Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: a SARS-CoV-2 replication kinetics in HAE from different donors, HCoV-NL63 was used as a control ( n = 3). b Transepithelial electrical resistance (TEER in Ω cm 2 ) between the apical and basal poles was measured at each time point ( n = 3). c SARS-CoV-2 infected both ciliated cells (72 h pi) and secretory cells (72 h pi). arrows: virus particles, arrowhead: cilium, asterisk: secretory vesicle, insets dashed-line squares indicate magnification of arrowed areas. d Costaining of SARS-CoV-2 N protein (green) with ciliated cell marker β-tubulin-IV (red), goblet cell marker Muc5AC (red), club cell marker CCSP (red), and ACE2 (red) positive cells. HCoV-NL63 N protein (green) staining was used as a control (72 h pi). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Data a , b are the means ± s.d. of three independent biological replicates. Source data a – d are provided as a Source Data file.

Article Snippet: ACE2 , Sino biologicals (10108-T56), rabbit polyclona (1:100).

Techniques: Infection, Marker, Staining

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: Source of antibodies and dyes with work concentration for immunofluorescence.

Article Snippet: ACE2 , Sino biologicals (10108-T56), rabbit polyclona (1:100).

Techniques: Concentration Assay, Immunofluorescence

Journal: Cardiovascular Research

Article Title: Blood flow patterns regulate PCSK9 secretion via MyD88-mediated pro-inflammatory cytokines

doi: 10.1093/cvr/cvz262

Figure Lengend Snippet: TLR4 signalling regulates distribution of PCSK9 expression along the aorta. (A) Effect of TLR4 inhibitor TAK-242, MyD88 inhibitor Pepinh-MYD, TRIF inhibitor Pepinh-TRIF, and NF-κB Helenalin on PCSK9 levels, measured by ELISA. (B) LPS injection induces expression of pro-inflammatory cytokines IL-1β, IL-18, MCP-1, IL-6, TNFα, IL-12, IFNγ, and GM-CSF. Measured by ELISA in serum on day 3 HFD group; rabbits treated with or without LPS. Bar graphs represent data compiled from three independent experiments (n = 7 rabbit per genotype), shown as mean ± standard deviation. The significances between two groups were tested by unpaired t-test; Multiple comparisons were analysed by one-way ANOVA, followed by Tukey’s post hoc comparisons test (**P < 0.01; ****P < 0.0001).

Article Snippet: Secretion of PCSK9, IL-1β, MCP-1, IL-6, TNFα, IL-12, and IFNγ was measured in rabbit sera or aorta by using a rabbit enzyme-linked immunosorbent assay (ELISA) kit for PCSK9, IL-1β, IL-18, MCP-1, IL-6, TNFα, IL-12, IFNγ, and GM-CSF (MyBioSource, Inc., San Diego, CA, USA); Rabbit ELISA kit for IL-18 and GM-CSF were from LSBio (Seattle, WA, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Injection, Standard Deviation

Journal: Nature Communications

Article Title: Human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 infection

doi: 10.1038/s41467-021-22781-1

Figure Lengend Snippet: a Representative expression of angiotensin-converting enzyme-II (ACE2) in kidney tissues from COVID-19 post-mortem (case #2) was detected by immunohistochemistry. Scale bars = 100 μm. b The co-expression of ACE2 and SARS-CoV-2 NP (nucleocapsid protein) or S (spike) antigens in kidney sections from COVID-19 post-mortem (case #2), hepatitis B virus-associated membranous nephropathy (HBV-MN) and trauma victims. Arrow indicates positive tubules. Scale bars = 100 μm. Data represent one of at least three technical replication each.

Article Snippet: For immunofluorescence double-staining, the sections were incubated with primary mouse originated antibodies including anti-SARS NP antibodies (ab273434, 1:500, mouse IgG), anti-SARS S antibodies (ab273433, 1:500, mouse IgG), or anti-DP2 (sc-271898, 1:200, mouse monoclonal C-5; Santa Cruz Biotechnology) and rabbit originated antibodies including anti-SARS NP antibodies (clone ID: 019, 1:200, rabbit IgG; Sino Biological, Beijing), anti-ACE2 (clone ID: 10108-RP01, 1:100, rabbit IgG; Sino Biological) or anti-PDS (ab182141, 1:100, rabbit IgG1; Abcam) at 4 °C overnight, respectively.

