Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: HuH7 cells were left untreated or were infected with HCoV-229E or MERS-CoV (MOI=1) for 3, 6, 12, or 24 h. Transcriptomic data (by RNA-seq, n=2) and proteomic data (by LC-MS/MS, n=2, three technical replicates per sample) were derived from samples obtained at the indicated time points p.i. and subsequently used to extract expression values for the KEGG pathway 04141 “protein processing in endoplasmic reticulum”. (A) Scatter plots show mean normalized protein / mRNA expression values for each component, fitted linear regression lines, confidence intervals and correlation coefficients for mock-infected HuH7 cells and HuH7 cells infected for 24 h. (B) Correlation matrix of Pearson’s r across all conditions. All p values are given in supplementary Table S1. (C) The heatmap shows mean ratio values of differentially expressed mRNAs or proteins based on significant differences (fold change ≥ 2, p ≤ 0.01) calculated from the two biological replicates. See also and Table S1.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Infection, RNA Sequencing Assay, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: Projection of normalized ratio values for transcriptomic (by RNA-seq) and proteomic (by LC-MS/MS) data derived in parallel from HuH7 cells infected for 24 h with HCoV-229E or MERS-CoV with a MOI=1 on the components of the KEGG pathway 04141 “protein processing in endoplasmic reticulum”. The left side of the boxes show mRNA values, right sides show protein values.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: RNA Sequencing Assay, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Infection

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: (A) Schematic overview of parameters used to monitor virus- and thapsigargin-mediated ER stress. (B) Schematic presentation of HCoV-229E infection of cells and/or treatment with thapsigargin for different periods of time as applied in this study. (C) HuH7 cells were left untreated or infected with HCoV-229E (MOI=1) for 24 h and treated with thapsigargin (1 μM) according to (B). Supernatants and RNA-isolated from the cell pellets were used to determine viral titers (upper graphs) and expression of HCoV-229E S gene-containing RNA (lower graphs). (D) Representative fluorescence microscopy images showing subcellular HCoV-229E replication sites (at 24 h p.i.) identified by nsp8- or double-strand RNA-specific antibodies in the presence or absence of thapsigargin. Cells were infected with virus and treated with thapsigargin (1 μM) for 24 h. (E) Total cell extracts from HuH7 cells infected with HCoV-229E (MOI=1) and treated with thapsigargin (1 μM) as shown in (B) were analyzed for the expression/modification of the indicated host cell and viral proteins by immunoblotting. (F) Quantification of immunoblot data from multiple experiments. (G) HuH7 cells were left untreated, or were infected with HCoV-229E (MOI=1) in the presence / absence of increasing concentrations of thapsigargin or DMSO as solvent control. After 24 h, cell viability was assessed by MTS assay. (H) Data from (G) were used to compute half maximal cellular cytotoxicity (CC 50 ) concentrations of thapsigargin. (I) HuH7 cells were treated and infected according to (B). 30 min before harvesting, puromycin (3 μM) was added to the cells as indicated. Total cell extracts were analyzed by immunoblotting. The blot membrane was stained with CBB to assess the steady state proteomes and then hybridized with anti-puromycin antibodies to detect de novo synthesized nascent polypeptides. The upper graphs show representative images and the lower graph shows the quantification of multiple replicates. Puromycin signals of each lane were normalized to the corresponding CBB staining and were background corrected by subtracting signals of samples in which puromycin had been omitted. Data points show values from independent biological replicates, error bars show s.d.. Asterisks indicate p values (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001) obtained by two-tailed unpaired t-tests.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Infection, Isolation, Expressing, Fluorescence, Microscopy, Modification, Western Blot, MTS Assay, Staining, Synthesized, Two Tailed Test

