ZBP1 induces inflammatory signaling via RIPK3 and promotes SARS-CoV-2-induced cytokine expression

Article activity feed

1. 

   ScreenIT

   Oct 5, 2021

   SciScore for 10.1101/2021.10.01.462460: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: Isolation of primary neutrophils: Primary human neutrophils were obtained from healthy donors with their written informed consent by The Oxford Radcliffe Biobank with project number ORB 20/A136. IRB: The study is authorized by South Central – Oxford C Research Ethics Committee (Ref# 19/SC/0173).
   Sex as a biological variable	not detected.
   Randomization	For cDNA synthesis, around 1 µg RNA (10 µl) was incubated with 1 µl 10 µM random pentadecamers (IDT 169190224) and 0.5 µl 100µM anchored oligo(dT)20 primer (Sigma, custom synthesized) at 65°C for 5 minutes, before supplemented with 0.5 µl RevertAid reverse transcriptase (Thermo Scientific EP0441) and 0.5 µl RiboLock RNase inhibitor (Thermo …

   SciScore for 10.1101/2021.10.01.462460: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: Isolation of primary neutrophils: Primary human neutrophils were obtained from healthy donors with their written informed consent by The Oxford Radcliffe Biobank with project number ORB 20/A136. IRB: The study is authorized by South Central – Oxford C Research Ethics Committee (Ref# 19/SC/0173).
   Sex as a biological variable	not detected.
   Randomization	For cDNA synthesis, around 1 µg RNA (10 µl) was incubated with 1 µl 10 µM random pentadecamers (IDT 169190224) and 0.5 µl 100µM anchored oligo(dT)20 primer (Sigma, custom synthesized) at 65°C for 5 minutes, before supplemented with 0.5 µl RevertAid reverse transcriptase (Thermo Scientific EP0441) and 0.5 µl RiboLock RNase inhibitor (Thermo Scientific EO0381) in a total volume of 20 µl RevertAid Reverse Transcriptase buffer (Thermo Scientific LT-02241).
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cells were routinely checked for Mycoplasma Spp. Authentication: Cell lines were not authenticated.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Primary antibodies used for Western blotting in this study include: anti-ZBP1 (Cell Signaling 60968), anti-Actin, clone C4 (Millipore MAB1501), anti-RIPK3 (Cell Signaling 13526), anti- phospho-p65 (Cell Signaling 3033), anti-p65 (Cell Signaling 8242), anti-phospho-p38 (Cell Signaling 4511), anti-p38 (Cell Signaling 9212), anti-phospho-JNK (BD 612540), anti-JNK (Cell Signaling 9258), anti-phospho-RIPK3 (S227) (Abcam ab209384), anti-RIPK1 (Cell Signaling 3493S), anti-Ubiquitin (rabbit, Cell Signaling, 43124), anti-Ubiquitin (mouse, Cell Signaling 3936), anti-GFP (Cell Signaling 2555), anti-CYLD (Cell Signaling 8462), anti- OTULIN (Cell Signaling 14127), anti-HOIP (R&D AF8039), anti-M1-Ub (kindly provided by David Komander and Rune Busk Damgaard), anti-HOIL-1L (Novus NBP1-88301), anti-SHARPIN (Protein tech 14626-1-AP), anti-TAK1 (Cell Signaling 4505), anti-IKK β (Cell Signaling 8943), anti-K63-Ub (Cell signaling 5621), anti-cIAP1 (Enzo ALX-803-335), anti-FLIP (Cell Signaling 3210), anti-Cleaved caspase-8 (Cell Signaling 9496), anti-Caspase-8 (D35G2) (Cell Signaling 4790) and anti-Caspase-8 (C15) (Adipogen, AG-20B-0057).	anti-ZBP1 suggested: (Cell Signaling Technology Cat# 60968, RRID:AB_2799599) anti-Actin suggested: (Millipore Cat# MAB1501, RRID:AB_2223041) anti-Actin, clone C4 (Millipore MAB1501), anti-RIPK3 (Cell Signaling 13526) suggested: None anti- phospho-p65 (Cell Signaling 3033) suggested: None phospho-p65 suggested: None anti-p65 suggested: None anti-phospho-p38 suggested: (Cell Signaling Technology Cat# 4511, RRID:AB_2139682) anti-p38 suggested: (Cell Signaling Technology Cat# 9212, RRID:AB_330713) anti-phospho-JNK suggested: (Proteintech Cat# 80024-1-RR, RRID:AB_2882943) anti-JNK suggested: (Cell Signaling Technology Cat# 9258, RRID:AB_2141027) anti-phospho-RIPK3 