Techniques: Expressing, Immunohistochemistry

Journal: Signal Transduction and Targeted Therapy

Article Title: Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection

doi: 10.1038/s41392-021-00800-3

Figure Lengend Snippet: SARS-CoV-2 infection activates the cGAS-STING pathway. a , b Representative Western blots of STING and IRF3 phosphorylation upon SARS-CoV-2 infection ( n = 2 independent experiments). Calu-3 ( a ) or HeLa-ACE2 ( b ) cells were mock-infected or infected with SARS-CoV-2 at an MOI of 0.5. At indicated time points after infection, cells were harvested and lysed. Lysates were then subjected to Western blot analysis using indicated antibodies. ✶Activated STING. c 2′3′-cGAMP levels in cells infected with SARS-CoV-2. HeLa-ACE2 were infected with SARS-CoV-2 as described in b . A competitive ELISA assay determined the 2′3′-cGAMP concentrations. Mean ± s.d., n = 3 independent experiments. ** P < 0.01, n.s. not significant. Two-tailed Student’s t -test on log-transformed data. d STING and IRF3 phosphorylation upon SeV infection. HeLa-ACE2 cells were mock-infected or infected with SeV. Cells were harvested at indicated times and analyzed by Western blot. e Semi-quantitative analysis on Western blot results from a and d by densitometry. f Calu-3 cells were treated with DMSO or 200 ng/ml of H-151. After 2 h, cells were mock-infected or infected with SARS-CoV-2 at an MOI of 0.5 for 24 h. RNA extracted from the cells was evaluated by quantitative PCR. The data are expressed as fold change of the IFNB , IFIT1 , ISG15 , CCL5 , and SARS-CoV-2 N mRNA levels relative to the GAPDH control. Mean ± s.d., n = 3 independent experiments. * P < 0.05, ** P < 0.01, two-tailed Student’s t -test

Article Snippet: The antibodies used for immunoblotting were: rabbit-anti-cGAS (D1D3G, #15102 S, Cell Signaling Technology, 1:500), rabbit-anti-STING (D2P2F, #13647 S, Cell Signaling Technology, 1:500), rabbit-anti-phospho-STING (Ser366) (D7C3S, #19781 S, Cell Signaling Technology, 1:500), rabbit-anti-IRF3 (ab76409, Abcam, 1:1000), rabbi-anti-phospho-IRF3 (ab76493, Abcam, 1:1000), rabbit-anti-GAPDH (BE0024, EASYBIO, 1:2000), mouse-anti-β-Tubulin (CW0098, CWBIO, 1:2000), rabbit-anti-SARS-CoV-2 spike protein (40589-T62, Sino Biological, 1:1000), rabbit-anti-SARS-CoV-2 nucleocapsid protein (NP) (40143-R019, Sino Biological, 1:1000), rabbit-anti-Sendai virus (PD029C1, MBL, 1:1000), rabbit-anti-ACE2 (10108-T60, Sino Biological, 1:1000), mouse-anti-MAVS (sc166583, Santa Cruz, 1:500), goat-anti-mouse IgG-HRP secondary antibody (115035003, Jackson ImmunoResearch), mouse-anti-Lamin A/C (MABT538, Sigma-Aldrich, 1:500), rabbit-anti-Lamin B1 (ab16048, Abcam, 1:500), goat-anti-rabbit IgG-HRP secondary antibody (111035003, Jackson ImmunoResearch).

Techniques: Infection, Western Blot, Competitive ELISA, Two Tailed Test, Transformation Assay, Real-time Polymerase Chain Reaction

Journal: Signal Transduction and Targeted Therapy

Article Title: Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection

doi: 10.1038/s41392-021-00800-3

Figure Lengend Snippet: cGAS colocalizes with cytosolic genomic DNA in SARS-CoV-2-induced syncytia. a Representative confocal immunofluorescence images of SARS-CoV-2-induced syncytia. HeLa-ACE2 cells were infected with SARS-CoV-2 at an MOI of 0.5. After 18 h, cells were fixed and stained for DNA with DAPI (blue) and viral nucleocapsid protein (NP) with anti-NP antibody (green). Triangles indicate budding chromatin (upper) or cytosolic chromatin (lower). Scale bar, 20 μm. b – d Quantification of cells for parameters as indicated. Cells were quantified for three different fields with at least 400 cells. Mean ± s.d., Data were pooled from 3 independent experiments. **** P < 0.0001, ** P < 0.01, two-tailed Student’s t -test. CC cytoplasmic chromatin. e Representative images of cells stained for NP, DNA, and cGAS-Flag. cGAS-null HeLa-ACE2 cells reconstituted with cGAS-Flag were infected with SARS-CoV-2 at an MOI of 0.5 for 18 h, followed by staining with anti-NP antibody (green), DAPI (blue), and anti-Flag antibody (red) as indicated. Scale bar, 20 μm. f Cellular distribution of endogenous cGAS. HeLa-ACE2 cells (left) or cGAS-null HeLa-ACE2 cells (right) were stained with anti-cGAS antibody (red) and DAPI (blue). Scale bar, 20 μm. g HeLa-ACE2 cells were infected with SARS-CoV-2, followed by staining with anti-NP antibody (green), DAPI (blue), and anti-cGAS antibody (red) as indicated. Scale bar, 20 μm. h HeLa-ACE2 cells were treated as described in e and were stained with anti-NP antibody (green), DAPI (blue), and anti-phospho-STING (Ser366) antibody (red). Scale bar, 20 μm. i K18-hACE2 transgenic mice were infected with 10 5 TCID 50 of SARS-CoV-2 for 3 days. Mouse lungs were harvested and subjected to immunohistochemistry analysis with anti-phospho-STING (Ser365) antibody (diaminobenzidine visualization). Nuclei were stained with hematoxylin (dark blue). Scale bar, 10 μm. j HeLa-ACE2 cells were treated as described in e and were stained with anti-NP antibody (green), DAPI (blue), and anti-IRF3 antibody (red). Scale bar, 20 μm