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: (A-D) Human embryonic MRC-5 lung fibroblasts were infected with HCoV-229E according to the scheme shown in . Viral titers (A, upper graph), and expression of viral S gene-containing RNAs (A, lower graph), viral and host cell proteins (B, C) and cell viability (D) were analyzed and quantified as described in the legend of . (E-K) Similarly, HuH7 cells or Vero E6 African green monkey kidney epithelial cells were infected with MERS-CoV (MOI=0.5) or SARS-CoV-2 (MOI=0.5) for 12 h or 24 in the presence / absence of 0.4 μM or 1 μM thapsigargin. (E, F) show viral titers and (G, H) the corresponding expression of MERS-CoV / SARS-CoV-2 nucleocapsid (N) and host cell proteins, respectively. (I) Dose-dependent suppression of MERS-CoV-2 replication by thapsigargin (upper graph) and the estimated effective inhibitory concentration (EC 50 ) in HuH7 cells infected with an MOI of 0.5 (lower graph). (J) Dose-dependent suppression of SARS-CoV-2 replication by thapsigargin (left graph) and the calculated effective inhibitory concentration (EC 50 ) in Vero E6 cells infected with an MOI of 0.5 (right graph). (K) The CC 50 of thapsigargin in Vero E6 cells was calculated by MTS assays as described in the legend of . Data points show values from independent biological replicates, error bars show s.d.. Asterisks indicate p values (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001) obtained by two-tailed unpaired t-tests. See for quantifications from replicates for MERS-CoV / SARS-CoV-2 immunoblot experiments.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Infection, Expressing, Concentration Assay, Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: Inhibiting coronavirus replication in cultured cells by chemical ER stress

doi: 10.1101/2020.08.26.266304

Figure Lengend Snippet: (A) Venn diagram demonstrating the overlap of orthologues proteins expressed in HuH7 and Vero cells based on the NCBI gene IDs corresponding to majority protein IDs. (B) The overlap of virus- and thapsigargin-regulated proteins common to HuH7 and Vero E6 cells was calculated based on gene IDs. This analysis identifies 180 proteins with higher and 61 proteins with lower expression in thapsigargin-treated infected cells compared to virus infection alone (ratio > 0, p ≥ −log 10 1.3). (C) Heatmaps showing individual mean ratio values of log 2 -transfomed normalized protein intensities for the top 50 up- or downregulated proteins in virus-infected and thapsigargin-treated cells. Ratio values of infected or thapsigargin-treated conditions compared to untreated cells (-) cells are shown for comparison. Note that ratios are sorted and color coded according to the virus plus thapsigargin conditions with infection alone conditions set as denominator. Green colors highlight HERPUD1 and BiP (HSPA5) values. Orange colors highlight SQSMT1 which was also identified as SARS-CoV-2-regulated protein in an independent study ( Stukalov et al ., 2020 ). (D) Overrepresentation analysis showing the top 10 pathways mapping to gene IDs with increased (180 proteins, red) or decreased (61 proteins, blue) expression levels in thapsigargin-treated and infected cells compared to virus infection alone. Gene ID lists were analyzed using Metascape software ( Zhou et al ., 2019 ). (E) Experimental evidence, co-occurrence, co-expression and confidence scores from the STRING database ( Szklarczyk et al , 2019 ) were used to identify protein:protein interactions (PPI) amongst the 180 up- and 61 downregulated thapsigargin-sensitive proteins. As shown, based on experimental evidence and combined STRING score criteria, 59 and 26 coregulated proteins are engaged in defined PPI networks; the remaining proteins are not known to interact. (F) Validation of thapsigargin-induced HERPUD1 and CTH upregulation by immunoblotting of HuH7 or Vero E6 whole cell extracts from cells treated as described above. BiP and IRE1α levels are shown for comparison. (G) Quantification of thapsigargin-mediated re-expression of HERPUD1 and CTH in cells infected with HCoV-229E, MERS-CoV or SARS-CoV-2 from two independent immunoblot experiments. Error bars show s.d.. (H) Heatmap showing thapsigargin-reprogrammed proteins of KEGG 04141 (mean ratio ≥ 1.5 fold) along with p values. See also for projection of thapsigargin-mediated protein changes on the KEGG pathway map. (I) Venn diagram showing the intersection of thapsigargin- / virus-regulated proteins with all novel ERAD components (FDR of 1 %) identified by ( Leto et al ., 2019 ). The regulation of 31 overlapping components is shown as a heatmap displaying mean ratio values in thapsigargin-treated or infected cells. Red colors highlight UBA5 and ZNF622 as discussed in the text. (J) Summary of the main findings of our study. Abbreviations: M, MERS-CoV; S, SARS-CoV-2; T, thapsigargin.

