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: 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: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Construction of HEK293T cells line that continuously expressing ACE2-GFP. HEK293Tcells were transfected with pCMV-ACE2-GFPSark tag plasmid and selected with hygromycin B to generate cell lines expressingACE2-GFP, cells were passaged 10 times to ensure stable expression. a Confocal imaging of HEK293T cell line with stably expressed ACE2-GFP. b Cell lysates were analyzed by Western blot with antibodies specific for ACE2 to confirm the expression of ACE2-GFP in HEK293T cells

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Expressing, Transfection, Plasmid Preparation, Imaging, Stable Transfection, Western Blot

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Establishment of the in vitro cell capturing system using immobilized spikeS1 protein. a - e , Optimization of the dose of immobilized spike S1 protein for capturing HEK293T/ACE2-GFP cells. Different amounts of S1 protein were coated on the 96-well microplate to capture the HEK293T/ACE2-GFP cells. Representative micrographs of captured cells are shown for 0 μg (A),0.125 μg(B),0.25 μg (C),0.5 μg(D),and 1.0 μg(E). F Quantification of captured cells using CCK8 test, data are presented as mean ± SD

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: In Vitro

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Competitive curve of cell capturing inhibition by RBD domain of Spike protein. 0.5 μg Spike S1 protein was coated on the microplate to capture the HEK293T/ACE2-GFP cells in presence of different concentrations of spike RBD protein. Amount of captured cells were determined by CCK8 test and expressed as relative value by setting the capability to capture cells of the non-RBD group as 100%. Data were plotted with a four-parameter logistic (4PL) regression curve fit of relative cell number (y-axis) versus the competitor concentration (x-axis)

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Inhibition, Concentration Assay

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Correlation between expression of ACE2 on target cells and cell capturing ability. Endogenous expression of ACE2 in different cell lines determined by A Western blotting and B qPCR. C The cell capture ability of immobilized S1 to different cell lines. Captured cells were counted manually under the optical microscope; D correlation between ACE2 RNA level and S1 captured cell number fit a linear relationship with R 2 = 0.82

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Expressing, Western Blot, Microscopy

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: The D614G mutation increases ACE2-expressing cell capturing ability of S protein. A S1 or D614G S1 variant was immobilized on 96 well microplates, and HEK293 cells stably expressing ACE2-GFP were incubated with ACE2-293 T cells. Captured cells were detached by Trypsin and counted by CCK8 test. Quantitation data are presented as mean ± SD; *** p < 0.05 by unpaired Student t-test. B FLAG-tagged ACE2 stable expressed cells lysate were incubate with Spike S1 or S1(D614G) proteins. After FLAG-IP, the immunocomplexes were blotted with anti-FLAG or anti-spike antibodies as indicated. Spike S1 monomer (apparent molecular weight ~ 116Kd) and trimer (apparent molecular weight ~ 300Kd) were found after the western blotting. Fluorescence intensity quantification of S1 or S1(D614G) indicated below each band were normalized by each input.

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Mutagenesis, Expressing, Variant Assay, Stable Transfection, Incubation, Quantitation Assay, Molecular Weight, Western Blot, Fluorescence

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Detachment and purification of captured cells. A A schematic overview of a 2-step cell purification protocol. In the first step, captured cells were invertedly centrifuged to remove the weakly-bound cell. In the second step, remaining cells were detached by treatment of trypsin. B Expression of ACE2 on transiently transfected ACE2-293 T cells before (left panel) and after (right panel) purification and gate M1 indicates the proportion of cells with high expression of ACE2-GFP. C Cell viability of ACE2-293 T cells before and after the purification. Cell viability was determined using Annexin-V/PI staining

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Purification, Expressing, Transfection, Staining

Journal: Biological Procedures Online

Article Title: Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

doi: 10.1186/s12575-021-00153-9

Figure Lengend Snippet: Competitive curve of cell capturing inhibition by serum purified from Spike-protein-immunized mice. Serum from S-protein immunized non-immunized mice were titrated and pre-incubated in SARS-CoV-2 Spike S1 coated wells for 1 h before the addition of ACE2-HEK293 cells. Data were plotted with a four-parameter logistic (4PL) regression curve fit of the relative amount of captured cells determined by CCK8 test (y-axis) versus 1:2n diluted serum samples (x-axis)

Article Snippet: ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen).