Article Snippet: The antibodies used for immunoblotting were: rabbit-anti-cGAS (D1D3G, #15102 S, Cell Signaling Technology, 1:500), rabbit-anti-STING (D2P2F, #13647 S, Cell Signaling Technology, 1:500), rabbit-anti-phospho-STING (Ser366) (D7C3S, #19781 S, Cell Signaling Technology, 1:500), rabbit-anti-IRF3 (ab76409, Abcam, 1:1000), rabbi-anti-phospho-IRF3 (ab76493, Abcam, 1:1000), rabbit-anti-GAPDH (BE0024, EASYBIO, 1:2000), mouse-anti-β-Tubulin (CW0098, CWBIO, 1:2000), rabbit-anti-SARS-CoV-2 spike protein (40589-T62, Sino Biological, 1:1000), rabbit-anti-SARS-CoV-2 nucleocapsid protein (NP) (40143-R019, Sino Biological, 1:1000), rabbit-anti-Sendai virus (PD029C1, MBL, 1:1000), rabbit-anti-ACE2 (10108-T60, Sino Biological, 1:1000), mouse-anti-MAVS (sc166583, Santa Cruz, 1:500), goat-anti-mouse IgG-HRP secondary antibody (115035003, Jackson ImmunoResearch), mouse-anti-Lamin A/C (MABT538, Sigma-Aldrich, 1:500), rabbit-anti-Lamin B1 (ab16048, Abcam, 1:500), goat-anti-rabbit IgG-HRP secondary antibody (111035003, Jackson ImmunoResearch).

Techniques: Immunofluorescence, Infection, Staining, Two Tailed Test, Transgenic Assay, Immunohistochemistry

Journal: Signal Transduction and Targeted Therapy

Article Title: Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection

doi: 10.1038/s41392-021-00800-3

Figure Lengend Snippet: Cell fusion activates the innate immune response via the cGAS-STING pathway. a Scheme of co-culture experiment. b Representative fluorescence images of co-culture experiment. HEK293T cells were transfected with vector control (Vec) or increasing amounts of plasmids expressing spike (S), along with plasmids expressing EGFP for 24 h. Cells were then detached and mixed with HeLa-ACE2 expressing mCherry (HeLa-ACE2-mCherry). After 8 h, cells were subjected to fluorescence microscopy analysis. Scale bar, 250 μm. c Cytokine genes/ISGs expression in co-cultured cells. Cells were co-cultured as indicated in b . RNA extracted from the cells was evaluated by quantitative PCR. The data are expressed as fold change of the IFNB , ISG15 , IL8 , and CCL5 mRNA levels relative to the GAPDH control. Mean ± s.d., n = 4 independent experiments. ** P < 0.01, *** P < 0.001, **** P < 0.0001, two-tailed Student’s t -test. d Western blot analysis of cells from co-culture experiment as described in b using indicated antibodies. STING blots were performed under non-reducing (top) or reducing conditions. ✶STING dimer. e HEK293T cells were transfected with vector control (Vec) or plasmids expressing spike. After 24 h, cells were detached and mixed with HeLa-ACE2-mCherry cells as indicated. cGAS KO , STING KO , and MAVS KO represent cells depleted of indicated genes. cGAS RE represents cGAS-null cells re-expressed cGAS. The expression of IFNB mRNA was assayed as described in c . Mean ± s.d., n = 3 independent experiments. ** P < 0.01, **** P < 0.0001, n.s. not significant. two-tailed Student’s t -test. f Western blot analysis of cells from the co-culture experiment as described in e . ✶ACE2 fragments generated during cell co-culture. S spike

