Journal: ACS nano

Article Title: Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography.

doi: 10.1021/acsnano.3c07265

Figure Lengend Snippet: Figure 1. Different distributions of SARS-CoV-2 N protein and dsRNA in infected human lung adenocarcinoma cells at different stages of the infection. A549-ACE2 cells were inoculated with SARS-CoV-2 (MOI 0.01), incubated at 37 °C for 24 h, chemically fixed and processed for immunofluorescence microscopy using antibodies against dsRNA (red) and SARS-CoV-2 N protein (green). Nuclei were counterstained with DAPI (blue). Scale bars represent 10 μm. (A−D) Examples of cells with increasing amounts of dsRNA signal (red) indicating different stages of the infection: (A) early, (B, C) intermediate, and (D) late stages of infection.

Article Snippet: A549 expressing hACE2 (A549-ACE2) was generated by transduction of parental A549 cells (ATCC CRM-CCL-185) with a retroviral vector expressing human ACE2 and a selection marker that confers resistance to blasticidin. pCMV3-hACE2 was obtained from Sino Biologicals S.L. (Beijing, China).

Techniques: Infection, Incubation, Immunofluorescence, Microscopy

Journal: ACS nano

Article Title: Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography.

doi: 10.1021/acsnano.3c07265

Figure Lengend Snippet: Figure 2. dsRNA-positive structures near centrosomes surrounded by vimentin during SARS-CoV-2 infection. A549-ACE2 cells were inoculated with SARS-CoV-2 (MOI 0.01), incubated at 37 °C for 24 h, chemically fixed, and processed for immunofluorescence microscopy using antibodies against dsRNA (red) and vimentin (A, B; green) or pericentrin (C; green). Nuclei and actin filaments were counterstained with DAPI (blue) and Alexa 660-conjugated phalloidin (A, B; cyan), respectively. (B) corresponds to a zoomed-in image of the indicated area in (A). Scale bars represent 10 μm.

Article Snippet: A549 expressing hACE2 (A549-ACE2) was generated by transduction of parental A549 cells (ATCC CRM-CCL-185) with a retroviral vector expressing human ACE2 and a selection marker that confers resistance to blasticidin. pCMV3-hACE2 was obtained from Sino Biologicals S.L. (Beijing, China).

Techniques: Infection, Incubation, Immunofluorescence, Microscopy

Journal: ACS nano

Article Title: Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography.

doi: 10.1021/acsnano.3c07265

Figure Lengend Snippet: Figure 3. Golgi apparatus dispersion in SARS-CoV-2-infected cells. A549-ACE2 cells were inoculated with SARS-CoV-2 (MOI 0.01), incubated at 37 °C for 24 h, chemically fixed, and processed for immunofluorescence microscopy using antibodies against dsRNA (red) and Golgi marker giantin (green). (A) and (B) show two different representative fields of infected cells. Arrowheads show late stages of infection and (n) dsRNA-negative, noninfected cells. Nuclei were counterstained with DAPI (blue). Scale bars represent 10 μm.

Article Snippet: A549 expressing hACE2 (A549-ACE2) was generated by transduction of parental A549 cells (ATCC CRM-CCL-185) with a retroviral vector expressing human ACE2 and a selection marker that confers resistance to blasticidin. pCMV3-hACE2 was obtained from Sino Biologicals S.L. (Beijing, China).

Techniques: Dispersion, Infection, Incubation, Immunofluorescence, Microscopy, Marker

Journal: ACS nano

Article Title: Three-Dimensional Remodeling of SARS-CoV2-Infected Cells Revealed by Cryogenic Soft X-ray Tomography.

doi: 10.1021/acsnano.3c07265

Figure Lengend Snippet: Figure 4. Transmission electron microscopy of SARS-CoV2-infected human cells. A549-ACE2 cells were inoculated at MOI 3 and incubated for 20 h before chemical fixation and processing for TEM. Representative images of (A) mock infected cells, where the inset shows the ultrastructure of a multilamellar body (MLB), and (B−F) SARS-CoV-2-infected cells. Definitions: N, nucleus; VRO, viral replication organelle; m, mitochondrion; VIR, putative virions; DMV, double-membrane vesicle; black arrowheads, enlarged ER cisternae; white arrowheads, centriole. Scale bars: (A, B) 5 μm; (C−E) 1 μm; (F) 0.5 μm. The scale bar in the inset to (A) is 1 μm. (H) corresponds to the area delimited by a dashed line in (G).

Article Snippet: A549 expressing hACE2 (A549-ACE2) was generated by transduction of parental A549 cells (ATCC CRM-CCL-185) with a retroviral vector expressing human ACE2 and a selection marker that confers resistance to blasticidin. pCMV3-hACE2 was obtained from Sino Biologicals S.L. (Beijing, China).

