For infection, Matrigel was liquefied and removed on ice and organoids were broken up by repeated resuspension using a disposable syringe with needle (27G). of samples was measured in (A) and (B), vaccinees = 19, non-VOC convalescent donors = 50, B1.1.7 patients = 13. PRNT, plaque reduction neutralization test; VOC, variant of concern. Observe S1 Rabbit Polyclonal to PDGFRb (phospho-Tyr771) Data.(TIF) pbio.3001871.s002.tif (247K) GUID:?EF724E3C-A7C8-4957-80CA-A09A812D886B S3 Fig: Relative sgRNA level normalized to total RNA reads and infection efficiency in B.1- and VOC Alpha-infected Calu-3 cells. (A) RNA-seq analysis was conducted from total cell lysates that were obtained 24 hours postinfection to quantify sgRNA proportions in SARS-CoV-2-infected cells (MOI of 2). Canonical, as well as ORF9b and N* sgRNAs were quantified from your RNA-seq dataset. Data were normalized to PK68 total RNA reads. (B, C) Quantity of SARS-CoV-2 nucleocapsid (N)-positive Calu-3 cells was determined by flow cytometry. Calu-3 were left either UI or were infected with B.1 and VOC Alpha (MOI of 2) for 24 hours, permeabilized and immunostained with rabbit-anti-SARS-CoV-2 nucleocapsid antibody, followed by goat anti-rabbit Alexa 488 secondary antibody. (B) Percentage of SARS-CoV-2 N-positive cells. (C) Gating strategy of living-, single-, and N-positive cells is usually depicted for UI, B.1-, and VOC Alpha-infected cells. MOI, multiplicity of contamination; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; sgRNA, subgenomic RNA; UI, uninfected; VOC, variant of concern. Observe S1 Data.(TIF) pbio.3001871.s003.tif (1.5M) GUID:?6863EA8F-6260-425E-AFFA-5044FDBFEE51 S4 Fig: Growth, replication, and gene expression in infected, parental, and ACE2/TMPRSS2-expressing A549 cells. (A) Computer virus growth was quantified in parental and ACE2/TMPRSS2-expressing A549 cells infected at an MOI of 0.01. Supernatant collected at the respective time points was titrated by plaque assay on Vero E6 cells. Growth kinetic experiments were performed once in triplicates. (B) Expression of cell-associated was decided in parental and ACE2/TMPRSS2-expressing A549 cells at 24 and 48 hours postinfection by Q-RT-PCR. (C) Expression of cell-associated sgN in Calu-3 cells at 24 and 48 hours postinfection was determined by Q-RT-PCR. TBP was utilized for normalization. (D) Expression of the indicated genes was determined by Q-RT-PCR. Shown is the mean fold switch +/? SD of 3 biologically impartial experiments that were each conducted in quadruplicates. RVFV cl.13, which is devoid of its IFN antagonist NSs, was included for the analysis of expression of and gene locus of NCI-H1299 and ACE2-KO-H1299 cells, expressing ACE2 protein from aa no. 220 ff. Knock-out of in ACE2-KO-H1299 cells was induced by CRISPR/Cas9 technology by the use of a synthetic guideline RNA (sgRNA) targeting the genomic region PK68 as indicated in the chromatogram. A frameshift mutation was generated through the insertion of 2 nucleotides, which leads to the expression of a PK68 truncated ACE2 protein (from aa 225 ff) due to PK68 a premature quit codon in the was utilized for normalization. (C) Expression of the indicated genes was determined by specific Q-RT-PCR. was utilized for normalization. Shown is the mean fold switch +/? SD of 3 biologically impartial experiments that were each conducted in quadruples. RVFV cl.13, which is devoid of its IFN antagonist NSs, was included for the expression of IFNs, ISGs, and pro-inflammatory cytokines. GE, genome equivalents; IFN, interferon; ISG, IFN-stimulated gene; Q-RT-PCR, quantitative real-time PCR; RVFV cl.13, Rift Valley Fever Computer virus clone 13; sgN, subgenomic nucleocapsid; TBP, TATA-binding protein. Observe S1 Data.(TIF) pbio.3001871.s010.tif (504K) GUID:?5E63C267-6299-4244-8BF4-9EA04E5839AB S11 Fig: Evaluation of titration method on Vero E6 and Calu-3 cells for the calibration of B.1 and VOC Alpha seeding doses and comparison of E gene copy figures versus infectious models (determined by Q-RT-PCR and plaque assay on Vero E6 cells) in PK68 SARS-CoV-2 stocks. (A) No significant differences in the titers were observed when titers of B.1 and VOC Alpha SARS-CoV-2 stocks were compared on Calu-3 versus Vero E6 using TCID50 titration method. Although plaque morphology of B.1- and VOC Alpha-infected Vero E6 cells differ, Vero E6 cells are suitable to determine titers by plaque titration assay. = 3 biologically impartial experiments each conducted in triplicates. (B) Overview on computer virus infectivity and viral RNA concentrations, determined by plaque assay (log10 PFU/ml) and E gene assay (log10 GE/ml), of all virus stocks is usually shown. Direct comparison of all B.1 and VOC Alpha stocks. Statistical analysis was.
For infection, Matrigel was liquefied and removed on ice and organoids were broken up by repeated resuspension using a disposable syringe with needle (27G)