Article Snippet: The antibodies used for immunoblotting were: rabbit-anti-cGAS (D1D3G, #15102 S, Cell Signaling Technology, 1:500), rabbit-anti-STING (D2P2F, #13647 S, Cell Signaling Technology, 1:500), rabbit-anti-phospho-STING (Ser366) (D7C3S, #19781 S, Cell Signaling Technology, 1:500), rabbit-anti-IRF3 (ab76409, Abcam, 1:1000), rabbi-anti-phospho-IRF3 (ab76493, Abcam, 1:1000), rabbit-anti-GAPDH (BE0024, EASYBIO, 1:2000), mouse-anti-β-Tubulin (CW0098, CWBIO, 1:2000), rabbit-anti-SARS-CoV-2 spike protein (40589-T62, Sino Biological, 1:1000), rabbit-anti-SARS-CoV-2 nucleocapsid protein (NP) (40143-R019, Sino Biological, 1:1000), rabbit-anti-Sendai virus (PD029C1, MBL, 1:1000), rabbit-anti-ACE2 (10108-T60, Sino Biological, 1:1000), mouse-anti-MAVS (sc166583, Santa Cruz, 1:500), goat-anti-mouse IgG-HRP secondary antibody (115035003, Jackson ImmunoResearch), mouse-anti-Lamin A/C (MABT538, Sigma-Aldrich, 1:500), rabbit-anti-Lamin B1 (ab16048, Abcam, 1:500), goat-anti-rabbit IgG-HRP secondary antibody (111035003, Jackson ImmunoResearch).

Techniques: Co-Culture Assay, Fluorescence, Transfection, Plasmid Preparation, Expressing, Microscopy, Cell Culture, Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, Generated

Journal: Signal Transduction and Targeted Therapy

Article Title: Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection

doi: 10.1038/s41392-021-00800-3

Figure Lengend Snippet: cGAS is colocalized with cytoplasmic chromatin in syncytial cells. a Representative confocal immunofluorescence images of co-cultured cells stained for DNA and cGAS-HA. HEK293T cells transfected with plasmids expressing spike (HEK293T(S)) were co-cultured with cGAS-null HeLa-ACE2 cells transfected with cGAS-HA. After 8 h, cells were stained with DAPI (blue) and anti-HA (red) antibody as indicated. Triangles indicate colocalization of cGAS with cytosolic chromatin. b Representative confocal immunofluorescence images of co-cultured cells stained for DNA, cGAS-HA, and Lamin B1. Co-culture experiments were performed as described in a . Cells were stained with DAPI (blue), anti-HA (red), and anti-Lamin B1 (green) antibodies. Three-dimensional reconstructed images based on z-stack images were displayed as volume view. c , d Extracted frames from live-cell imaging of co-cultured cells using confocal microscopy. HEK293T(S) cells transfected with plasmids expressing cGAS-GFP were co-cultured with cGAS-null HeLa-ACE2 cells transfected with plasmids expressing cGAS-GFP. After 1 h, cells were stained with DRAQ5 (purple) for visualizing DNA and subjected to live-cell imaging. e Co-culture experiments were performed as described in a . Cells were stained with DAPI (blue), anti-HA (red) antibody, and anti-γH2AX (green) antibody. Scale bars, 20 μm or 5 μm (inset)

Article Snippet: The antibodies used for immunoblotting were: rabbit-anti-cGAS (D1D3G, #15102 S, Cell Signaling Technology, 1:500), rabbit-anti-STING (D2P2F, #13647 S, Cell Signaling Technology, 1:500), rabbit-anti-phospho-STING (Ser366) (D7C3S, #19781 S, Cell Signaling Technology, 1:500), rabbit-anti-IRF3 (ab76409, Abcam, 1:1000), rabbi-anti-phospho-IRF3 (ab76493, Abcam, 1:1000), rabbit-anti-GAPDH (BE0024, EASYBIO, 1:2000), mouse-anti-β-Tubulin (CW0098, CWBIO, 1:2000), rabbit-anti-SARS-CoV-2 spike protein (40589-T62, Sino Biological, 1:1000), rabbit-anti-SARS-CoV-2 nucleocapsid protein (NP) (40143-R019, Sino Biological, 1:1000), rabbit-anti-Sendai virus (PD029C1, MBL, 1:1000), rabbit-anti-ACE2 (10108-T60, Sino Biological, 1:1000), mouse-anti-MAVS (sc166583, Santa Cruz, 1:500), goat-anti-mouse IgG-HRP secondary antibody (115035003, Jackson ImmunoResearch), mouse-anti-Lamin A/C (MABT538, Sigma-Aldrich, 1:500), rabbit-anti-Lamin B1 (ab16048, Abcam, 1:500), goat-anti-rabbit IgG-HRP secondary antibody (111035003, Jackson ImmunoResearch).

