Journal: Nature Aging

Article Title: Removal of senescent cells reduces the viral load and attenuates pulmonary and systemic inflammation in SARS-CoV-2-infected, aged hamsters

doi: 10.1038/s43587-023-00442-w

Figure Lengend Snippet: A , Immunofluorescence analysis of Ace2 in the lung from uninfected (mock) young hamsters and aged hamsters. Bars: 25 μm. Right panel, The histograms indicate the mean ± s.d fold change relative to average intensity in young animals (n = 3). Significant differences were determined using the two tailed Mann Whitney U test . B and C , Representative photomicrographs of lungs from SARS-CoV-2- − infected young hamsters and aged hamsters (H&E staining) at 7 dpi ( B ), and 22 dpi ( C ). C , Arrow: inflammatory cell infiltrate; sun: type II pneumocyte hyperplasia. D , The lungs were stained with Sirius Red. Representative images are depicted (22 dpi). Stars indicate altered structure of epithelial and vascular basal membranes. B - D , Scales are indicated. One of two representative experiments is shown.

Article Snippet: For p16, ACE2 and viral nucleoprotein labeling (immunofluorescence), the following antibodies were used: p16 (sc-377412, 1:50, Santa Cruz Biotechnology), ACE2 (NBP1-76611, 1:50, Bio-Techne) and SARS-CoV nucleocapsid (NB100-56576, 1:50, Bio-Techne).

Techniques: Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Infection, Staining

Journal: Nature Aging

Article Title: Removal of senescent cells reduces the viral load and attenuates pulmonary and systemic inflammation in SARS-CoV-2-infected, aged hamsters

doi: 10.1038/s43587-023-00442-w

Figure Lengend Snippet: Young hamsters and aged hamsters were infected with SARS-CoV-2. Lungs were collected at 3 dpi, 7 dpi and 22 dpi ( n = 3–6). Left and middle: quantification of viral RdRp and E protein transcript levels using RT–PCR assays. The data are expressed as Δ C t and genome copy per microgram of RNA. Right: number of infectious virus particles per lung (50% tissue culture infectious dose, TCID 50 ). b , Immunohistochemistry analysis of spike. Scale bars, 20 μm. c , Leftl: immunofluorescence staining for DAPI (blue) and viral nucleoprotein (red) is shown. Scale bars, 25 μm. Right: the intensity of nucleoprotein signals was normalized by DAPI count. The histograms indicate the fold change relative to average intensity in young animals ( n = 6). d , Gene expression was quantified by RT–PCR (fold change relative to average gene expression in young animals) ( n = 3–6). e , Expression of AC2 and β-tubulin in young and aged whole lung homogenates as assessed by western blotting. Right: the relative protein levels normalized to β-tubulin are shown ( n = 6–8). f , Body weight loss curves (four aged hamsters and six young hamsters). g , Histopathological examination of lung sections (H&E staining). The mean sum of the subscores is shown ( n = 3–6). h , Representative photomicrographs at 7 dpi. Arrowhead: inflammatory cell infiltrate; star: alveolar wall rupture; sun: type II pneumocyte hyperplasia; thunderbolt: necrosis. Scale bars, 50 μm. i , Numbers of inflammatory foci per lung section ( n = 6). j , Percentage of Sirius Red labeling ( n = 5–10). a – i , One of two representative experiments is shown. j , A pool of two independent experiments is depicted. For all graphs, errors indicate mean ± s.d. Significant differences were determined using the two-tailed Mann–Whitney U- test ( a , c , d , e , g , i ) and the one-way ANOVA Kruskal–Wallis test (non-parametric), followed by Dunn’s post test ( j ). Significance of body weight regain (area under the curve) in infected young hamsters was calculated using the Wilcoxon matched-pairs signed-rank test ( f ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For p16, ACE2 and viral nucleoprotein labeling (immunofluorescence), the following antibodies were used: p16 (sc-377412, 1:50, Santa Cruz Biotechnology), ACE2 (NBP1-76611, 1:50, Bio-Techne) and SARS-CoV nucleocapsid (NB100-56576, 1:50, Bio-Techne).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Virus, Immunohistochemistry, Immunofluorescence, Staining, Expressing, Western Blot, Labeling, Two Tailed Test, MANN-WHITNEY