Article Snippet: Primary antibodies against the following proteins or peptides were used: anti β-actin (Santa Cruz, #sc-4778), anti PERK (Santa Cruz, #sc-377400), anti PERK (Abcam, #ab65142), anti BiP (Cell Signaling, #3177), anti eIF2α (Cell Signaling #9722), anti P(S51)-eIF2α (Cell Signaling #9721), anti P(S724)-IRE1α (Novus Biologicals, #NB100-2323), anti IRE1α (Santa Cruz, #sc-390960), anti ATF4 (Santa Cruz, #sc-390063), anti ATF3 (Santa Cruz, #sc-188), anti HERPUD1 antibody (Abnova, #H00009709-A01), anti CTH antibody (Cruz, #sc-374249), anti HCoV-229E N protein ((Ingenasa, Batch 250609), mouse anti HCoV-229E nsp12 (gift from Carsten Grötzinger), rabbit anti HCoV-229E nsp8 , anti MERS-CoV N protein (Sinobiological, #100213-RP02), rabbit anti SARS-CoV N protein cross-reacting with SARS-CoV-2 N protein (gift from Friedemann Weber), anti SARS-CoV-2 N protein (Rockland, #200-401-A50), anti puromycin (Kerafast Inc., 3RH11, #EQ 0001), anti J2 (SCICONS, English & Scientific Consulting Kft, #10010200).

Techniques: Expressing, Infection, Software, Western Blot

Journal: Frontiers in Immunology

Article Title: Immune responses during COVID-19 breakthrough cases in vaccinated children and adolescents

doi: 10.3389/fimmu.2024.1372193

Figure Lengend Snippet: Specific IgG levels against different SARS-CoV-2 proteins in plasma of breakthrough cases of children and adolescents. Titers of specific IgG levels against different SARS-CoV-2 in twelve breakthrough cases. (A) Antibody titer against S1 protein. (B) Antibody titer against N protein. (C) Antibody titer against M protein. (D) Antibody titer against E protein. (E) Antibody titer against NSP8 protein. An in-house indirect ELISA assay was used to assess plasma IgG specific antibody titers against these proteins. Data were transformed, plotted as reciprocal dilution in log base 2 on a linear scale. Circles, triangles and squares correspond to subjects 3-5 yo, 6-11 yo and 12-17 yo, respectively. On the other hand, the black, blue and red colors represent the samples evaluated at pre-immune, 2 nd dose + 4 weeks and 2 to 8 weeks post-infection, respectively, by before-after graph. Black values below the significance line indicate geometric mean titers (GMT), blue values above the significance line indicate an increase in GMT of the two time points compared, and red values above the significance line indicate a decrease in GMT of the two time points compared. The data was analyzed using a mixed effects model, ***p<0.001; **p<0.01; *p<0.05, not significant (ns).

Article Snippet: Total IgG titers for the structural proteins Spike (S1: subunit 1) (SinoBiological, #40591-V08B1), Nucleocapsid (N) (R&D systems, #10474-CV), Membrane (M) (R&D systems, #10690-CV), and Envelope (E) (SinoBiological, #40609-V10E3), as well as the Non-Structural Protein 8 (NSP8) (R&D systems, #10633-CV) of SARS-CoV-2, were tested by in-house indirect ELISA assays, as previously described ( ).

Techniques: Clinical Proteomics, Indirect ELISA, Transformation Assay, Infection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Incipient functional SARS-CoV-2 diversification identified through neural network haplotype maps

doi: 10.1073/pnas.2317851121

Figure Lengend Snippet: Haplotype composition of the master neuron in the nsp12 and spike amplicons that define seven SOM classes

Article Snippet: The pRSFDuet-1 plasmid [(14his-nsp8-nsp7)(nsp12), from the Wuhan isolate] optimized for expression in Escherichia coli , and obtained from Addgene (Plasmid #165451) , was used to coexpress 14his-nsp8-nsp7-nsp12 complex.