Techniques: Immunofluorescence, Cell Culture, Staining, Transfection, Expressing, Co-Culture Assay, Live Cell Imaging, Confocal Microscopy

Journal: Signal Transduction and Targeted Therapy

Article Title: Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection

doi: 10.1038/s41392-021-00800-3

Figure Lengend Snippet: Syncytia formation disrupts actin cytoskeleton and nucleoskeleton. a Representative fluorescence images of co-culture experiment. HEK293T cells were transfected with vector control (Vec) or plasmids expressing spike (S) for 24 h. Cells were then detached and mixed with HeLa-ACE2. After 4.5 h, cells were stained with DAPI (blue) and fluorescently labeled phalloidin (green) as indicated. Scale bar, 20 μm. b Three-dimensional reconstructed images of a were displayed as volume view. c Quantification of cell thickness. Cells were measured for height based on z-stack images. Mean ± s.d. Data were pooled from 3 independent experiments. **** P < 0.0001, two-tailed Student’s t -test. d Cells were treated as described in a and subjected to Western blot analysis using indicated antibodies. e Cells were treated as described in a and stained with DAPI (blue) and anti-lamin A/C antibody (red) as indicated. Scale bar, 20 μm. f Quantification of Lamin A/C positive cells from experiments described in e . Mean ± s.d., n = 3 independent experiments. *** P < 0.001. Two-tailed Student’s t -test. g HEK293T cells were transfected with plasmids expressing S for 24 h. Cells were then detached and mixed with HeLa-ACE2. After 4.5 h, cells were stained with DAPI (blue), anti-γH2AX (green) antibody, and anti- Lamin A/C antibody (red) as indicated. Scale bar, 20 μm

Article Snippet: The antibodies used for immunoblotting were: rabbit-anti-cGAS (D1D3G, #15102 S, Cell Signaling Technology, 1:500), rabbit-anti-STING (D2P2F, #13647 S, Cell Signaling Technology, 1:500), rabbit-anti-phospho-STING (Ser366) (D7C3S, #19781 S, Cell Signaling Technology, 1:500), rabbit-anti-IRF3 (ab76409, Abcam, 1:1000), rabbi-anti-phospho-IRF3 (ab76493, Abcam, 1:1000), rabbit-anti-GAPDH (BE0024, EASYBIO, 1:2000), mouse-anti-β-Tubulin (CW0098, CWBIO, 1:2000), rabbit-anti-SARS-CoV-2 spike protein (40589-T62, Sino Biological, 1:1000), rabbit-anti-SARS-CoV-2 nucleocapsid protein (NP) (40143-R019, Sino Biological, 1:1000), rabbit-anti-Sendai virus (PD029C1, MBL, 1:1000), rabbit-anti-ACE2 (10108-T60, Sino Biological, 1:1000), mouse-anti-MAVS (sc166583, Santa Cruz, 1:500), goat-anti-mouse IgG-HRP secondary antibody (115035003, Jackson ImmunoResearch), mouse-anti-Lamin A/C (MABT538, Sigma-Aldrich, 1:500), rabbit-anti-Lamin B1 (ab16048, Abcam, 1:500), goat-anti-rabbit IgG-HRP secondary antibody (111035003, Jackson ImmunoResearch).

Techniques: Fluorescence, Co-Culture Assay, Transfection, Plasmid Preparation, Expressing, Staining, Labeling, Two Tailed Test, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection

doi: 10.1038/s41392-021-00800-3

Figure Lengend Snippet: Targeting cGAS-STING pathway as potential therapeutics against SARS-CoV-2. a Effect of cGAS expression on SARS-CoV-2 replication. Wildtype HeLa-ACE2 (WT), HeLa-cGAS KO -ACE2, and HeLa-cGAS RE -ACE2 cells were infected with SARS-CoV-2 at an MOI of 0.5. At indicated times, total RNA extracted from cells was evaluated by quantitative PCR. The data are expressed as fold changes of the RNA levels of the viral N gene relative to the GAPDH control. Mean ± s.d., n = 3. * P < 0.05, ** P < 0.01, **** P < 0.0001, n.s. not significant. Two-tailed Student’s t -test. b The chemical structure of diABZI. c , d Antiviral effect of diABZI on SARS-CoV-2. Calu-3 ( c ) or HeLa-ACE2 ( d ) cells were treated with serially diluted diABZI for 24 h (Calu-3) or 1 h (HeLa-ACE2). Cells were then subjected to viability assay or infected with SARS-CoV-2 at an MOI of 0.2. After 24 h (Calu-3) or 48 h (HeLa-ACE2), supernatants were harvested for RNA extraction, followed by absolute quantification of viral N mRNA by PCR. Mean ± s.d., n = 4. The IC 50 (the half-maximal inhibitory concentration) and CC 50 (the half-maximal cytotoxic concentration) values were calculated using Prism software. Lower panels showed diABZI-induced STING and IRF3 activation. Calu-3 ( c ) or HeLa-ACE2 ( d ) cells were treated with serially diluted diABZI for 24 h and 6 h, respectively, followed by Western blot analysis using indicated antibodies