Techniques: Inhibition, Purification, Incubation

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: bioRxiv

Article Title: Co-infection of influenza A virus enhances SARS-CoV-2 infectivity

doi: 10.1101/2020.10.14.335893

Figure Lengend Snippet: ( A and B ) A549 cells were mock-infected or infected with WSN at MOI 0.1. At 12 hours post-infection (h.p.i.), total RNAs were extracted from cells, and mRNA of ACE2, TMPRSS2, Furin, CatL ( A ), or mRNA of NP, Mx1, ISG54 ( B )was evaluated by quantitative real-time PCR (qRT-PCR) using SYBR green method. The data were expressed as fold changes relative to the Mock infections. ( C ) A549 cells were infected with WSN at MOI 0.1. IAV NP proteins (red) and ACE2 (green) were detected by an immunofluorescence assay using a confocal microscope at 12 hours-post-infection. Scale bars were shown. A549 ( D ), Calu-3 ( E ), and NHBE ( F ) cells were pre-infected with WSN at MOI 0.1 for 12 hours. Cells were then infected with live SARS-CoV-2 at MOI 0.01 for another 48 hours. Total RNAs were extracted from cells and mRNA of ACE2 was evaluated by quantitative real-time PCR (qRT-PCR) using SYBR green method. The protein expression levels of ACE2, SARS-CoV-2 N gene, IAV NP, and β-actin were measured by western blot. ( G ) The relative mRNA levels of ACE2 were measured from lung homogenates in indicated groups and the protein expression of IAV NP and ACE2 were detected by western blot accordingly. ( D-G and J ) The data were expressed as fold changes relative to the non-IAV infection control. ( H-K ) To establish ACE2 knock-down cells, A549 cell mixture was transduced with lentivirus encoding CRISPR-Cas9 system with two guide RNAs targeting ACE2 (sgRNA1 and sgRNA2) or control guide RNA respectively. Cells were infected with live SARS-CoV-2 at MOI 0.01 with or without IAV infection under the same procedure as above. The mRNA levels of ACE2 (qRT-PCR) ( H ) and SARS-CoV-2 E gene (Taqman-qRT-PCR) ( I ) expression were detected. ( J ) The mRNA level of ACE2 was detected by qRT-PCR in live SARS-CoV-2-infected cells transfected with either vector of WSN segment-2 respectively. ( K ) The mRNA levels of the SARS-CoV-2 E gene from either vector- or segment2-transfected cells were measured by Taqman-qRT-PCR at 48 hours post-live-SARS-CoV-2-infection in the present of control sgRNA or ACE2 sgRNAs. The data were expressed as fold change relative to non-IAV infection control. Values are mean ± s.d. of three independent results. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

Article Snippet: The primary antibodies used in this study were rabbit polyclonal antibody against ACE2 for immunofluorescence (Sino Biological, 10108-T26) and anti-influenza virus-NP (kindly provided by Prof. Ningshao Xia).

Techniques: Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay, Immunofluorescence, Microscopy, Expressing, Western Blot, Transduction, CRISPR, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Co-infection of influenza A virus enhances SARS-CoV-2 infectivity

doi: 10.1101/2020.10.14.335893

Figure Lengend Snippet: Calu-3 cells were mock-infected or infected with WSN at MOI of 0.1. At 12 hours h.p.i., total RNAs were extracted from cells, and mRNA of ACE2, TMPRSS2, Furin, CatL, NP, Mx1, and ISG54 were evaluated by qRT-PCR using the SYBR green method. The data were expressed as fold changes relative to the Mock infections. Values are mean ± s.d. of three independent results. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

Article Snippet: The primary antibodies used in this study were rabbit polyclonal antibody against ACE2 for immunofluorescence (Sino Biological, 10108-T26) and anti-influenza virus-NP (kindly provided by Prof. Ningshao Xia).

Techniques: Infection, Quantitative RT-PCR, SYBR Green Assay