Techniques: Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Incipient functional SARS-CoV-2 diversification identified through neural network haplotype maps

doi: 10.1073/pnas.2317851121

Figure Lengend Snippet: Composite self-organized haplotype maps (SOMs) of amplicons of the nsp12-coding region ( A ) and of the spike (S)-coding region ( B ) of SARS-CoV-2 from the Madrid cohort. The coding region, amplicon number, and SOM class are indicated in the boxes at the Left of each panel. The number of haplotypes that compose each composite map and the number of neurons and the peak size distribution (plots on the Right ) are shown. For plot numbers 1, 3, 4, 5, 7, and 11 (numbers encircled in each plot), the distributions could be fitted to an exponential decay function (purple lines) of the form y = y 0 + A × e (−x/t) (R 2 ≥ 0.9). However, for plot numbers 2, 8, and 10, the distributions could not fit this function optimally (R 2 < 0.9). For plot numbers 6, 9, and 12, the method did not converge. The SOMs for each individual patient are depicted in SI Appendix , Figs. S2–S7 in https://saco.csic.es/index.php/s/smeN9oSSgMMxDLw ).

Article Snippet: The pRSFDuet-1 plasmid [(14his-nsp8-nsp7)(nsp12), from the Wuhan isolate] optimized for expression in Escherichia coli , and obtained from Addgene (Plasmid #165451) , was used to coexpress 14his-nsp8-nsp7-nsp12 complex.

Techniques: Amplification

Journal: Journal of Molecular Biology

Article Title: Fidelity of Ribonucleotide Incorporation by the SARS-CoV-2 Replication Complex

doi: 10.1016/j.jmb.2023.167973

Figure Lengend Snippet: Biochemical determinants of SARS-CoV-2 nsp12 activity. (A) Purified recombinant nsp12, nsp7 and nsp8. (B) Schematic showing the structure of the double-stranded RNA substrate that served as a template for primer extension. (C) Enzyme activity is highest at 10 mM-50 mM KCl. Subsequent steady-state reactions were carried out with 50 mM KCl. (D and E) The effect of pH or Mg 2+ /Mn 2+ concentration on nsp12 activity. All reactions are carried out with 5 mM Mn 2+ at pH 7.5. (F) Single rUTP incorporation with double-stranded RNA template. Results showed a ∼7.5-fold higher activity with Mn 2+ than Mg 2+ . Error bars in C, D, E and F represent standard deviation of the mean (n = 3)

Article Snippet: SARS-CoV-2 nsp7 (Addgene #154757) and nsp8 (Addgene #154758) were overexpressed separately in Escherichia Coli BL21(DE3) Arctic Express cells at 16 °C for 16hrs.

Techniques: Activity Assay, Purification, Recombinant, Concentration Assay, Standard Deviation

Journal: Journal of Molecular Biology

Article Title: Fidelity of Ribonucleotide Incorporation by the SARS-CoV-2 Replication Complex

doi: 10.1016/j.jmb.2023.167973

Figure Lengend Snippet: Mutations on nsp12 and nsp14 that correlate with a higher mutational load. (A) V435 and P918 (green) are on the region of nsp12 (red) that interacts with nsp14 (orange) to form a trans dimer for proofreading (PDB code: 7EGQ). (B-E) PDB code: 7EIZ. (B) G228 (green) is on the region of nsp12 (red) that interacts with nsp9 (blue). (C) V330, V675 and S384 (green) are on the interaction region between nsp12 (red) and nsp8 (blue). (D) H26, A23 and M57 (green) on nsp14 (orange) that interact with nsp10 (blue). (E) Y154 (green) on nsp14 (orange) interacts with nsp12 (red). (F) M501 (green) is on the region of nsp14 (orange) that interacts with nsp12 (magenta) to form a trans dimer for proofreading (PDB code: 7EGQ).