Article Snippet: The antibodies used for immunoblotting were: rabbit-anti-cGAS (D1D3G, #15102 S, Cell Signaling Technology, 1:500), rabbit-anti-STING (D2P2F, #13647 S, Cell Signaling Technology, 1:500), rabbit-anti-phospho-STING (Ser366) (D7C3S, #19781 S, Cell Signaling Technology, 1:500), rabbit-anti-IRF3 (ab76409, Abcam, 1:1000), rabbi-anti-phospho-IRF3 (ab76493, Abcam, 1:1000), rabbit-anti-GAPDH (BE0024, EASYBIO, 1:2000), mouse-anti-β-Tubulin (CW0098, CWBIO, 1:2000), rabbit-anti-SARS-CoV-2 spike protein (40589-T62, Sino Biological, 1:1000), rabbit-anti-SARS-CoV-2 nucleocapsid protein (NP) (40143-R019, Sino Biological, 1:1000), rabbit-anti-Sendai virus (PD029C1, MBL, 1:1000), rabbit-anti-ACE2 (10108-T60, Sino Biological, 1:1000), mouse-anti-MAVS (sc166583, Santa Cruz, 1:500), goat-anti-mouse IgG-HRP secondary antibody (115035003, Jackson ImmunoResearch), mouse-anti-Lamin A/C (MABT538, Sigma-Aldrich, 1:500), rabbit-anti-Lamin B1 (ab16048, Abcam, 1:500), goat-anti-rabbit IgG-HRP secondary antibody (111035003, Jackson ImmunoResearch).

Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Two Tailed Test, Viability Assay, RNA Extraction, Concentration Assay, Software, Activation Assay, Western Blot

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Sequences of small-interfering RNAs

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques:

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Sequences of the primers used for RT-qPCR

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques: Sequencing

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Correlation analysis between LPS, inflammatory factors, and RAS pathway members. A Heat map showing the correlation between LPS, inflammatory cytokines and RAS members. Blue dots represent positive correlation and red dots represent negative correlation. Larger dots and darker colour correspond to greater correlation. Lower correlation is represented by smaller dots and lighter colour. B Ratio of ACE/ACE2 after treatment of BMEC with different concentrations of LPS. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques:

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Distribution of ACE2 in BMEC. Cells were stained with antibodies against ACE2 and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Representative images were visualized by confocal laser microscopy. Red, ACE2; blue, DAPI. Scale bar: 10 μm.

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques: Staining, Microscopy

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: DAD3 induces ACE2 expression in LPS-induced BMEC. BMEC were treated with LPS (0.5 μg/mL), DA (20 μg/mL), DAD3 (20 μg/mL), LPS+DA (0.5 μg/mL+20 μg/mL) and LPS+DAD3 (0.5 μg/mL+20 μg/mL) for 24 h. A Relative ACE2 mRNA levels were determined by RT-qPCR. B ACE2 protein expression levels were determined by Western blotting. β-actin was used as a control. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Effects of ACE2 silencing and DAD3 treatments on the transcriptional or expression levels of RAS members. A-E BMEC were treated with either control siRNA (scrambled) or ACE2-siRNA for 24 h, followed by treatment with 0.5 μg/mL LPS for 24 h. After washing with PBS, cells were treated with 20 μg/mL DA or 20 μg/mL DAD3 for 24 h. The ACE2, AT1R and AT2R relative mRNA levels (transcriptional levels) of RAS members were determined by RT-qPCR. The Ang II and Ang-(1–7) protein expression levels of RAS members were determined by ELISA. A Relative ACE2 mRNA levels. B Expression levels of Ang II. C Expression levels of Ang-(1–7). D Relative AT1R mRNA levels. E Relative AT2R mRNA levels. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Effects of ACE2 silencing and DAD3 treatments on the transcriptional levels of pro-inflammatory factors. A–D BMEC were treated with either control siRNA (scrambled) or ACE2-siRNA for 24 h, followed by treatment with 0.5 μg/mL LPS for 24 h. After washing with PBS, cells were treated with 20 μg/mL DA or 20 μg/mL DAD3 for 24 h. Relative mRNA levels of pro-inflammatory factors were determined by RT-qPCR. A Relative IL-1β mRNA levels. B Relative IL-6 mRNA levels. C Relative IL-8 mRNA levels. D Relative TNF-α mRNA levels. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques: Quantitative RT-PCR

