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MedChemExpress diphyllin

<t>Diphyllin</t> inhibits SARS-CoV-2 infections. (A-C) Caco-2, Calu-3 and A549 cells were transfected with plasmid hACE2. At 72 h post transfection, the cells were treated with diphyllin in different concentrations of diphyllin (0.01 to 10 μM) or control DMSO for 2 h, followed by infection with SARS-CoV-2-S-pseudotyped viruses (7.6*10 5 copies/ml) for 24 h At 72 h post-pseudovirus inoculation, RLUs were measured using luciferase reporter assay and cell viability was detected by using CCK-8 assay. (D-F) Caco-2-hACE2, Calu-3-hACE2, and A549-hACE2 cells were pre-incubated with 2 μM diphyllin for 2 h, followed by infection with SARS-CoV-2-S-pseudotyped viruses. The fluorescent signal of viral spike expression was imaged by confocal microscopy at 48 h post-virus infection. Scale bar: 10 μm; magnification: 630 × . (G, H) Caco-2 and Calu-3 cells were pre-treated with diphyllin at 1 μM, 3.3 μM, and 10 μM for 1.5 h and subsequently infected with a clinical SARS-CoV-2 isolate wild-type strain B.1 (MOI =0.1). At 2 h post-infection, the supernatant was removed and replaced with fresh culture medium containing the respective amount of diphyllin. At 24 h post-virus inoculation, viral S and N proteins in the infected cells were quantified by in-cell-ELISA. * P < 0.05; ** P < 0.01; ns, not significant. (K, L) Fully differentiated human primary tracheal epithelial cells in air liquid interface culture were washed with Hanks’ salt solution (HSS) and pretreated apically and basolaterally with diphyllin at 1 μM, 3.3 μM, and 10 μM for 1.5 h before the cells were infected apically with 50,000 PFU per well with the respective amount of diphyllin. The virus solution was removed after 1 h of incubation and the cells were washed 3 times with 250 µl HSS per well for 10 min. The third wash was collected and stored at -80 °C as 2 aliquots of 100 µl. These samples were analyzed as 0 dpi samples. At 1, 2, and 3 days post-infection, cells were incubated apically with 250 µl of HSS for 15 min before the solution was removed and stored at -80 °C as 2 aliquots of 100 µl. The samples were inactivated for 10 min at 70 °C and for 10 min at 95 °C before they were used for quantification of viral genomes. The samples were diluted 1:15 in HSS and SARS-CoV-2 genome copy numbers were determined by use of the diagnostic Alinity m SARS-CoV-2 Amp Kit. Human primary tracheal epithelial cells isolated from 3 donors were analyzed in triplicate. Quantification of viral genomes was performed in duplicate. The diphyllin-treated conditions were compared to the DMSO-treated control by one-way ANOVA. * P < 0.05; ** P < 0.01.

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Article Title: Diphyllin elicits a doubled-pronged attack on the entry of SARS-CoV-2 by inhibiting cathepsin L and furin