Journal: Nature Aging

Article Title: Removal of senescent cells reduces the viral load and attenuates pulmonary and systemic inflammation in SARS-CoV-2-infected, aged hamsters

doi: 10.1038/s43587-023-00442-w

Figure Lengend Snippet: a , Lungs from mock-infected and SARS-CoV-2-infected, aged hamsters and young hamsters were stained with a p16 antibody. Representative photomicrographs showing labeling of p16 are shown at 3 dpi, 7 dpi and 22 dpi. Scale bars, 20 μm. b , p16 labeling was performed on lung sections collected at 3 dpi. Scale bars, 25 μm. Bottom: the histograms indicate the fold change relative to average intensity in young animals ( n = 4–5). c , The mRNA expression level of Cdkn2a (encoding p16) was quantified by RT–PCR. The data are expressed as fold increase relative to average gene expression in mock-infected young hamsters ( n = 3–6). d , Aged hamsters and young hamsters were treated (or not) with ABT-263 and then infected with SARS-CoV-2. Arrows indicate p16-positive cells (3 dpi). Scale bars, 20 μm. e , Effect of ABT-263 treatment on p16 expression as assessed by immunofluorescence (3 dpi). Scale bars, 25 μm. Right: the intensity of p16 signals was normalized by DAPI count. The histograms indicate the fold change relative to average intensity in vehicle-treated infected, aged animals ( n = 5–10). f , The Cdkn2a transcript levels are indicated. The data are expressed as the fold increase relative to average gene expression in vehicle-treated infected young hamsters ( n = 5–6). g , Effect of ABT-263 treatment on the expression of genes related to SASP factors in infected, aged hamsters ( n = 3–4, 7 dpi). Heat map (hierarchical clustering) of the differences in expression of SASP factors, calculated using the difference between log intensity of ABT-263 and the control (fold change > 1.5, P < 0.01). a – f , One of two representative experiments is shown. g , One experiment performed. For all graphs, errors indicate mean ± s.d. Significant differences were determined using the two-tailed Mann–Whitney U- test ( b , e ), moderated t -test after Benjamani–Hochberg correction ( g ) and one-way ANOVA Kruskal–Wallis test (non-parametric), followed by Dunn’s post test ( c , f ). * P < 0.05, ** P < 0.01.

Article Snippet: For p16, ACE2 and viral nucleoprotein labeling (immunofluorescence), the following antibodies were used: p16 (sc-377412, 1:50, Santa Cruz Biotechnology), ACE2 (NBP1-76611, 1:50, Bio-Techne) and SARS-CoV nucleocapsid (NB100-56576, 1:50, Bio-Techne).

Techniques: Infection, Staining, Labeling, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Aging

Article Title: Removal of senescent cells reduces the viral load and attenuates pulmonary and systemic inflammation in SARS-CoV-2-infected, aged hamsters

doi: 10.1038/s43587-023-00442-w

Figure Lengend Snippet: Aged hamsters and young hamsters were treated (or not) with ABT-263 and then infected with SARS-CoV-2. A , Body weight loss of vehicle-treated and ABT-263-treated aged hamsters and young hamsters following a SARS-CoV-2 infection (n = 6). B , Effect of ABT-263 treatment on the frequency of p16-positive cells (7 dpi) as assessed by immunohistochemistry. Arrows indicate p16-positive cells. Bars: 20 μm. C , The p16 transcript levels are indicated (n = 3-6). The data are expressed as the fold increase relative to average gene expression in infected (7 dpi) vehicle-treated young hamsters (n = 3-6). For all graphs, errors indicate mean ± s.d. One of two representative experiments are shown (A-C). Significance of body weight loss between animal groups was calculated using wilcoxon matched-pairs signed rank test ( A ). Significant differences were determined using One-way ANOVA Kruskal-Wallis test (nonparametric), followed by the Dunn’s posttest test ( C ).

Article Snippet: For p16, ACE2 and viral nucleoprotein labeling (immunofluorescence), the following antibodies were used: p16 (sc-377412, 1:50, Santa Cruz Biotechnology), ACE2 (NBP1-76611, 1:50, Bio-Techne) and SARS-CoV nucleocapsid (NB100-56576, 1:50, Bio-Techne).