Journal: Veterinary Research

Article Title: DAD3 targets ACE2 to inhibit the MAPK and NF-κB signalling pathways and protect against LPS-induced inflammation in bovine mammary epithelial cells

doi: 10.1186/s13567-022-01122-0

Figure Lengend Snippet: Effects of ACE2 silencing and DA and DAD3 treatment on the expression levels of components of the MAPK and NF-κB pathways. A, B BMEC were treated with either control siRNA (scrambled) or ACE2-siRNA for 24 h, followed by treatment with 0.5 μg/mL LPS for 24 h. After washing with PBS, cells were treated with 20 μg/mL DA or 20 μg/mL DAD3 for 24 h. The phosphorylation levels of p38, JNK1/2/3, ERK1/2 and IκB-α (p-p38, p-JNK, p-ERK1/2 and p-IκB-α) were determined by Western blotting. Activation of NF-κB pathway was indicated by p-IκB-α, an indicator for the activation of NF-κB. The band intensity of all detected proteins was normalized to β-actin and the expressions of p-p38, p-JNK, and p-ERK1/2 were normalized to p38, JNK, and ERK1/2, respectively. A DA treatments on the expression levels of components of the MAPK and NF-κB pathways; B DAD3 treatments on the expression levels of components of the MAPK and NF-κB pathways. All data were represented as the mean ± SEM ( n = 3). ns, represents no significant difference ( p > 0.05); * represents p < 0.05; ** represents p < 0.01; and *** represents p < 0.001.

Article Snippet: Anti-phospho-IκB-α (bs-2513R/Polyclonal/Rabbit), anti-ERK(bs-0022R/Polyclonal/Rabbit), anti-phospho-ERK(bs-1522R/Polyclonal/Rabbit), anti-JNK(bs-2592R/Polyclonal/Rabbit), anti-phospho-JNK (bs-1640R/Polyclonal/Rabbit), anti-ACE2 (bs-23444R/Polyclonal/Rabbit) and anti-β-actin (bs-0061R/Polyclonal/Rabbit) were from Bioss (Beijing, China).

Techniques: Expressing, Western Blot, Activation Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells

doi: 10.1038/s41392-020-00426-x

Figure Lengend Snippet: Identification of the interaction and co-localization between CD147 and spike protein. a – c The interaction of CD147 and spike was detected by SPR assay ( a ), ELISA ( b ), and Co-IP assay ( c ). The mouse IgG (mIgG) and rabbit IgG (rIgG) were served as negative controls. d OpNS-EM images of CD147, spike(RBD) and CD147-spike(RBD) complexes. Left panels showed the survey view of the micrograph. Right panels showed 8 class averages were selected from a total of more than 300 class averages which were respectively calculated from a total of 6,681 CD147 particles; 5,073 particles of spike(RBD); 12,426 particles of CD147-spike(RBD) complexes. Scale bars: 10 nm. e The co-localization of CD147 and spike protein was observed by immuno-electron microscope. Virions (orange arrows) were observed in virus-infected Vero E6 cells and lung and kidney tissues from COVID-19 patient. The co-localization of CD147 (20 nm-gold colloid, red arrows) and spike protein (10 nm-gold colloid, yellow arrows) in SARS-CoV-2 infected Vero E6 cells and lung and kidney tissues from a patient with COVID-19. Scale bars: 200 nm

Article Snippet: After blocked with 5% goat serum, the slide was sequentially incubated with anti-spike (40150-R007, Sino Biological, China), anti-CD147 (Jiangsu Pacific Meinuoke Biopharmceutical Co. Ltd, China), anti-ACE2 (80031-R003, Sino Biological, China), anti-Rab5 (3547s, Cell Signaling Technology, USA), and anti-CD3 (ab5690, Abcam, UK) antibodies.

Techniques: SPR Assay, Enzyme-linked Immunosorbent Assay, Co-Immunoprecipitation Assay, Microscopy, Infection

Journal: Signal Transduction and Targeted Therapy

Article Title: CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells

doi: 10.1038/s41392-020-00426-x

Figure Lengend Snippet: SARS-CoV-2 employs the CD147 receptor for host cell entry. a , b Left, Vero E6 and BEAS-2B cells were transfected with shRNA for CD147 gene silence; the gene expression level was detected by real-time PCR. Right, SARS-CoV-2 infection test was performed in Vero E6-shCD147 and BEAS-2B-shCD147 cells. At 48 h after infection, the virus copy number was detected with quantitative PCR (** p < 0.01, *** p < 0.001, two-tailed t -test, mean ± SEM). c , d Left, CD147 expression level was detected by real-time PCR in BEAS-2B-CD147 and BHK-21-CD147 cells. Right, SARS-CoV-2 infection test was performed in BEAS-2B-CD147 and BHK-21-CD147 cells. At 48 h after infection, the virus copy number was detected with quantitative PCR (** p < 0.01, *** p < 0.001, two-tailed t -test, mean ± SEM). e , f The infection efficiency of SARS-CoV-2 pseudovirus was detected by luciferase reporter assay in BEAS-2B-shCD147 and BEAS-2B-CD147 cells (** p < 0.01, *** p < 0.001, two-tailed t -test, mean ± SEM). g SARS-CoV-2 pseudovirus infection of BHK-21-CD147 cells was neutralized by recombinant human CD147 extracellular fragment (** p < 0.01, *** p < 0.001, two-tailed t -test, mean ± SEM)

