Journal: Biomedicines

Article Title: Anti-SARS-CoV-2 Activity of Extracellular Vesicle Inhibitors: Screening, Validation, and Combination with Remdesivir

doi: 10.3390/biomedicines9091230

Figure Lengend Snippet: The dose-dependent effect, the half maximal inhibitory concentration (IC50), and the half maximal cytotoxicity (CC50) of calpeptin against SARS-CoV-2 infected Vero E6 cells. Vero E6 cells were infected with SARS-CoV-2 at 25TCID50 for 2 h and then post-infection treated with calpeptin, at concentrations ranging from 0.008 to 100 µM, for 48 h. Positive convalescent serum of a COVID-19 patient was included as a positive control. The supernatant was collected for viral output quantification. The infected cells were fixed and stained for viral nucleoproteins with anti-SARS-CoV NP mAb. The SARS-CoV-2 infected cells were detected by high-content imaging. ( a ) The high-content images of calpeptin treatment in SARS-CoV-2 infected Vero E6 cells are demonstrated. Scale bar: 20 µm. The percentage of inhibition was calculated as the percentage of the control conditions. The cytotoxicity assay was performed in parallel to evaluate the cell viability at each concentration. ( b ) The percentage of virus inhibition (blue) and cell viability (red) is shown. The data are presented as mean ± SEM of three biological replicates. ( c ) Viral output was examined by plaque reduction assay, and data are presented as % of the control ( n = 2 biological replicates).

Article Snippet: We decided to evaluate calpeptin (1 and 25 µM for Vero E6 and Calu-3, respectively) and remdesivir (0.5 and 0.1 µM for Vero E6 and Calu-3, respectively) at the concentrations below the IC50 values based on the high-content imaging studies in the corresponding cell lines ( b and b).

Techniques: Concentration Assay, Infection, Positive Control, Staining, Imaging, Inhibition, Cytotoxicity Assay

Journal: Biomedicines

Article Title: Anti-SARS-CoV-2 Activity of Extracellular Vesicle Inhibitors: Screening, Validation, and Combination with Remdesivir

doi: 10.3390/biomedicines9091230

Figure Lengend Snippet: Anti-SARS-CoV-2 activity of calpeptin in human-lung epithelial cells Calu-3. Calu-3 cells were infected with SARS-CoV-2 at 25TCID50 for 2 h and then post-infection treated with calpeptin at concentrations ranging from 0.008 to 100 µM for 48 h. Positive convalescent serum of a COVID-19 patient was included as a positive control. The supernatant was collected for viral output quantification. The infected cells were fixed and stained for viral nucleoproteins with anti-SARS-CoV NP mAb. The SARS-CoV-2 infected cells were detected by high-content imaging. ( a ) The high-content images of calpeptin treatment in SARS-CoV-2 infected Calu-3 cells are demonstrated. Scale bar: 20 µm. The percentage of inhibition was calculated as the percentage of the control conditions. The cytotoxicity assay was performed in parallel to evaluate the cell viability at each concentration. The data are presented as the mean ± SEM of three biological replicates. ( b ) The percentage of virus inhibition (blue) and cell viability (red) is shown. ( c ) Viral output was examined by plaque reduction assay, and data are presented as % of the control ( n = 2 biological replicates).

Article Snippet: We decided to evaluate calpeptin (1 and 25 µM for Vero E6 and Calu-3, respectively) and remdesivir (0.5 and 0.1 µM for Vero E6 and Calu-3, respectively) at the concentrations below the IC50 values based on the high-content imaging studies in the corresponding cell lines ( b and b).

Techniques: Activity Assay, Infection, Positive Control, Staining, Imaging, Inhibition, Cytotoxicity Assay, Concentration Assay

Journal: Biomedicines

Article Title: Anti-SARS-CoV-2 Activity of Extracellular Vesicle Inhibitors: Screening, Validation, and Combination with Remdesivir

doi: 10.3390/biomedicines9091230

Figure Lengend Snippet: Effects of calpeptin and remdesivir combination on anti-SARS-CoV-2 activity in Vero E6 and Calu-3 cells. Cells were infected with SARS-CoV-2 at 25TCID50 for 2 h and then post-infection treated-indicated concentration of remdesivir (µM):calpeptin (µM). Positive convalescent serum of a COVID-19 patient was included as the positive control, and mock infection was performed in parallel as a negative control. The supernatant was collected for viral output quantification. The infected cells were fixed and stained for viral nucleoproteins with anti-SARS-CoV NP mAb. The SARS-CoV-2-infected cells were detected by high-content imaging. The high-content images of combination treatment in SARS-CoV-2 infected Vero E6 ( a ) and Calu-3 ( b ) cells are shown. The percentage of infected Vero E6 ( c ) and Calu-3 ( f ) was calculated and present at the indicated concentrations. The amounts of infectious virions in the supernatant of infected Vero E6 ( d ) and Calu-3 ( g ) cells were quantified by plaque assay, and data were presented as the percentage of the control. The percentage of cell viability of Vero E6 ( e ) and Calu-3 ( h ) are shown. The data are presented as the mean ± SEM of three biological replicates. Statistical analysis was performed by using one-way ANOVA with Tukey post hoc test: * p < 0.05, ** p < 0.005, and *** p < 0.001, ns, not significant. Scale bar: 20 µm.

Article Snippet: We decided to evaluate calpeptin (1 and 25 µM for Vero E6 and Calu-3, respectively) and remdesivir (0.5 and 0.1 µM for Vero E6 and Calu-3, respectively) at the concentrations below the IC50 values based on the high-content imaging studies in the corresponding cell lines ( b and b).

Techniques: Activity Assay, Infection, Concentration Assay, Positive Control, Negative Control, Staining, Imaging, Plaque Assay

Journal: Biomedicines

Article Title: Anti-SARS-CoV-2 Activity of Extracellular Vesicle Inhibitors: Screening, Validation, and Combination with Remdesivir

doi: 10.3390/biomedicines9091230

Figure Lengend Snippet: Proposed mechanisms of SARS-CoV-2 inhibition by remdesivir and calpeptin combination. Remdesivir, a virus-targeting drug, inhibits RNA-dependent RNA polymerase (RdRp), leading to the inhibition of SARS-CoV-2 replication. Calpeptin, a host-targeting compound, suppresses the viral release via inhibiting EV trafficking and shedding microvesicles, resulting in the inhibition of SARS-CoV-2 production and release. This figure was created with BioRender.com (accessed on 13 August 2021).

Article Snippet: We decided to evaluate calpeptin (1 and 25 µM for Vero E6 and Calu-3, respectively) and remdesivir (0.5 and 0.1 µM for Vero E6 and Calu-3, respectively) at the concentrations below the IC50 values based on the high-content imaging studies in the corresponding cell lines ( b and b).

Techniques: Inhibition