Article Snippet: SARS-CoV-2 nsp7 (Addgene #154757) and nsp8 (Addgene #154758) were overexpressed separately in Escherichia Coli BL21(DE3) Arctic Express cells at 16 °C for 16hrs.

Techniques:

Journal: Microbiology Spectrum

Article Title: Expression Profile and Localization of SARS-CoV-2 Nonstructural Replicase Proteins in Infected Cells

doi: 10.1128/spectrum.00744-22

Figure Lengend Snippet: Construction of SARS-CoV-2 nonstructural replicase genes

Article Snippet: The following commercial antibodies were used in the study: mouse anti-dsRNA monoclonal antibody J2 (SCICONS #10010200, 1:1,000); rabbit anti-SARS nsp8 (Rockland #100-401-A53, 1:600); rabbit anti-SARS nsp13 (Rockland #100-401-A54, 1:600); rabbit anti-SARS N protein (Rockland #200-401-A50, 1:1,000); goat anti-mouse antibody conjugated with Alexa Fluor 488 (Thermo #A-28175, 1:1,000); goat anti-mouse antibody conjugated with Alexa Fluor 555 (Thermo #A-21424, 1:1,000); goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Thermo #A-11034, 1:1,000); and goat anti-rabbit antibody conjugated with Alexa Fluor 555 (Thermo #A-32732, 1:1,000).

Techniques: Clone Assay

Journal: Microbiology Spectrum

Article Title: Expression Profile and Localization of SARS-CoV-2 Nonstructural Replicase Proteins in Infected Cells

doi: 10.1128/spectrum.00744-22

Figure Lengend Snippet: Expression and subcellular distribution of SARS-CoV-2 nonstructural proteins in A549-ACE2 cells. A549-ACE2 cells were mock infected or SARS-CoV-2-infected at an MOI of 2. (A) Cells were harvested at 24-h postinfection (hpi), total cell lysates were prepared, and proteins were separated by 10% SDS-PAGE and analyzed by Western blot with home-made mouse anti-SARS-CoV-2 nsp1, nsp2, nsp3, nsp5, nsp8, and nsp9 serum, followed by incubating with horseradish peroxidase (HRP)-conjugated goat anti-rabbit and developed using SuperSignal West Pico chemiluminescent substrate. Numbers on the right indicate protein sizes in kDa. (B) At 6 hpi, the cells were fixed, permeabilized, and costained with rabbit anti-SARS nucleocapsid (N) protein polyclonal antibody (pAb) and the appropriate home-made mouse anti-SARS-CoV-2 nsp sera, followed by staining with Alexa Fluor 488 conjugated goat anti-rabbit Ab (green) and Alexa Fluor 555 conjugated goat anti-mouse Ab (red). Cell nuclei were stained with DAPI (blue) and examined by confocal microscopy; scale bars represent 10 μm.

Article Snippet: The following commercial antibodies were used in the study: mouse anti-dsRNA monoclonal antibody J2 (SCICONS #10010200, 1:1,000); rabbit anti-SARS nsp8 (Rockland #100-401-A53, 1:600); rabbit anti-SARS nsp13 (Rockland #100-401-A54, 1:600); rabbit anti-SARS N protein (Rockland #200-401-A50, 1:1,000); goat anti-mouse antibody conjugated with Alexa Fluor 488 (Thermo #A-28175, 1:1,000); goat anti-mouse antibody conjugated with Alexa Fluor 555 (Thermo #A-21424, 1:1,000); goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Thermo #A-11034, 1:1,000); and goat anti-rabbit antibody conjugated with Alexa Fluor 555 (Thermo #A-32732, 1:1,000).