Journal: Virus Research

doi: 10.1016/j.virusres.2024.199485

Diphyllin inhibits SARS-CoV-2 infections. (A-C) Caco-2, Calu-3 and A549 cells were transfected with plasmid hACE2. At 72 h post transfection, the cells were treated with diphyllin in different concentrations of diphyllin (0.01 to 10 μM) or control DMSO for 2 h, followed by infection with SARS-CoV-2-S-pseudotyped viruses (7.6*10 5 copies/ml) for 24 h At 72 h post-pseudovirus inoculation, RLUs were measured using luciferase reporter assay and cell viability was detected by using CCK-8 assay. (D-F) Caco-2-hACE2, Calu-3-hACE2, and A549-hACE2 cells were pre-incubated with 2 μM diphyllin for 2 h, followed by infection with SARS-CoV-2-S-pseudotyped viruses. The fluorescent signal of viral spike expression was imaged by confocal microscopy at 48 h post-virus infection. Scale bar: 10 μm; magnification: 630 × . (G, H) Caco-2 and Calu-3 cells were pre-treated with diphyllin at 1 μM, 3.3 μM, and 10 μM for 1.5 h and subsequently infected with a clinical SARS-CoV-2 isolate wild-type strain B.1 (MOI =0.1). At 2 h post-infection, the supernatant was removed and replaced with fresh culture medium containing the respective amount of diphyllin. At 24 h post-virus inoculation, viral S and N proteins in the infected cells were quantified by in-cell-ELISA. * P < 0.05; ** P < 0.01; ns, not significant. (K, L) Fully differentiated human primary tracheal epithelial cells in air liquid interface culture were washed with Hanks’ salt solution (HSS) and pretreated apically and basolaterally with diphyllin at 1 μM, 3.3 μM, and 10 μM for 1.5 h before the cells were infected apically with 50,000 PFU per well with the respective amount of diphyllin. The virus solution was removed after 1 h of incubation and the cells were washed 3 times with 250 µl HSS per well for 10 min. The third wash was collected and stored at -80 °C as 2 aliquots of 100 µl. These samples were analyzed as 0 dpi samples. At 1, 2, and 3 days post-infection, cells were incubated apically with 250 µl of HSS for 15 min before the solution was removed and stored at -80 °C as 2 aliquots of 100 µl. The samples were inactivated for 10 min at 70 °C and for 10 min at 95 °C before they were used for quantification of viral genomes. The samples were diluted 1:15 in HSS and SARS-CoV-2 genome copy numbers were determined by use of the diagnostic Alinity m SARS-CoV-2 Amp Kit. Human primary tracheal epithelial cells isolated from 3 donors were analyzed in triplicate. Quantification of viral genomes was performed in duplicate. The diphyllin-treated conditions were compared to the DMSO-treated control by one-way ANOVA. * P < 0.05; ** P < 0.01.

Figure Legend Snippet: Diphyllin inhibits SARS-CoV-2 infections. (A-C) Caco-2, Calu-3 and A549 cells were transfected with plasmid hACE2. At 72 h post transfection, the cells were treated with diphyllin in different concentrations of diphyllin (0.01 to 10 μM) or control DMSO for 2 h, followed by infection with SARS-CoV-2-S-pseudotyped viruses (7.6*10 5 copies/ml) for 24 h At 72 h post-pseudovirus inoculation, RLUs were measured using luciferase reporter assay and cell viability was detected by using CCK-8 assay. (D-F) Caco-2-hACE2, Calu-3-hACE2, and A549-hACE2 cells were pre-incubated with 2 μM diphyllin for 2 h, followed by infection with SARS-CoV-2-S-pseudotyped viruses. The fluorescent signal of viral spike expression was imaged by confocal microscopy at 48 h post-virus infection. Scale bar: 10 μm; magnification: 630 × . (G, H) Caco-2 and Calu-3 cells were pre-treated with diphyllin at 1 μM, 3.3 μM, and 10 μM for 1.5 h and subsequently infected with a clinical SARS-CoV-2 isolate wild-type strain B.1 (MOI =0.1). At 2 h post-infection, the supernatant was removed and replaced with fresh culture medium containing the respective amount of diphyllin. At 24 h post-virus inoculation, viral S and N proteins in the infected cells were quantified by in-cell-ELISA. * P < 0.05; ** P < 0.01; ns, not significant. (K, L) Fully differentiated human primary tracheal epithelial cells in air liquid interface culture were washed with Hanks’ salt solution (HSS) and pretreated apically and basolaterally with diphyllin at 1 μM, 3.3 μM, and 10 μM for 1.5 h before the cells were infected apically with 50,000 PFU per well with the respective amount of diphyllin. The virus solution was removed after 1 h of incubation and the cells were washed 3 times with 250 µl HSS per well for 10 min. The third wash was collected and stored at -80 °C as 2 aliquots of 100 µl. These samples were analyzed as 0 dpi samples. At 1, 2, and 3 days post-infection, cells were incubated apically with 250 µl of HSS for 15 min before the solution was removed and stored at -80 °C as 2 aliquots of 100 µl. The samples were inactivated for 10 min at 70 °C and for 10 min at 95 °C before they were used for quantification of viral genomes. The samples were diluted 1:15 in HSS and SARS-CoV-2 genome copy numbers were determined by use of the diagnostic Alinity m SARS-CoV-2 Amp Kit. Human primary tracheal epithelial cells isolated from 3 donors were analyzed in triplicate. Quantification of viral genomes was performed in duplicate. The diphyllin-treated conditions were compared to the DMSO-treated control by one-way ANOVA. * P < 0.05; ** P < 0.01.

