C910 chemical compound inhibits the traffiking of several bacterial AB toxins with cross-protection against influenza virus

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1. Version published to 10.1016/j.isci.2022.104537

   Jul 1, 2022
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   Aug 19, 2021

   SciScore for 10.1101/2021.08.13.454991: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: In vivo IAV infection model: All mice were housed under specific-pathogen-free conditions at Seattle Children’s Research Institute and all animal experiments performed at Seattle Children’s Research Institute were approved by the Institutional Animal Care and Use Committee (IACUC00580). IRB: The project was submitted to the French Ethics Committee CEEA (Comité d’Ethique en Expérimentation Animale) and obtained the authorization APAFIS#10108-2017060209348158 v3.
   Sex as a biological variable	A pilot experiment showed a protective effect of C910 in male animals therefore we used male mice for further experiments (data not shown).
   Randomization	Cells in each condition were randomly selected …

   SciScore for 10.1101/2021.08.13.454991: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: In vivo IAV infection model: All mice were housed under specific-pathogen-free conditions at Seattle Children’s Research Institute and all animal experiments performed at Seattle Children’s Research Institute were approved by the Institutional Animal Care and Use Committee (IACUC00580). IRB: The project was submitted to the French Ethics Committee CEEA (Comité d’Ethique en Expérimentation Animale) and obtained the authorization APAFIS#10108-2017060209348158 v3.
   Sex as a biological variable	A pilot experiment showed a protective effect of C910 in male animals therefore we used male mice for further experiments (data not shown).
   Randomization	Cells in each condition were randomly selected and analyzed under the same threshold.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antibodies and reagents: C910 was purchased from Chembridge (ID: 5454910, San Diego, CA, USA) or Synthenova (ID: SN0218L3, Hérouville Saint-Clair, France).	C910 suggested: (GenWay Biotech Inc. Cat# GWB-82C910, RRID:AB_10513344)
   , mouse anti-Rac1 (610651 [clone 102]), mouse anti-Rab4 (610888), mouse anti-Lamp1 (611043) was from BD Bioscience; mouse anti-TGN38 (sc-101273), rabbit anti-MEK2 (SC-524) from Santa Cruz; rabbit anti-Calnexin (ADI-SPA-860-D) from Enzo; mouse-anti-LBPA (Z-PLBPA) was from Echelon Biosciences; Paraformaldehyde (PFA) (15710) from Electron Microscopy science.	anti-Calnexin suggested: (Enzo Life Sciences Cat# ADI-SPA-860-D, RRID:AB_2038898)
   Horseradish peroxidase (HRP)-conjugated goat anti-mouse (P0399) or swine anti-rabbit secondary antibodies (P0447) were from Dako.	anti-mouse ( P0399 suggested: None
   Cells were next labelled with an anti-Rac1 mouse antibody [clone 102] for 1 h and revealed with Alexa Fluor 488-coupled anti-mouse secondary antibody.	anti-mouse suggested: None
   In parallel, cell lysates from intoxicated cells were examined for glucosylated-Rac1 that no longer react to anti-Rac1 monoclonal antibody [clone 102].	anti-Rac1 suggested: None
   Signals were revealed using horseradish peroxidase-conjugated goat anti-mouse or swine anti-rabbit secondary antibodies (DAKO) followed by chemiluminescence using Immobilon® Western (Millipore).	anti-rabbit suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   HeLa, L929, Vero, A549, Vero E6 and U2OS-ACE2 cell lines were cultured at 37°C in 5% CO2 in DMEM/GlutaMax (Invitrogen) supplemented with 10% heat-inactivated FBS (F9665, Sigma-Aldrich) and 1% penicillin/streptomycin (Invitrogen).	Vero E6 suggested: None U2OS-ACE2 suggested: None