Techniques: Expressing, Infection, SDS Page, Western Blot, Staining, Confocal Microscopy

Journal: Microbiology Spectrum

Article Title: Expression Profile and Localization of SARS-CoV-2 Nonstructural Replicase Proteins in Infected Cells

doi: 10.1128/spectrum.00744-22

Figure Lengend Snippet: Time course of nsp3, nsp5, and nsp8 detection. A549-ACE2 cells were mock infected or infected with SARS-CoV-2 (MOI = 2), fixed at 2-, 3-, 4-, 6-, 8-, and 24-h postinfection (hpi), and costained with rabbit anti-SARS nucleocapsid (N) protein pAb and home-made mouse anti-SARS-CoV-2 nsp3 (A), nsp5 (B), or nsp8 (C). Staining was carried out with Alexa Fluor 488 conjugated goat anti-rabbit Ab (green) and Alexa Fluor 555 conjugated goat anti-mouse Ab (red). Cell nuclei were stained with DAPI (blue) and examined by confocal microscopy; scale bars represent 10 μm. The intensity distribution describes the timing of expression of nsp3, nsp5 or nsp8 for specific fluorescence along the indicated line. (D) Pearson’s correlation was used to analyze the changes in nsp3, nsp5, or nsp8 over time. One-way analysis of variance (ANOVA) was used for multiple comparisons between different times among the nsp8 or nsp13 in GraphPad Prism 8.4.3 software. ****, P < 0.0001; **, P < 0.01.

Article Snippet: The following commercial antibodies were used in the study: mouse anti-dsRNA monoclonal antibody J2 (SCICONS #10010200, 1:1,000); rabbit anti-SARS nsp8 (Rockland #100-401-A53, 1:600); rabbit anti-SARS nsp13 (Rockland #100-401-A54, 1:600); rabbit anti-SARS N protein (Rockland #200-401-A50, 1:1,000); goat anti-mouse antibody conjugated with Alexa Fluor 488 (Thermo #A-28175, 1:1,000); goat anti-mouse antibody conjugated with Alexa Fluor 555 (Thermo #A-21424, 1:1,000); goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Thermo #A-11034, 1:1,000); and goat anti-rabbit antibody conjugated with Alexa Fluor 555 (Thermo #A-32732, 1:1,000).

Techniques: Infection, Staining, Confocal Microscopy, Expressing, Fluorescence, Software

Journal: Microbiology Spectrum

Article Title: Expression Profile and Localization of SARS-CoV-2 Nonstructural Replicase Proteins in Infected Cells

doi: 10.1128/spectrum.00744-22

Figure Lengend Snippet: Time course of nsp8 and nsp13 detection. A549-ACE2 cells were mock infected or infected with SARS-CoV-2 (MOI 2), fixed at 2-, 3-, 4-, 6-, 8-, and 24-h postinfection (hpi), and costained with home-made mouse anti-SARS-CoV-2 N protein serum and rabbit anti-SARS nsp8 (A) or nsp13 (B) polyclonal, followed by staining with goat anti-mouse secondary antibody conjugated with Alexa Fluor 488 (green) and goat anti-rabbit secondary antibody conjugated with Alexa Fluor 555 (red). The cell nuclei were stained with DAPI (blue) and examined by confocal microscopy. No specific signal was observed at 2 and 3 hpi (not shown); scale bars represent 10 μm. The intensity distribution describes the timing of expression of nsp8 or nsp13 for specific fluorescence along the indicated line. (C) Pearson’s correlation was used to analyze changes in nsp8 or nsp13 over time. One-way analysis of variance (ANOVA) was used for multiple comparisons between different times among the nsp8 or nsp13 in GraphPad Prism 8.4.3 software. ****, P < 0.0001.

Article Snippet: The following commercial antibodies were used in the study: mouse anti-dsRNA monoclonal antibody J2 (SCICONS #10010200, 1:1,000); rabbit anti-SARS nsp8 (Rockland #100-401-A53, 1:600); rabbit anti-SARS nsp13 (Rockland #100-401-A54, 1:600); rabbit anti-SARS N protein (Rockland #200-401-A50, 1:1,000); goat anti-mouse antibody conjugated with Alexa Fluor 488 (Thermo #A-28175, 1:1,000); goat anti-mouse antibody conjugated with Alexa Fluor 555 (Thermo #A-21424, 1:1,000); goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Thermo #A-11034, 1:1,000); and goat anti-rabbit antibody conjugated with Alexa Fluor 555 (Thermo #A-32732, 1:1,000).