Article Snippet: After blocked with 5% goat serum, the slide was sequentially incubated with anti-spike (40150-R007, Sino Biological, China), anti-CD147 (Jiangsu Pacific Meinuoke Biopharmceutical Co. Ltd, China), anti-ACE2 (80031-R003, Sino Biological, China), anti-Rab5 (3547s, Cell Signaling Technology, USA), and anti-CD3 (ab5690, Abcam, UK) antibodies.

Techniques: Transfection, shRNA, Expressing, Real-time Polymerase Chain Reaction, Infection, Two Tailed Test, Luciferase, Reporter Assay, Recombinant

Journal: Signal Transduction and Targeted Therapy

Article Title: CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells

doi: 10.1038/s41392-020-00426-x

Figure Lengend Snippet: CD147 is an alternative receptor for SARS-CoV-2 infection in ACE2-deficient cell types. a No interaction of CD147 and ACE2 was detected by Co-IP assay. The mIgG and rIgG were served as negative controls. b No co-localization was found between CD147 and ACE2 by FRET. The color bar denotes FRET ratio. Scale bars: 10 μm. c No co-localization of CD147-ACE2 and the co-localizations of spike-ACE2 and spike-CD147 were observed by immuno-electron microscope (scale bars: 200 nm) and multicolor immunofluorescence (magnification: ×200) in lung tissues from COVID-19 patient. Spike protein, 10 nm-gold colloid, purple arrows; CD147, 20 nm-gold colloid, blue arrows; and ACE2, 40 nm-gold colloid, green arrows. d Virions (red arrows) were observed in lymphocytes of lung tissues from COVID-19 patient. Scale bars: 500 nm. The localization of spike protein and CD3 was analyzed by multicolor immunofluorescence staining. Magnification: ×200. e The gene expressions of CD147 and ACE2 in CD4+ and CD8+ T cells were detected by real-time PCR (*** p < 0.001, two-tailed t -test, mean ± SEM). f The expressions of CD147 and ACE2 in unactivated or activated T cells were detected by real-time PCR (* p < 0.05, Mann–Whitney test, mean ± SEM). g The infection efficiency of SARS-CoV-2 pseudovirus was detected by luciferase reporter assay in unactivated and activated T cells (* p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed t -test, mean ± SEM). h SARS-CoV-2 pseudovirus infection of CD4+ and CD8+ T cells was inhibited by Meplazumab (*** p < 0.001, two-tailed t -test, mean ± SEM). i The gene expressions of CD147 and ACE2 in Vero E6 and BEAS-2B cells were detected by real-time PCR (*** p < 0.001, two-tailed t -test, mean ± SEM)

Article Snippet: After blocked with 5% goat serum, the slide was sequentially incubated with anti-spike (40150-R007, Sino Biological, China), anti-CD147 (Jiangsu Pacific Meinuoke Biopharmceutical Co. Ltd, China), anti-ACE2 (80031-R003, Sino Biological, China), anti-Rab5 (3547s, Cell Signaling Technology, USA), and anti-CD3 (ab5690, Abcam, UK) antibodies.

Techniques: Infection, Co-Immunoprecipitation Assay, Microscopy, Immunofluorescence, Multicolor Immunofluorescence Staining, Real-time Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY, Luciferase, Reporter Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells

doi: 10.1038/s41392-020-00426-x

Figure Lengend Snippet: SARS-CoV-2 enters the host cells through CD147-mediated endocytosis. a The sequential endocytosis of SARS-CoV-2 was observed in Vero E6 cells by electron microscope. Scale bars: 200 nm. b The co-localization of spike protein, CD147, and Rab5 were analyzed in BHK-21-CD147 cells and lung tissues from COVID-19 patient by multicolor immunofluorescence staining. Magnification: ×200.

Article Snippet: After blocked with 5% goat serum, the slide was sequentially incubated with anti-spike (40150-R007, Sino Biological, China), anti-CD147 (Jiangsu Pacific Meinuoke Biopharmceutical Co. Ltd, China), anti-ACE2 (80031-R003, Sino Biological, China), anti-Rab5 (3547s, Cell Signaling Technology, USA), and anti-CD3 (ab5690, Abcam, UK) antibodies.

Techniques: Microscopy, Multicolor Immunofluorescence Staining