Techniques: Infection, Immunohistochemistry, Expressing

Journal: Nature Aging

Article Title: Removal of senescent cells reduces the viral load and attenuates pulmonary and systemic inflammation in SARS-CoV-2-infected, aged hamsters

doi: 10.1038/s43587-023-00442-w

Figure Lengend Snippet: Aged hamsters and young hamsters were treated (or not) with ABT-263 and then infected with SARS-CoV-2. Animals were euthanized at 3 dpi. a , Determination of viral loads in the lungs. Left: the number of infectious particles was determined in a TCID 50 assay. The data are expressed as the number of infectious virus particles per lung ( n = 6–11). Middle and right: quantification of viral RdRp and E protein transcript levels in the whole lungs, using RT–PCR. The data are expressed as Δ C t and genome copy per microgram of RNA ( n = 5–8). b , mRNA copy numbers (for IFNs and ISGs) were quantified by RT–PCR. The data are expressed as the fold change relative to average gene expression in mock-infected animals ( n = 5–6). c , Immunohistochemistry analysis of spike in the lung from SARS-CoV-2-infected, aged hamsters treated (or not) with ABT-263. Scale bars, 100 μm and 20 μm. d , Viral nucleoprotein labeling (immunofluorescence) was performed on lung sections. Scale bars, 25 μm. Right: the histograms indicate the fold change relative to average intensity in vehicle-treated infected, aged animals ( n = 6). e , f , Expression of the viral nucleoprotein, ACE2 and β-tubulin (western blotting) in vehicle-treated and ABT-263-treated SARS-CoV-2-infected, aged hamsters (whole lung homogenates). The relative protein levels normalized to β-tubulin are shown ( n = 3–8). For all graphs, errors indicate mean ± s.d. Pooled results from two independent experiments ( a ) and one of two representative experiments ( b – e ) are shown. Significant differences were determined using the two-tailed Mann–Whitney U- test ( b , d , e , f ) or one-way ANOVA Kruskal–Wallis test (non-parametric), followed by Dunn’s post test ( a ). * P < 0.05, ** P < 0.01.

Article Snippet: For p16, ACE2 and viral nucleoprotein labeling (immunofluorescence), the following antibodies were used: p16 (sc-377412, 1:50, Santa Cruz Biotechnology), ACE2 (NBP1-76611, 1:50, Bio-Techne) and SARS-CoV nucleocapsid (NB100-56576, 1:50, Bio-Techne).

Techniques: Infection, Virus, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Labeling, Immunofluorescence, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Nature Aging

Article Title: Removal of senescent cells reduces the viral load and attenuates pulmonary and systemic inflammation in SARS-CoV-2-infected, aged hamsters

doi: 10.1038/s43587-023-00442-w

Figure Lengend Snippet: Aged hamsters and young hamsters were treated (or not) with ABT-263 and infected with SARS-CoV-2. A , Quantification of viral RdRp and E protein transcript levels in the whole lungs using RT-PCR assays (7 dpi). The data are expressed as ΔCt and genome copy/μg RNA (n = 5-6) . B , mRNA copy numbers of genes (interferons and ISGs), as quantified with RT-PCR. The data are expressed as the fold change, relative to average gene expression in mock-infected animals (n = 5-6). C , Immunohistochemistry analysis of spike in the lung from SARS-CoV-2-infected young hamsters treated (or not) with ABT-263 (3 dpi). Bars: 100 μm and 20 μm. D , Nucleoprotein labelling (immunofluorescence) was performed on lung sections collected at 3 dpi. Bars: 25 μm. Right panel, The histograms indicate the fold change relative to average intensity in vehicle-treated young animals (n = 6). A-D , One of two representative experiments is shown. For all graphs, errors indicate mean ± s.d. Significant differences were determined using the two tailed Mann Whitney U test ( B and D ) and One-way ANOVA Kruskal-Wallis test (nonparametric), followed by the Dunn’s posttest test ( A ). * P < 0.05, ** P < 0.01.

Article Snippet: For p16, ACE2 and viral nucleoprotein labeling (immunofluorescence), the following antibodies were used: p16 (sc-377412, 1:50, Santa Cruz Biotechnology), ACE2 (NBP1-76611, 1:50, Bio-Techne) and SARS-CoV nucleocapsid (NB100-56576, 1:50, Bio-Techne).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Immunofluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Nature Aging