Techniques Used: Transfection, Plasmid Preparation, Control, Infection, Luciferase, Reporter Assay, CCK-8 Assay, Incubation, Expressing, Confocal Microscopy, Virus, In-Cell ELISA, Diagnostic Assay, Isolation

Diphyllin inhibits various SARS-CoV-2 variant pseudoviral infections in vitro. Calu-3 and Caco-2 cells were transfected with plasmid hACE2. (A, B) At 72 h post transfection, Calu-3-hACE2 and Caco-2-hACE2 cells were pre-treated with various concentrations of diphyllin (0.033 to 10 μM) for 2 h, followed by infection with equivalent doses of each SARS-CoV-2-S-pseudotyped viruses (7.6*10 5 copies/mL), including WT, Alpha, Beta, Delta, and Omicron. (C, D) At 72 h post hACE2 transfection, Calu-3-hACE2 and Caco-2-hACE2 cells were pre-treated with 2 μM diphyllin for 2 h and subsequently infected with different SARS-CoV-2-S-pseudotyped viruses as described above. The fluorescent signal of viral spike expression was imaged by confocal microscopy at 48 h post-virus infection. Scale bar: 10 μm; magnification: 630 × . * P < 0.05; ** P < 0.01; ns, not significant.

Figure Legend Snippet: Diphyllin inhibits various SARS-CoV-2 variant pseudoviral infections in vitro. Calu-3 and Caco-2 cells were transfected with plasmid hACE2. (A, B) At 72 h post transfection, Calu-3-hACE2 and Caco-2-hACE2 cells were pre-treated with various concentrations of diphyllin (0.033 to 10 μM) for 2 h, followed by infection with equivalent doses of each SARS-CoV-2-S-pseudotyped viruses (7.6*10 5 copies/mL), including WT, Alpha, Beta, Delta, and Omicron. (C, D) At 72 h post hACE2 transfection, Calu-3-hACE2 and Caco-2-hACE2 cells were pre-treated with 2 μM diphyllin for 2 h and subsequently infected with different SARS-CoV-2-S-pseudotyped viruses as described above. The fluorescent signal of viral spike expression was imaged by confocal microscopy at 48 h post-virus infection. Scale bar: 10 μm; magnification: 630 × . * P < 0.05; ** P < 0.01; ns, not significant.

Techniques Used: Variant Assay, In Vitro, Transfection, Plasmid Preparation, Infection, Expressing, Confocal Microscopy, Virus

Diphyllin has no effect on ACE2 expression and distribution as well as the binding of ACE2 and SARS-CoV-2 S protein. (A, B) Calu-3 and A549 cells were treated with 2 μM diphyllin for 48 h The fluorescent signal of ACE2 expression was imaged by confocal microscopy using an anti-ACE2 primary antibody. Scale bar: 25 μm; magnification: 630 × . (C, D) The levels of intracellular ACE2 and dynamin expression were measured by western blot with actin as a loading control. The ACE2 /actin ratio was calculated by densitometry analysis. E HEK293T cells were co-transfected with plasmid 3 × Flag-hACE2 and 2 × Strep-S for 48 h Whole-cell extracts were immunoprecipitated with anti-Flag antibody. F HEK293T cells were co-transfected with plasmid 3 × Flag-hACE2 and 2 × Strep-S, followed by treatment with 2 μM diphyllin or DMSO for 48 h Cell lysates were purified using anti-Flag antibody. Immunoprecipitated and input proteins were probed with the indicated antibodies. * P < 0.05; ** P < 0.01; ns, not significant.

Figure Legend Snippet: Diphyllin has no effect on ACE2 expression and distribution as well as the binding of ACE2 and SARS-CoV-2 S protein. (A, B) Calu-3 and A549 cells were treated with 2 μM diphyllin for 48 h The fluorescent signal of ACE2 expression was imaged by confocal microscopy using an anti-ACE2 primary antibody. Scale bar: 25 μm; magnification: 630 × . (C, D) The levels of intracellular ACE2 and dynamin expression were measured by western blot with actin as a loading control. The ACE2 /actin ratio was calculated by densitometry analysis. E HEK293T cells were co-transfected with plasmid 3 × Flag-hACE2 and 2 × Strep-S for 48 h Whole-cell extracts were immunoprecipitated with anti-Flag antibody. F HEK293T cells were co-transfected with plasmid 3 × Flag-hACE2 and 2 × Strep-S, followed by treatment with 2 μM diphyllin or DMSO for 48 h Cell lysates were purified using anti-Flag antibody. Immunoprecipitated and input proteins were probed with the indicated antibodies. * P < 0.05; ** P < 0.01; ns, not significant.