suggested: (Affinity Biosciences Cat# AF3894, RRID:AB_2847208) S227 suggested: (Abcam Cat# ab209384, RRID:AB_2714035) anti-RIPK1 suggested: (Cell Signaling Technology Cat# 3493, RRID:AB_2305314) anti-Ubiquitin (rabbit, Cell Signaling, 43124) suggested: None anti-Ubiquitin suggested: (Cell Signaling Technology Cat# 3936, RRID:AB_331292) anti-GFP suggested: (Cell Signaling Technology Cat# 2555, RRID:AB_10692764) anti-CYLD (Cell Signaling 8462), suggested: (Cell Signaling Technology Cat# 8462, RRID:AB_10949157) anti-HOIP suggested: (Proteintech Cat# 16289-1-AP, RRID:AB_2878239) anti-M1-Ub suggested: None anti-HOIL-1L suggested: None anti-SHARPIN suggested: None anti-TAK1 suggested: (Cell Signaling Technology Cat# 4505, RRID:AB_490858) anti-IKK suggested: None anti-K63-Ub suggested: None anti-cIAP1 suggested: None anti-FLIP suggested: (Cell Signaling Technology Cat# 3210, RRID:AB_2080940) anti-Cleaved caspase-8 suggested: (Cell Signaling Technology Cat# 9496, RRID:AB_561381) anti-Caspase-8 suggested: (Cell Signaling Technology Cat# 4790, RRID:AB_10545768) D35G2) (Cell Signaling 4790) suggested: (Cell Signaling Technology Cat# 4790, RRID:AB_10545768) C15 suggested: (AdipoGen Cat# AG-20B-0057, RRID:AB_2490271)
   Secondary antibodies used for Western blotting in this study include: anti-Mouse IgG-HRP (Dako P0447), anti-Rabbit IgG-HRP (Bio-Rad 1706515), anti-Sheep IgG-HRP (R&D HAF016), anti-human IgG-HRP (Bio-Rad 172-1033) and anti-Rat IgG-HRP (Thermo Fisher 31470).	anti-Mouse IgG-HRP suggested: None anti-Rabbit IgG-HRP suggested: None anti-Sheep IgG-HRP suggested: (Santa Cruz Biotechnology Cat# sc-2924, RRID:AB_656969) anti-human IgG-HRP suggested: None anti-Rat IgG-HRP suggested: (Thermo Fisher Scientific Cat# PA1-31470, RRID:AB_1954744)
   Experimental Models: Cell Lines
   Sentences	Resources
   After selection with 1 µg/ml puromycin (Invitrogen ant-pr) in the presence of 5 µg/ml blasticidin, HCT116 / Tet-On-GFP, HCT116 / Tet-On-GFP-K63-SUB and HCT116 / Tet-On-GFP-M1-SUB cells were transduced with retroviral particles produced from LZRS-zeo-RIPK3-2xFV plasmids and selected with the combination of 5 µg/ml blasticidin, 1 µg/ml puromycin and 250 ng/µl zeocin.	HCT116 suggested: None
   Production of lentiviral particles: For the production of lentiviral particles, HEK293FT cells were plated at a density of 3.5x106 cells in 10 cm dishes in 15 ml complete growth media.	HEK293FT suggested: RRID:CVCL_6911)
   Generation of HT29 cells with Dox-inducible expression of ZBP1: For lentivirus production, HEK293T cells were transfected with C-terminally FLAG-tagged wild-type or Zα1α2-mutant human ZBP1-expressing transducing vectors in the doxycycline- inducible Tet-On pDG2 backbone (De Groote et al, 2016) together with the pCMV delta R8.91 gag-pol–expressing packaging plasmids and pMD2.G VSV-G-expressing envelope plasmid.	HEK293T suggested: None
   HT29 and HT29 / RIPK1-KO clones were transduced using 100 µl 0.45 µm syringe-filtered lentivirus-containing supernatant in 12-well plate.	HT29 suggested: None
   For the transduction of lentiviral particles, HCT116 / RIPK3-2xFV cells were seeded in 12-well plates at a density of 0.5-1x105 cells per well and transduced by incubation with 10-20 μl precipitated virus in 1 ml complete growth medium containing 10 μg/ml polybrene for 24 h.	RIPK3-2xFV suggested: None
   SARS-CoV-2 infection of Calu-3 cells: The Wuhan-like early European SARS-CoV-2 B.1, Freiburg isolate (FR4286, kindly provided by Professor Georg Kochs, University of Freiburg) (Hoffmann et al, 2020), was propagated in Vero cells expressing human TMPRSS2 (Olagnier et al, 2020) and virus titer determined by TCID50% as previously described (Fougeroux et al, 2021).	Vero suggested: None