Techniques: Infection, Staining, Confocal Microscopy, Expressing, Fluorescence, Software

Journal: Microbiology Spectrum

Article Title: Expression Profile and Localization of SARS-CoV-2 Nonstructural Replicase Proteins in Infected Cells

doi: 10.1128/spectrum.00744-22

Figure Lengend Snippet: Colocalization of nsp8 with other replicase proteins and dsRNA in SARS-CoV-2-infected cells. (A) A549-ACE2 cells were infected with SARS-CoV-2 (MOI = 2), fixed at 6-h postinfection (hpi), and costained with rabbit anti-SARS nsp8 and appropriate home-made mouse anti-SARS-CoV-2 nsp sera or mouse anti-dsRNA MAb, followed by staining with Alexa Fluor 488-conjugated goat anti-rabbit (green) and Alexa Fluor 555-conjugated goat anti-mouse (red). Cell nuclei were stained with DAPI (blue) and examined by confocal microscopy. Images in the fifth column were obtained at higher magnification to show single-cell details of fluorescence labeling; scale bars represent 10 μm. The intensity distribution describes the colocalization of nsp8 with other replicase proteins for specific fluorescence along the indicated line. (B) Pearson’s correlation analysis demonstrated colocalization of nsp8 with other replicase proteins. One-way analysis of variance (ANOVA) was used for multiple comparisons on the colocalization between nsp8 and different nsps in GraphPad Prism 8.4.3 software. ****, P < 0.0001; ***, P < 0.001.

Article Snippet: The following commercial antibodies were used in the study: mouse anti-dsRNA monoclonal antibody J2 (SCICONS #10010200, 1:1,000); rabbit anti-SARS nsp8 (Rockland #100-401-A53, 1:600); rabbit anti-SARS nsp13 (Rockland #100-401-A54, 1:600); rabbit anti-SARS N protein (Rockland #200-401-A50, 1:1,000); goat anti-mouse antibody conjugated with Alexa Fluor 488 (Thermo #A-28175, 1:1,000); goat anti-mouse antibody conjugated with Alexa Fluor 555 (Thermo #A-21424, 1:1,000); goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Thermo #A-11034, 1:1,000); and goat anti-rabbit antibody conjugated with Alexa Fluor 555 (Thermo #A-32732, 1:1,000).

Techniques: Infection, Staining, Confocal Microscopy, Fluorescence, Labeling, Software

Journal: Microbiology Spectrum

Article Title: Expression Profile and Localization of SARS-CoV-2 Nonstructural Replicase Proteins in Infected Cells

doi: 10.1128/spectrum.00744-22

Figure Lengend Snippet: Interaction of nsp8 with other replicase proteins. HEK-293T cells were transfected with equal amounts (2 μg) of the empty vector pRK5-Flag alone (as a control), pRK5-nsp8-Flag, and pRK5-nsp7 (A)/nsp8-Myc (B). Whole-cell lysates (WCLs) were pre-adsorbed onto Dynabeads protein G and incubated with anti-Flag antibodies for coimmunoprecipitation analysis. The protein complex was detected by a horseradish peroxidase (HRP)-conjugated anti-Flag MAb and HRP-conjugated anti-Myc MAb.

Article Snippet: The following commercial antibodies were used in the study: mouse anti-dsRNA monoclonal antibody J2 (SCICONS #10010200, 1:1,000); rabbit anti-SARS nsp8 (Rockland #100-401-A53, 1:600); rabbit anti-SARS nsp13 (Rockland #100-401-A54, 1:600); rabbit anti-SARS N protein (Rockland #200-401-A50, 1:1,000); goat anti-mouse antibody conjugated with Alexa Fluor 488 (Thermo #A-28175, 1:1,000); goat anti-mouse antibody conjugated with Alexa Fluor 555 (Thermo #A-21424, 1:1,000); goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Thermo #A-11034, 1:1,000); and goat anti-rabbit antibody conjugated with Alexa Fluor 555 (Thermo #A-32732, 1:1,000).

Techniques: Transfection, Plasmid Preparation, Incubation