Techniques Used: Expressing, Binding Assay, Confocal Microscopy, Western Blot, Control, Transfection, Plasmid Preparation, Immunoprecipitation, Purification

Diphyllin inhibits SARS-CoV-2 endocytosis. Calu-3 and A549 cells were transfected with plasmid hACE2. (A, B) At 72 h post hACE2 transfection, Calu-3-hACE2 and A549-hACE2 cells were pre-treated with 1 μM diphyllin and 10 μM chlorpromazine for 2 h, followed by SARS-CoV-2-S-pseudotyped virus infection (7.6*10 5 copies/mL) for 24 h At 72 h post-pseudovirus inoculation, RLUs were measured using a luciferase reporter assay and normalized to that of the DMSO control. (C, D) At 72 h post hACE2 transfection, Calu-3-hACE2 and A549-hACE2 cells were treated with 2 μM diphyllin for 48 h EEA1, CD63, and LAMP1 expression levels were measured by western blot with actin as a loading control. (E, F) Internal LAMP1 expression was detected by confocal microscopy using an anti-LAMP1 antibody. (G, H) Calu-3-hACE2 and A549-hACE2 cells were treated as described above, followed by incubation with 10 μg/ml DQ-BSA Red for 30 min. Cells treated with Earle's balanced salt solution (EBSS) for 2 h were used as a positive control. The accumulating fluorescent signal of DQ-BSA Red was analyzed by confocal microscopy. (I, J) Calu-3-hACE2 and A549-hACE2 cells were treated with 2 μM diphyllin for 24 h and subsequently stained using 100 nM LysoTracker Red for 1 h Cells treated with EBSS and starved for 2 h were used as a positive control. The fluorescence intensity of LysoTracker Red was imaged by confocal microscopy. Scale bar: 10 μm; magnification: 630 × . * P < 0.05; ** P < 0.01; ns, not significant.

Figure Legend Snippet: Diphyllin inhibits SARS-CoV-2 endocytosis. Calu-3 and A549 cells were transfected with plasmid hACE2. (A, B) At 72 h post hACE2 transfection, Calu-3-hACE2 and A549-hACE2 cells were pre-treated with 1 μM diphyllin and 10 μM chlorpromazine for 2 h, followed by SARS-CoV-2-S-pseudotyped virus infection (7.6*10 5 copies/mL) for 24 h At 72 h post-pseudovirus inoculation, RLUs were measured using a luciferase reporter assay and normalized to that of the DMSO control. (C, D) At 72 h post hACE2 transfection, Calu-3-hACE2 and A549-hACE2 cells were treated with 2 μM diphyllin for 48 h EEA1, CD63, and LAMP1 expression levels were measured by western blot with actin as a loading control. (E, F) Internal LAMP1 expression was detected by confocal microscopy using an anti-LAMP1 antibody. (G, H) Calu-3-hACE2 and A549-hACE2 cells were treated as described above, followed by incubation with 10 μg/ml DQ-BSA Red for 30 min. Cells treated with Earle's balanced salt solution (EBSS) for 2 h were used as a positive control. The accumulating fluorescent signal of DQ-BSA Red was analyzed by confocal microscopy. (I, J) Calu-3-hACE2 and A549-hACE2 cells were treated with 2 μM diphyllin for 24 h and subsequently stained using 100 nM LysoTracker Red for 1 h Cells treated with EBSS and starved for 2 h were used as a positive control. The fluorescence intensity of LysoTracker Red was imaged by confocal microscopy. Scale bar: 10 μm; magnification: 630 × . * P < 0.05; ** P < 0.01; ns, not significant.