Techniques: Transmission Assay, Electron Microscopy, Infection, Incubation, Membrane

Journal: PLoS ONE

Article Title: Phenolic compounds disrupt spike-mediated receptor-binding and entry of SARS-CoV-2 pseudo-virions

doi: 10.1371/journal.pone.0253489

Figure Lengend Snippet: (A) Dose-dependent binding of RBD-SARS-CoV-2 to immobilized hACE2 receptor. Control– 0.025% DMSO, positive and negative controls were provided by the manufacturer; data are presented as % of control ± SD. (B) Dose-dependent binding of A546 cells expressing SARS-CoV-2 eGFP-spike protein, in the presence of indicated polyphenols at different concentrations, to soluble hACE2 receptor. Control– 0.25% DMSO; positive and negative controls were provided by the manufacturer; data are presented as % of control ± SD. (C) Viability of A549 cells, positive control—100% dead cells, negative control—addition-free sample; TF-3—theaflavin-3,3’-digallate; # p ≤ 0.05, Δ p ≤ 0.01, * p ≤ 0.001.

Article Snippet: Human alveolar epithelial cell line A549, stably overexpressing hACE2 receptor (hACE2/A549), and eGFP-luciferase-SARS-CoV-2 spike glycoprotein pseudo-typed particles were obtained from GenScript (Piscataway, NJ).

Techniques: Binding Assay, Control, Expressing, Positive Control, Negative Control

Journal: PLoS ONE

Article Title: Phenolic compounds disrupt spike-mediated receptor-binding and entry of SARS-CoV-2 pseudo-virions

doi: 10.1371/journal.pone.0253489

Figure Lengend Snippet: (A) Dose-dependent binding of SARS-CoV-2 spike protein-encapsulated pseudo-virions to A549 cells stably overexpressing human ACE2 receptor evaluated after 1h incubation. (B) Dose-dependent binding of SARS-CoV-2 spike protein-encapsulated pseudo-virions to A549 cells stably overexpressing hACE2 receptor evaluated after 3h incubation. Data are presented as % of control ± SD; control– 0.025% DMSO, positive and negative controls were provided by the manufacturer; TF-3 –theaflavin-3,3’-digallate # p ≤ 0.05, Δ p ≤ 0.01, * p ≤ 0.001.

Article Snippet: Human alveolar epithelial cell line A549, stably overexpressing hACE2 receptor (hACE2/A549), and eGFP-luciferase-SARS-CoV-2 spike glycoprotein pseudo-typed particles were obtained from GenScript (Piscataway, NJ).

Techniques: Binding Assay, Stable Transfection, Incubation, Control

Journal: PLoS ONE

Article Title: Phenolic compounds disrupt spike-mediated receptor-binding and entry of SARS-CoV-2 pseudo-virions

doi: 10.1371/journal.pone.0253489

Figure Lengend Snippet: (A) Cell-cell fusion of A549 cells expressing eGFP spike protein with A549 cells stably expressing human ACE2 receptor. The scale bar indicates 250 μm. (B) Quantitative analysis of formed syncytia. Experiments were done in triplicate and repeated three times. Data are presented as % of control ± SD; TF-3 –theaflavin-3,3’-digallate Δ p ≤ 0.01, * p ≤ 0.001. Control– 0.025% DMSO, positive control– 20 μg/ml anti-ACE2 antibody.

Article Snippet: Human alveolar epithelial cell line A549, stably overexpressing hACE2 receptor (hACE2/A549), and eGFP-luciferase-SARS-CoV-2 spike glycoprotein pseudo-typed particles were obtained from GenScript (Piscataway, NJ).

Techniques: Expressing, Stable Transfection, Control, Positive Control

Journal: PLoS ONE

Article Title: Phenolic compounds disrupt spike-mediated receptor-binding and entry of SARS-CoV-2 pseudo-virions

doi: 10.1371/journal.pone.0253489

Figure Lengend Snippet: (A) Binding of indicated polyphenols at different concentrations to hACE2 receptor. Data are presented as % of control ± SD; control– 0.025% DMSO, positive control– 50% DMSO. (B) Activity of recombinant hACE2 upon treatment with indicated polyphenols at different concentrations. (left panel). Activity of cellular hACE2 upon treatment with indicated polyphenols at different concentrations. (right panel). Data are presented as % of control ± SD; * p ≤ 0.001. Control– 0.025% DMSO, positive control– 10% DMSO. (C) Activity of recombinant TMPTSS2 upon treatment with indicated polyphenols at different. (left panel). Activity of cellular TMPTSS2 upon treatment with indicated polyphenols at different concentrations (right panel). Data are presented as % of control ± SD; # p ≤ 0.05, Δ p ≤ 0.01, * p ≤ 0.001. Control– 0.025% DMSO, positive control– 50–100 μM camostat mesylate. (D) Western blot analysis of hACE2 and TMPRSS2 expression in A549 cells upon treatment with indicated polyphenols with different concentration for 48h period. Data are presented as % of control ± SD; control– 0.025% DMSO, TF-3 –theaflavin-3,3’-digallate.

Article Snippet: Human alveolar epithelial cell line A549, stably overexpressing hACE2 receptor (hACE2/A549), and eGFP-luciferase-SARS-CoV-2 spike glycoprotein pseudo-typed particles were obtained from GenScript (Piscataway, NJ).

Techniques: Binding Assay, Control, Positive Control, Activity Assay, Recombinant, Western Blot, Expressing, Concentration Assay