Article Title: Removal of senescent cells reduces the viral load and attenuates pulmonary and systemic inflammation in SARS-CoV-2-infected, aged hamsters

doi: 10.1038/s43587-023-00442-w

Figure Lengend Snippet: Aged hamsters and young hamsters were treated (or not) with ABT-263 and then infected with SARS-CoV-2. A , Representative micrographs showing H&E-stained lungs from aged hamsters and young hamsters treated (or not) with ABT-263 (magnification: x 2.5). The percentage of lung sections affected by subacute bronchointerstitial pneumonia (consolidation) is depicted for each group of animals (lower panel, (n = 11-12 aged and n = 6 young). B , Body weight loss curves for infected animals (n = 5-6). C , Heatmaps of the differential expressed components in the lungs of vehicle-treated and ABT-263-treated aged hamsters, in a mass-spectrometry analysis of the proteome (fold change in protein abundance >2, P < 0.05) (n = 4). Pooled results from two independent experiments (A left, B and C) and one of two representative experiments (A right) are shown. For all graphs, errors indicate mean ± SD. A and C , Significant differences were determined using the two tailed Mann Whitney U test. ** P < 0.01.

Article Snippet: For p16, ACE2 and viral nucleoprotein labeling (immunofluorescence), the following antibodies were used: p16 (sc-377412, 1:50, Santa Cruz Biotechnology), ACE2 (NBP1-76611, 1:50, Bio-Techne) and SARS-CoV nucleocapsid (NB100-56576, 1:50, Bio-Techne).

Techniques: Infection, Staining, Mass Spectrometry, Two Tailed Test, MANN-WHITNEY

Journal: Nature Aging

Article Title: Removal of senescent cells reduces the viral load and attenuates pulmonary and systemic inflammation in SARS-CoV-2-infected, aged hamsters

doi: 10.1038/s43587-023-00442-w

Figure Lengend Snippet: Aged hamsters and young hamsters were treated (or not) with ABT-263 and then infected with SARS-CoV-2. Animals were euthanized at 7 dpi and 22 dpi. a , Left: histopathological examination of lung sections (H&E staining, 7 dpi). The sum of the subscores is shown ( n = 11–12 aged and n = 6 young). Right: photomicrographs showing lower alveolar destruction in ABT-263-treated aged hamsters (but not ABT-263-treated young hamsters). Arrowhead: inflammatory cell infiltrate; star: alveolar wall rupture; sun: type II pneumocyte hyperplasia; thunderbolt: necrosis; arrow: activated blood vessel. Scale bars, 50 μm. b , Heat maps of the differential expressed prothrombotic and inflammatory factors in the serum of vehicle-treated and ABT-263-treated aged hamsters, in a mass spectrometry analysis of the proteome (fold change in protein level > 1.2, P < 0.05) ( n = 5–6). c , Histopathological examination of lung sections (H&E staining, 22 dpi). Lower panels: The total histology score (left) and the numbers of inflammatory foci (inflammation and type II hyperplasia) per lung section (right) are shown ( n = 4–6). d , Sirius Red labeling in the lungs of vehicle-treated and ABT-263-treated aged hamsters and young hamsters at 22 dpi. Top: representative images showing (stars) the destructured basal membranes in vehicle-treated aged animals. Bottom: the percentages of Sirius Red labeling are shown ( n = 4–6). e , TMT-based proteomic analysis of lung extracts (vehicle-treated and ABT-263-treated aged hamsters). Heat maps of the differentially expressed components, in a mass spectrometry analysis of the proteome, are depicted (fold change in protein abundance > 2, P < 0.05) ( n = 4). For all graphs, errors indicate mean ± s.d. Pooled results from two independent experiments ( a , left) and one of two representative experiments ( a , right, and b – e ) are shown. Significant differences were determined using the two-tailed Mann–Whitney U- test ( a , b , e ) and one-way ANOVA Kruskal–Wallis test (non-parametric), followed by Dunn’s post test ( c , d ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For p16, ACE2 and viral nucleoprotein labeling (immunofluorescence), the following antibodies were used: p16 (sc-377412, 1:50, Santa Cruz Biotechnology), ACE2 (NBP1-76611, 1:50, Bio-Techne) and SARS-CoV nucleocapsid (NB100-56576, 1:50, Bio-Techne).

