated killing of target cells. The observed sti...

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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 pages 64, 38, 39, 40, 57, 58, 29, 62 and 63. 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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2. 

   Version published to 10.1101/2020.08.02.230839 on bioRxiv

   Aug 3, 2020