Inhibiting LSD1 suppresses coronavirus-induced inflammation but spares innate antiviral activity

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   ScreenIT

   May 8, 2021

   SciScore for 10.1101/2021.05.02.441948: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: Blood donors provided written informed consent for the collection of samples and subsequent analysis.
   Sex as a biological variable	BMDM were obtained from bone marrow of 6-10 week old female C57Bl6 mice (Charles River).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: To assure mycoplasma-free conditions, all cells were routinely tested.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   CD14 cells were purified by anti-CD14 monoclonal antibody (mAb)-conjugated magnetic microbeads (Miltenyi Biotec) and then cultured for 6 days in RPMI 1640 medium (Life Technologies Invitrogen), supplemented with heat-inactivated 10% …

   SciScore for 10.1101/2021.05.02.441948: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: Blood donors provided written informed consent for the collection of samples and subsequent analysis.
   Sex as a biological variable	BMDM were obtained from bone marrow of 6-10 week old female C57Bl6 mice (Charles River).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: To assure mycoplasma-free conditions, all cells were routinely tested.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   CD14 cells were purified by anti-CD14 monoclonal antibody (mAb)-conjugated magnetic microbeads (Miltenyi Biotec) and then cultured for 6 days in RPMI 1640 medium (Life Technologies Invitrogen), supplemented with heat-inactivated 10% lipopolysaccharide-free FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin (all from EuroClone) in the presence of human recombinant M-CSF (100 ng/µL; Peprotech).	anti-CD14 suggested: None
   Slides were stained with primary antibodies diluted in 1% BSA in PBS (NF-kB and IRF1 1:400, NSP9 1:1000) for 90 min.	IRF1 suggested: None NSP9 suggested: None
   Cells labelled with DAPI and anti-NF-kB/NSP9 or anti-IRF1/NSP9 antibodies were imaged with a 60x 1.4 NA oil immersion objective lens on a CSU-W1 Yokogawa Spinning Disk confocal system with a 50 μm pinhole disk mounted on an Eclipse Ti2 stative and equipped with a motorized xyz stage, 6 solid state lasers, a multi-dichroic mirror, single emission filters and a Prime BSI sCMOS camera (TELEDYNE PHOTOMETRICS, Tucson, AZ 85706, USA).	anti-NF-kB/NSP9 suggested: None anti-IRF1/NSP9 suggested: None
   The immunoprecipitation was performed using magnetic Dynabeads Protein G. Beads were blocked with 0,5% BSA in PBS and then mixed with the different antibodies (15 µg antibody:100 µl beads ratio) and incubated overnight on a rotating platform at 4°C. 1% of Triton X-100 was added to the sonicated lysates and lysates were centrifuged in microfuge (8000g, 10 min. at 4C) to pellet debris.	antibody:100 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, supernatants from infected cells were 10-fold serially diluted and titrated on target cells plated at 80-90% confluence (for MHV, L929 cells; for SARS-CoV2, VeroE6 cells) in 96 wells in a total volume of 100 µl, (8 replicate wells for MHV, 10 replicate wells for SARS-CoV2) and incubated at 37 °C under 5% carbon dioxide.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   The plasmid was used to produce lentiviral particles in 293T cells.	293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
   Bone marrow derived macrophages or L929 cells were seeded on a 12 glass-bottom wells (MatTek Corporation, Ashland, MA 01721, USA), coated with poly-D-Lysine 0.1% (w/v) in water (3×105 cells/well).	L929 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Cell culture: Raw264.7, L929, LA4, Calu3 and VeroE6 cell lines were purchased by American Type Culture Collection and grown according to ATCC recommendations.	Raw264.7 suggested: None
   BMDM were obtained from bone marrow of 6-10 week old female C57Bl6 mice (Charles River).	C57Bl6 suggested: None
   Recombinant DNA
   Sentences	Resources
   Generation of LSD1 KO cells by CRISPR/Cas9: The sgRNA oligo (see Reagents table) 10, targeting exon 3 of Lsd1, was cloned into pSpCas9(BB)-2A-GFP, a gift from Feng Zhang (Addgene plasmid # 48138; http://n2t.net/addgene:48138; RRID:Addgene_48138) 52.	pSpCas9 suggested: RRID:Addgene_48137) detected: RRID:Addgene_48138)
   Forty-eight hours later, GFP-positive cells were single-cell sorted in 96-well plates and after clonal expansion, sublines were screened by Western blot against LSD1. RNAi Knockdown: To knock down LSD1, one short hairpin RNA (shRNA) sequences was tested. shLSD1#1 (see reagents table for sequence) was cloned into the pLKO vector by AgeI–Eco RI double digestion.	pLKO suggested: RRID:Addgene_52920)
   Software and Algorithms
   Sentences	Resources
   PolyIC was purchased from Cytiva and dissolved in PBS to a concentration of 1mg/ml.	PolyIC suggested: None
   UV inactivation was performed on ice, using Agilent Genomics/Stratagene Stratalinker 2400 UV Crosslinker, by delivering an energy dose equivalent to 0,3 Joules.	Agilent Genomics/Stratagene suggested: None
   Widefield images of the DAPI and Lysosensor were quantified using a custom-made ImageJ macro.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   RNA-seq data were mapped using STAR aligner 54 against the mouse genome (mm10).	STAR suggested: (STAR, RRID:SCR_004463)
   Counts were obtained by htseq-counts 55 and differential expression analysis was performed with DESeq2 package hosted in Galaxy online platform 56 using a false discovery rate (FDR) cut-off of 1 x 10-4 9.	Galaxy suggested: (Galaxy, RRID:SCR_006281)
   Then, BAM files for all samples were pooled and the coordinates of mapped reads were intersected (bedtools intersect 57) with those of TEs annotated in RepeatMasker (v405).	bedtools suggested: (BEDTools, RRID:SCR_006646) RepeatMasker suggested: (RepeatMasker, RRID:SCR_012954)
   Cluster counts for each sample were first normalised using size factors estimated from the number of reads uniquely mapping to Ensembl genes, then differential expression analysis was performed using the Wald Test and a design formula capturing the interaction between DDP treatment and MHV infection status (∼Infection*Treatment).	Cluster suggested: (Cluster, RRID:SCR_013505)
   For the remaining TE clusters, we then plotted the log2 fold changes estimated by DESeq2 as boxplots.	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   Viral genome analysis and assembly: To identify the MHV viral strain we used by the SPADES software 60.	SPADES suggested: (SPAdes, RRID:SCR_000131)

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   Results from OddPub: Thank you for sharing your data.

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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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   Results from scite Reference Check: We found no unreliable references.

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   Version published to 10.1101/2021.05.02.441948 on bioRxiv

   May 3, 2021