Techniques Used: Transfection, Plasmid Preparation, Virus, Infection, Luciferase, Reporter Assay, Control, Expressing, Western Blot, Confocal Microscopy, Incubation, Positive Control, Staining, Fluorescence

Diphyllin blocks SARS-CoV-2 endocytosis by inhibiting endosomal cathepsin L activity. (A, B) Calu-3 and A549 cells were transfected with plasmid Strep-S expressing SARS-CoV-2 S protein and empty vector pLVX-EF1α, followed by treating with 2 μM diphyllin for 48 h. Analysis of SARS-CoV-2 S expression by western blot using anti-Strep antibody directed against the C-terminal Strep tag added to the viral S proteins. (C-F) Calu-3 and A549 cells were transfected with plasmid hACE2. At 72 h post hACE2 transfection, Calu-3-hACE2 or A549-hACE2 cells were treated with 2 μM diphyllin for 48 h (C, D) CTSB (mature, 38 kDa; inactive, 44 kDa), CTSD, CTSL (mature, 26 kDa; inactive, 29 kDa), ATP6V1B2, and ATP6V1D expression levels were measured by western blot. The ratios of mature CTSL: actin were calculated by densitometry analysis. (E, F) CTSL expression levels inside the cells were imaged by confocal microscopy using an anti-CTSL antibody. Scale bar: 25 μm; magnification: 630 × . (G, H) Calu-3-hACE2 and A549-hACE2 cells were pre-treated with 1 μM diphyllin and 10 μM MDL28170 for 2 h, followed by SARS-CoV-2-S-pseudotyped virus infection (7.6*10 5 copies/mL) for 24 h At 72 h post-pseudovirus inoculation, CTSL expression was measured by western blot and (I, J) relative luminescence units (RLUs) were measured using a luciferase reporter assay and normalized to that of the DMSO control. (K) The enzymatic activity of CTSL was assessed using cathepsin L inhibitor screening assay kit. * P < 0.05; ** P < 0.01; ns, not significant.

Figure Legend Snippet: Diphyllin blocks SARS-CoV-2 endocytosis by inhibiting endosomal cathepsin L activity. (A, B) Calu-3 and A549 cells were transfected with plasmid Strep-S expressing SARS-CoV-2 S protein and empty vector pLVX-EF1α, followed by treating with 2 μM diphyllin for 48 h. Analysis of SARS-CoV-2 S expression by western blot using anti-Strep antibody directed against the C-terminal Strep tag added to the viral S proteins. (C-F) Calu-3 and A549 cells were transfected with plasmid hACE2. At 72 h post hACE2 transfection, Calu-3-hACE2 or A549-hACE2 cells were treated with 2 μM diphyllin for 48 h (C, D) CTSB (mature, 38 kDa; inactive, 44 kDa), CTSD, CTSL (mature, 26 kDa; inactive, 29 kDa), ATP6V1B2, and ATP6V1D expression levels were measured by western blot. The ratios of mature CTSL: actin were calculated by densitometry analysis. (E, F) CTSL expression levels inside the cells were imaged by confocal microscopy using an anti-CTSL antibody. Scale bar: 25 μm; magnification: 630 × . (G, H) Calu-3-hACE2 and A549-hACE2 cells were pre-treated with 1 μM diphyllin and 10 μM MDL28170 for 2 h, followed by SARS-CoV-2-S-pseudotyped virus infection (7.6*10 5 copies/mL) for 24 h At 72 h post-pseudovirus inoculation, CTSL expression was measured by western blot and (I, J) relative luminescence units (RLUs) were measured using a luciferase reporter assay and normalized to that of the DMSO control. (K) The enzymatic activity of CTSL was assessed using cathepsin L inhibitor screening assay kit. * P < 0.05; ** P < 0.01; ns, not significant.

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Strep-tag, Confocal Microscopy, Virus, Infection, Luciferase, Reporter Assay, Control, Screening Assay

Diphyllin blocks SARS-CoV-2 cell surface entry by decreasing furin expression. (A, B) HEK293T cells were transfected with plasmid GFP-SARS-CoV-2-S or empty vector pAdTrack-TO4-GFP as effector cells; Calu-3 and A549 cells were transfected with plasmid hACE2 as target cells. The cell–cell fusion rate was analyzed by confocal microscopy in the presence of 2 μM diphyllin, 10 μM chloroquine (CQ), or DMSO (control). The fusion rate was set as 100% in a DMSO-treated group. Scale bar: 10 μm; magnification: 200 × . (C, D) Calu-3 and A549 cells were transfected with plasmid hACE2. At 72 h post-hACE2 transfection, Calu-3-hACE2 or A549-hACE2 cells were treated with 2 μM diphyllin for 48 h The levels of furin and TMPRSS2 expression were measured using western blot. The ratios of furin: actin were calculated by densitometry analysis. Calu-3-hACE2 and A549-hACE2 cells were treated with 1 μM diphyllin and 30 μM Decanoyl-RVKR-CMK (CMK) for 2 h, followed by SARS-CoV-2-S-pseudotyped virus infection for 24 h At 72 h post-pseudovirus inoculation, (E, F) furin expression was measured using western blot and (G, H) RLUs of SARS-CoV-2-S were measured using luciferase reporter assay and normalized to that of the DMSO control. (I) The enzymatic activities of furin and TMPRSS2 were assessed using the dedicated furin protease assay kit and TMPRSS2 fluorogenic assay kit, respectively. * P < 0.05; ** P < 0.01; ns, not significant.