Techniques: Infection, Staining, Mass Spectrometry, Labeling, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: A . Human HAE cells (MucilAir™, Epithelix) were mock infected (N.I.) or incubated with SARS-CoV-2 on the apical side at MOI 0,01 and 0,1 for 2 h. Viral input was removed and the apical side washed with PBS1X before incubation for 48 h and 72 h. Cells were harvested at the indicated time points and lysed for RNA extraction and RT-qPCR analysis using RdRp primers and probe. B . Human HAE cells were mock infected (N.I.) or incubated with SARS-CoV-2 on the apical side at MOI 0,1 for 2 h. Viral input was removed and the apical side washed with PBS1X before incubation for 48 h. Cells were fixed and stained for Actin with Phalloidin conjugated to Alexa Fluor 488 (shown here in magenta) and with an anti-double stranded RNA and a secondary antibody conjugated to Alexa Fluor 546 (shown in green). Images were acquired with an LSM880 Airyscan microscope. Representative images are shown; scale bar 10 μm. C . Human HAE cells were mock infected (N.I.) or incubated with SARS-COV-2 (MOI 0,1), as in A. Cytokine concentrations in the basal media were measured using the Human Anti-Virus Response Panel LEGENDplex™ at 72 h post-infection (top) and the fold difference in cytokine concentration in the basal media from infected compared to N.I. cells is represented as a heat map (bottom; log2 scale). D . An Antiviral Response RT 2 profiler PCR array analysis was performed using the RNAs extracted at 72 h (MOI 0,1). The mean of 4 (A) or 3 (C and D) independent experiments is shown, with error bars representing one standard deviation (s.d.) from the mean.

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Infection, Incubation, RNA Extraction, Quantitative RT-PCR, Staining, Microscopy, Virus, Concentration Assay, Standard Deviation

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: A . Human HAE cells of nasal, tracheal and bronchial origins were mock infected (N.I.) or incubated with SARS-CoV-2 on the apical side at MOI 0,01 for 2 h as in . Cells were harvested at the indicated time points and lysed for RNA extraction and RT-qPCR analysis using RdRp primers and probe. B . Differential gene expression was measured in RNAs from A. using the indicated taqmans, and data were normalized to both ActinB and GAPDH. C . Data from Human Anti-Virus Response Panel LEGENDplex™ as performed in with supernatants from cells of nasal, tracheal and bronchial origins. D . Data from Antiviral Response RT 2 profiler PCR array analysis as in for RNAs from cells of nasal, tracheal and bronchial origins. The light blue line (sets at 1) indicates no change in cytokine production or in gene expression (C and D). The mean of 6 (A and B) or 3 (C and D) independent experiments is shown (apart for the 24 h time point in A and B, n=3), with error bars representing one standard deviation (s.d.) from the mean (A and B).

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Infection, Incubation, RNA Extraction, Quantitative RT-PCR, Expressing, Virus, Standard Deviation

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: Caco-2 and A549 cells were transduced or not with lentiviral vectors to stably overexpress ACE2, or ACE2 and TMPRSS2. The indicated (unmodified and modified) cell lines were infected with SARS-CoV-2 at MOI 0,05 and lysed 48 h later for RNA extraction and RdRp RT-qPCR analysis. A representative experiment (with technical triplicates) is shown.

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Stable Transfection, Modification, Infection, RNA Extraction, Quantitative RT-PCR

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: A . Human Calu-3 cells were non-infected (N.I.) or incubated with SARS-CoV-2 at MOIs of 0,0005, 0,005 and 0,05. Cells were harvested at the indicated time points and lysed for RNA extraction and RT-qPCR analysis using RdRp primers and probe. B . The cell supernatants from (A) were harvested at the indicated time points and cytokine concentrations were measured using the Human Anti-Virus Response Panel LEGENDplex™ at 24 h and 48 h. Concentrations are shown (top), and the fold difference in cytokine concentration in supernatants from infected compared to non-infected cells was calculated and represented as a heat map at 24 h and 48 h (bottom; log2 scale). C . An Antiviral Response RT 2 profiler PCR array analysis was performed using the RNAs extracted at 48 h (MOI 0,005). The mean of 4 (A) or 3 (B and C) independent experiments is shown, with error bars representing one s.d. from the mean.

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Infection, Incubation, RNA Extraction, Quantitative RT-PCR, Virus, Concentration Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: A . Human Calu-3 cells were mock infected or incubated with SARS-CoV-2 at the indicated MOIs. Cells were harvested at 24 h or 48 h and lysed for RNA extraction. Relative expression levels of the indicated IFN genes and ISGs were analysed by RT-qPCR analysis using both ActinB and GAPDH for normalization. B . The cell supernatants from (A) were harvested at the indicated time points and type I and type III IFN concentrations were measured using HEK-Blue™ IFN-α/β and IFN-γ reporter cells, respectively. The mean of 3 to 4 (A) or 3 (B and C) independent experiments is shown, with error bars representing one s.d. from the mean.