   Cytotoxic effects of the LCGTs TcdA and TcdB on Vero cells (Sigma-Aldrich) were evaluated by measure of cell rounding.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   IAV infection: For single-cycle IAV infection assays, A549 cells (70,000/ well) were infected with a reporter H1N1 A/WSN/33 (H1N1WSN PB2-2A-mCitrine), at a MOI of 5 PFU/cell for 6 h.	A549 suggested: None
   For multicycle growth assays, A549 cells were infected with a seasonal A/Bretagne/7608/2009 (H1N1pdm09) strain adapted to human cell lines (50), at the MOI of 10−3 infective particles/cell and the production of infectious particles in the culture supernatant was determined at 24 h using a standard plaque assay on the highly sensitive canine MDCK-SIAT cells, as described previously(72).	MDCK-SIAT suggested: ECACC Cat# 05071502, RRID:CVCL_Z936)
   Determination of CC50s of C910 on HUVECs and HeLa cells: Cells seeded in Corning™ 96 well Flat Clear Bottom Black Microplates (#3603) were incubated with increasing doses of C910 for 6 h, medium was replaced with fresh medium including 10% of Alamar Blue (final concentration 1:10).	HeLa suggested: None
   Medisch Centrum Utrecht, Netherland), HUVECs were fixed at room temperature for 2 h in 2.5% glutaraldehyde in PHEM buffer, pH 7.2 (60 mM 1,4 piperazine diethylsulfonic acid (PIPES), 25 mM N-2-hydroxyethylpiperazine	HUVECs suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Viral challenges were carried out on groups of 9~10 week-old C57BL/6 male mice (Charles River Laboratories).	C57BL/6 suggested: None
   Experiments were conducted on adult male C57BL/6J mice (23.5 ± 0.3 g) purchased from Janvier Labs (Le Genest St Isle, France).	C57BL/6J suggested: RRID:IMSR_JAX:000664)
   Software and Algorithms
   Sentences	Resources
   Cell culture and bacterial toxins: Human umbilical vein endothelial cells (HUVECs) (PromoCell, Heidelberg, Germany) were cultured as described (24).	PromoCell suggested: None
   HIS-tagged B subunit of Shiga toxin 1(SML0655) and Apilimod (A149227) from Sigma; rabbit anti-EEA1 (#3288), rabbit anti-Rab7 (#9367), rabbit-anti-his tag (#12698) from Cell Signaling; rabbit anti-Rab7 (ab137029), mouse anti-Giantin (ab37266) and Cathepsin B Activity Assay Kit (ab65300) from Abcam; mouse-anti-6x-his (R930-25 [clone 3D5]), DQTM Red BSA (D-112051) from ThermoFisher Scientific; mouse-anti-EEA1 (BD610457), mouse-anti-Rab5 (610724)	ThermoFisher Scientific suggested: None
   , mouse anti-Rac1 (610651 [clone 102]), mouse anti-Rab4 (610888), mouse anti-Lamp1 (611043) was from BD Bioscience; mouse anti-TGN38 (sc-101273), rabbit anti-MEK2 (SC-524) from Santa Cruz; rabbit anti-Calnexin (ADI-SPA-860-D) from Enzo; mouse-anti-LBPA (Z-PLBPA) was from Echelon Biosciences; Paraformaldehyde (PFA) (15710) from Electron Microscopy science.	BD Bioscience suggested: (BD Biosciences, RRID:SCR_013311)
   8 software (GraphPad, San Diego, CA)	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Data were analyzed with FlowJo (BD Bioscience).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Fiji ImageJ software (National Institutes of Health) was used for image processing and quantification.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Briefly, each individual cell was selected from a single mid-z stack of the confocal image by hand-drawing a “region of interest” (ROI) from Fiji software.	Fiji suggested: (Fiji, RRID:SCR_002285)
   Measures of the size of EEA1-positive vesicles were performed from the single mid-z stack of confocal images that were next quantified with the Analyze particles tool from Fiji Software.	Analyze suggested: (ANALYZE, RRID:SCR_009120)
   Data were fitted with Prism v8 software (Graphpad Inc., San Diego, CA) to obtain the concentrations giving 50% toxicity.	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 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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 57 and 50. 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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   Version published to 10.1101/2021.08.13.454991v1 on bioRxiv

   Aug 16, 2021