Figure Legend Snippet: Diphyllin blocks SARS-CoV-2 cell surface entry by decreasing furin expression. (A, B) HEK293T cells were transfected with plasmid GFP-SARS-CoV-2-S or empty vector pAdTrack-TO4-GFP as effector cells; Calu-3 and A549 cells were transfected with plasmid hACE2 as target cells. The cell–cell fusion rate was analyzed by confocal microscopy in the presence of 2 μM diphyllin, 10 μM chloroquine (CQ), or DMSO (control). The fusion rate was set as 100% in a DMSO-treated group. Scale bar: 10 μm; magnification: 200 × . (C, D) Calu-3 and A549 cells were transfected with plasmid hACE2. At 72 h post-hACE2 transfection, Calu-3-hACE2 or A549-hACE2 cells were treated with 2 μM diphyllin for 48 h The levels of furin and TMPRSS2 expression were measured using western blot. The ratios of furin: actin were calculated by densitometry analysis. Calu-3-hACE2 and A549-hACE2 cells were treated with 1 μM diphyllin and 30 μM Decanoyl-RVKR-CMK (CMK) for 2 h, followed by SARS-CoV-2-S-pseudotyped virus infection for 24 h At 72 h post-pseudovirus inoculation, (E, F) furin expression was measured using western blot and (G, H) RLUs of SARS-CoV-2-S were measured using luciferase reporter assay and normalized to that of the DMSO control. (I) The enzymatic activities of furin and TMPRSS2 were assessed using the dedicated furin protease assay kit and TMPRSS2 fluorogenic assay kit, respectively. * P < 0.05; ** P < 0.01; ns, not significant.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Control, Western Blot, Virus, Infection, Luciferase, Reporter Assay, Protease Assay

Proposed model of diphyllin inhibiting SARS-CoV-2 entry. Diphyllin elicits a doubled-pronged attack on the entry of SARS-CoV-2 by blocking the endocytosis route through inhibiting CTSL activity and the cell surface route through inhibition of furin activity.

Figure Legend Snippet: Proposed model of diphyllin inhibiting SARS-CoV-2 entry. Diphyllin elicits a doubled-pronged attack on the entry of SARS-CoV-2 by blocking the endocytosis route through inhibiting CTSL activity and the cell surface route through inhibition of furin activity.

Techniques Used: Blocking Assay, Activity Assay, Inhibition

CCK-8 Assay:

Article Title: Diphyllin elicits a doubled-pronged attack on the entry of SARS-CoV-2 by inhibiting cathepsin L and furin.
Article Snippet: The samples were diluted 1:15 in HSS and SARS-CoV-2 genome copy numbers were determined by use of the diagnostic Alinity m SARS-CoV-2 Amp Kit (Abbott, Illinois, USA) according to the manufactureŕs instructions. .. Potential cytotoxic effects of diphyllin on various cells were assessed by the Cell Counting Kit-8 (CCK-8; MedChemExpress, HY-K0301) according to the manufacturer’s instructions (Mao et al., 2022). ..

Article Title: Diphyllin elicits a doubled-pronged attack on the entry of SARS-CoV-2 by inhibiting cathepsin L and furin
Article Snippet: The samples were diluted 1:15 in HSS and SARS-CoV-2 genome copy numbers were determined by use of the diagnostic Alinity m SARS-CoV-2 Amp Kit (Abbott, Illinois, USA) according to the manufacturer ́s instructions. .. Potential cytotoxic effects of diphyllin on various cells were assessed by the Cell Counting Kit-8 (CCK-8; MedChemExpress, HY-K0301) according to the manufacturer's instructions ( ). ..

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