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Infection, Incubation, RNA Extraction, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: Human A549-ACE2 ( A ) and Caco-2-ACE2 ( B ) cells were non-infected or incubated with SARS-CoV-2 at the indicated MOIs. Cells were harvested at 48 h and 24 h, respectively (i.e. the time points at which replication reached similar levels without a major impact on cell survival, not shown), and lysed for RNA extraction. Relative expression levels of the indicated IFN genes and ISGs were analysed by RT-qPCR analysis using both ActinB and GAPDH for normalization. The mean of 3 independent experiments is shown, with error bars representing one s.d. from the mean.

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Infection, Incubation, RNA Extraction, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: Calu-3-Cas9 cells were transduced with lentiviral vectors expressing CRISPR non-targeting single guide RNAs (Ctrl #1, #2) or single guide RNAs targeting RIG-I, MDA-5 or MAVS. Cells were antibiotic selected for at least 15 days and challenged with SARS-CoV-2 at MOI 0,05. The cell supernatants were harvested at 24 h and 48 h post-infection, as indicated, and the concentrations of type I ( A ) and type III ( B ) IFNs produced analysed using HEK-Blue™ IFN-α/β and IFN-γ reporter cells, respectively. The mean of 3 independent experiments (performed with 2 series of KO cell populations generated independently) is shown, with error bars representing one s.d. from the mean. C . A representative immunoblot is shown, Actin served as a loading control.

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Transduction, Expressing, CRISPR, Infection, Produced, Generated, Western Blot

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: Vero E6 cells were pre-treated or not with increasing concentrations of type I IFN, as indicated, for 16 h prior to SARS-CoV-2 infection at MOI 0,0005. 72 h later, the cells were lysed and the supernatants collected, the RNAs were extracted and viral replication was monitored in cells ( A , left panel) and viral production in the supernatants ( B , left panel) by RdRp RT-qPCR. Right panels. The fold inhibition by IFN is shown ( A and B , right panels). The mean of 3 independent experiments is shown, with error bars representing one s.d. from the mean.

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Infection, Quantitative RT-PCR, Inhibition

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: A . Human HAE cells of nasal, tracheal and bronchial origins (as indicated) were pre-treated or not with type I IFN for 20 h, and mock infected (N.I.) or incubated with SARS-CoV-2 on the apical side at MOI 0,1 for 1-2 h. The viral input was removed and the apical side washed in PBS1X. Cells were harvested and lysed for RNA extraction and RT-qPCR analysis using RdRp primers and probe at 72 h post-infection. B . Washes of the apical side of the HAE cells from (A) were performed at 24 h, 48 h and 72 h, harvested and frozen down for subsequent titrations. Plaque assays were then performed in technical duplicates to determine the number of plaque forming units (PFU) per mL of supernatant in each condition. The grey, dotted line indicates the detection threshold. C . Human HAE cells of bronchial origin were pre-treated or not with IFN for 20 h, and mock infected (N.I.) or incubated with SARS-CoV-2 on the apical side at MOI 0,1 and 0,25, as indicated, for 2 h. Viral input was removed and the apical side washed with PBS1X before incubation for 48 h. Cells were fixed and stained for Actin with Phalloidin conjugated to Alexa 488 (shown in magenta on the merge images) and with an anti-double stranded RNA and a secondary antibody conjugated to Alexa 546 (shown in green on the merge images). Images were acquired with an LSM880 Airyscan microscope. Representative images are shown; scale bar 10 μm. D . Human Calu-3 cells were pre-treated or not with IFN for 16-20 h, the media was replaced and the cells were mock-infected (N.I.) or incubated with SARS-CoV-2 at the indicated MOIs. Cells were harvested at the indicated time points and lysed for RNA extraction and RT-qPCR analysis using RdRp primers and probe. E . Aliquots of the supernatants from D were harvested at 24 h and 48 h post infection and plaque assays were performed to evaluate the production of infectious viruses in the different conditions. F . Calu-3 cells were pre-treated or not with IFN and infected as in D, and the cells were fixed with PFA at 24 h post-infection, permeabilized and stained with an anti-Spike antibody conjugated to an Alexa fluorochrome. The percentage of Spike + cells was scored by flow cytometry. G . Calu-3 cells were pre-treated or not with IFN and infected as in D and lysed 24 h post-infection for immunoblot analysis of SARS-CoV-2 Nucleoprotein (N) and Spike, and IFITM3, RIG-I and MX1 expression levels, Actin serving as a loading control. A representative immunoblot is shown. H . CTRL and IRF9 Calu-3 knockout cells were infected with SARS-CoV-2 at the indicated MOIs and viral replication was measured 48 h later by RdRp RT-qPCR. I . CTRL and IRF9 knockout cells were pre-treated or not with IFN for 48 h, lysed and the expression levels of IFITM3, RIG-I and MX1 were analysed by immunoblot, Actin served as a loading control. A representative immunoblot is shown. The mean of 3 (A-B, H) or 4 (D-F) independent experiments is shown, with error bars representing one standard deviation (s.d.) from the mean.