   Calu-3 cells were seeded in flat-bottom 12-well plates (2x105 cells/well in 1 ml media) or flat-bottom 6-well plates (5x105 cells/well in 2 ml media).	Calu-3 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   HCT116/RIPK1-KO clones used in this study were all generated with gRNAc.	HCT116/RIPK1-KO suggested: None
   Recombinant DNA
   Sentences	Resources
   The next day, transfection was carried out with a mixture of 1.5 ml OptiMEM, 36 μl FuGENE HD (Promega E2311), 1.2 μg pMD.G (VSVG) plasmid and 10.8 µg of the retroviral vector, and media was replaced 24h after transfection.	pMD.G (VSVG) suggested: None
   Generation of HCT116 cells with inducible GFP-SUB expression: To generate HCT116 / Tet-On-GFP-SUB / RIPK3-2xFV cells, HCT116 cells were first transduced with lentiviral particles generated from pLenti-CMV-Blast plasmids carrying the Tet Repressor gene.	pLenti-CMV-Blast suggested: None
   HCT116 / Tet-On clone C4 was plated at a density of 5x104 cells per well in 12-well plates, and transduced with 3 µl precipitated lentiviral particles generated with pLVX-tight-puro plasmids encoding GFP, GFP-K63-SUB or GFP-M1-SUB sequences (Hrdinka et al, 2016).	pLVX-tight-puro suggested: RRID:Addgene_64844)
   After selection with 1 µg/ml puromycin (Invitrogen ant-pr) in the presence of 5 µg/ml blasticidin, HCT116 / Tet-On-GFP, HCT116 / Tet-On-GFP-K63-SUB and HCT116 / Tet-On-GFP-M1-SUB cells were transduced with retroviral particles produced from LZRS-zeo-RIPK3-2xFV plasmids and selected with the combination of 5 µg/ml blasticidin, 1 µg/ml puromycin and 250 ng/µl zeocin.	LZRS-zeo-RIPK3-2xFV suggested: None
   The next day, they were transfected with a mixture of 1.5 ml OptiMEM, 36 μl FuGENE HD, 6 μg psPAX2 vector, 1.5 μg pMD.G (VSVG) and 4.5 μg lentiviral vector.	psPAX2 suggested: RRID:Addgene_12260) pMD.G suggested: None
   Generation of HT29 cells with Dox-inducible expression of ZBP1: For lentivirus production, HEK293T cells were transfected with C-terminally FLAG-tagged wild-type or Zα1α2-mutant human ZBP1-expressing transducing vectors in the doxycycline- inducible Tet-On pDG2 backbone (De Groote et al, 2016) together with the pCMV delta R8.91 gag-pol–expressing packaging plasmids and pMD2.G VSV-G-expressing envelope plasmid.	pDG2 suggested: None pCMV delta R8.91 gag-pol–expressing suggested: None pMD2.G VSV-G-expressing suggested: None
   shRNA plasmids used for this study are pLKO.	pLKO suggested: RRID:Addgene_52920)
   The next day, they were transfected using per well 125 ng pBIIX-Luc (NF-κB reporter plasmid) (Saksela & Baltimore, 1993), 25 ng SV40- Renilla luciferase plasmid, 0.5 ng ZBP1-expressing pLenti6.3 plasmid (with pBabe-puro or pLenti6.3-GFP as control), and, where indicated, 100 ng pBabe-CYLD plasmids (with pBabe- puro as EV control), 100 ng pcDNA3-OTULIN plasmids (with pcDNA3 as EV control), or 10 ng pcDNA3-GFP-SUB plasmids, in a mixture with 20 μl OptiMEM and 1μl FuGENE HD or FuGENE 6 (Promega E2691) per well.	pBIIX-Luc suggested: None pLenti6.3 suggested: RRID:Addgene_120848) pBabe-puro suggested: RRID:Addgene_1764) pLenti6.3-GFP suggested: None pBabe-CYLD suggested: None pBabe- suggested: None pcDNA3-OTULIN suggested: None pcDNA3 suggested: RRID:Addgene_15475) pcDNA3-GFP-SUB suggested: None
   Software and Algorithms
   Sentences	Resources
   After stimulation, cells were washed twice with PBS and lysed in 500 µl/dish TBSN buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40) supplemented with protease inhibitor cocktail, phosphatase inhibitor cocktail and 50 mM N- Ethylmaleimide (NEM; Sigma Aldrich E1271) by ice incubation for a minimum of 15min.	NEM; Sigma Aldrich suggested: None
   Generation of standard curves and interpolation of data were performed in GraphPad Prism.	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 did not find any issues relating to the usage of bar graphs.

   ---

   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 10.1101/2021.10.01.462460 on bioRxiv

   Oct 1, 2021