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Infection, Incubation, RNA Extraction, Quantitative RT-PCR, Staining, Microscopy, Flow Cytometry, Western Blot, Expressing, Knock-Out, Standard Deviation

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: Human A549-ACE2 (left) and Caco-2-ACE2 (right) cells were pre-treated or not with IFN for 16-20 h, the media was replaced and the cells were mock-infected (N.I.) or incubated with SARS-CoV-2 at the indicated MOIs. Cells were harvested at 48 h (A549-ACE2) or 24 h (Caco-2-ACE2). A . The cells were lysed for RNA extraction and RT-qPCR analysis using RdRp primers and probe. B . The cells were fixed with PFA at 24 h post-infection, permeabilized and stained with an anti-Spike antibody conjugated to an Alexa fluorochrome. The percentage of Spike + cells was scored by flow cytometry. C . The cells were lysed for Immunoblot analysis of SARS-CoV-2 Nucleoprotein (N) and Spike, IFITM3, RIG-I and MX1 ISG expression levels, Actin serving as a loading control. Of note, MX1 was not detected in Caco-2-ACE2 cell lysates. A representative immunoblot is shown. The mean of 3 independent experiments is shown (A and B), with error bars representing one standard deviation (s.d.) from the mean.

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Infection, Incubation, RNA Extraction, Quantitative RT-PCR, Staining, Flow Cytometry, Western Blot, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: CTRL, RIG-I, MDA-5 and MAVS Calu-3 knockout cells were infected with SARS-CoV-2 at MOI 0,05 (as in ) and viral production was measured 48 h later by plaque assays on Vero E6 cells. The mean of 2 independent experiments is shown, with error bars representing one s.d. from the mean.

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Knock-Out, Infection

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: A . CTRL and JAK1 A549-ACE2 knockout cells were infected with SARS-CoV-2 at MOI 0,0005 and viral replication was measured 48 h later using RdRp RT-qPCR. The mean of 2 independent experiments is shown, with error bars representing one s.d. from the mean. B . CTRL and JAK1 knockout cells were pre-treated or not with IFN for 48 h, lysed and the expression levels of IFITM3, RIG-I and MX1 were analysed by immunoblot, Actin serving as a loading control. A representative immunoblot is shown.

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Knock-Out, Infection, Quantitative RT-PCR, Expressing, Western Blot

Journal: bioRxiv

Article Title: SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

doi: 10.1101/2020.10.28.358945

Figure Lengend Snippet: Calu-3 cells were infected with SARS-CoV-2 at MOI 0,0005, 0,005 and 0,05 after a 24h pre-treatment with IFN or not, or were subsequently treated with IFN at 4 h, 8 h or 24 h post infection. Viral replication was measured 48 h post infection using RdRp RT-qPCR. The mean of 3 independent experiments is shown, with error bars representing one s.d. from the mean.

Article Snippet: Cells were lysed in lysis buffer (10 mM TRIS 1M pH7.6, NaCl 150 mM, Triton X100 1%, EDTA 1 mM, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromphenol blue), resolved by SDS-PAGE and analysed by immunoblotting using primary antibodies against SARS-CoV Nucleocapsid (Bio-Techne NB100-56683), SARS-CoV Spike (GeneTex GTX632604), Actin (Sigma-Aldrich A1978), IFITM3 (Proteintech 11714-1-AP), MX1 (ThermoFisher Scientific PA5-22101), RIG-I (Covalab mab10110), MDA-5 (Ozyme D74E4), and MAVS (ProteinTech 14341-1-AP), followed by secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad).

Techniques: Infection, Quantitative RT-PCR