huh-7 cell line cl-0120  (Procell Inc)

Journal: The EMBO Journal

Article Title: Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

doi: 10.15252/embj.2020106765

Figure Lengend Snippet: A ACE2 expression was measured for different Namalwa Syndecan cell lines in comparison to Huh7.5 cells and ACE2‐transfected 293T cells. B Huh7.5 cells were left untreated or treated with heparinase III for 1 h at 37°C and heparan sulfate or digested heparan sulfate expression was determined by flow cytometry. Cells were stained for HS and diHS respectively or an isotype control mAb, directly corresponding to the specific antibody (mouse IgM and IgG2b). One representative donor out of 3 is depicted. C Cell viability of infected Huh7.5 cells with SARS‐CoV‐2 pseudovirus in presence of different concentrations of UF heparin and LMWH enoxaparin ( n = 4 in duplicate). D Cell surface expression of ACE2 on 293T cells (control and ACE2‐transfected) was determined by quantitative real‐time PCR. E SARS‐CoV‐2 pseudovirus infection on 293T cells (control vs ACE2‐transfected cells) was measured by luciferase reporter activity. F SARS‐CoV‐2 isolate binding capacity to hACE2 was measured by quantitative real‐time PCR (ORFb1). SARS‐CoV‐2 isolate (10,000 TCID/ml) was pre‐incubated in presence or absence of different LMWH enoxaparin concentrations (250 IU/ml and 5 IU/ml) and added to a high binding ELISA plate coated with recombinant hACE2 (2 µg/ml). LMWH enoxaparin ( n = 3 measured in triplicate). Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (D) unpaired, parametric, Student's t ‐test. * P ≤ 0.05, ** P ≤ 0.01, (E) 2‐way ANOVA with Dunnett’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01 ( n = 3 in triplicate), HS: Heparan sulfate, diHS: digested Heparan sulfate, FI: fluorescent intensity, RLU: relative light units, ND: not determined.

Article Snippet: HuH7.5 cells were treated in D‐PBS/0.25% BSA with 46 milliunits heparinase III (Amsbio) for 1 h at 37°C, washed, and used in subsequent experiments.

Techniques: Expressing, Transfection, Flow Cytometry, Staining, Infection, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: The EMBO Journal

Article Title: Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

doi: 10.15252/embj.2020106765

Figure Lengend Snippet: A Huh7.5 cells were pre‐incubated with neutralizing antibody to ACE2 and SARS‐CoV‐2 pseudovirus was pre‐incubated with patient isolated mAb COVA1‐18, COVA1‐21 and COVA2‐15 (10 µg/ml) or UF heparin (250 IU/ml) for 30 min at 37°C. SARS‐CoV‐2 pseudovirus alone or with blocks was added to the cells for 4 h at 4°C and binding was determined by ELISA. B Heparan sulfates were removed from Huh7.5 cells by enzymatic treatment with heparinase III for 1 h at 37°C, then washed, and exposed to SARS‐CoV‐2 pseudovirus for 4 h at 4°C. Treated and untreated cells were subsequently lysed and binding was determined by ELISA. C Flow cytometry analysis of cell surface expression of heparan sulfates (HS) in control transduced cells or upon CRISPR/Cas9‐mediated EXT1 KO (EXT1 −/− ). D Control and EXT1 −/− XG1 cells were exposed to SARS‐CoV‐2 pseudovirus or SARS‐CoV‐2 pseudovirus pre‐treated with 250 IU/ml UF heparin for 30 min at 37°C. After incubation for 4 h at 4°C, cells were lysed and binding was measured by ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) ordinary one‐way ANOVA with Tukey multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 ( n = 3), (B) unpaired Student’s t ‐test with Welch’s correction. * P ≤ 0.05, ** P ≤ 0.01 ( n = 3), (D) two‐way ANOVA with Dunnett’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01 ( n = 3).

Article Snippet: HuH7.5 cells were treated in D‐PBS/0.25% BSA with 46 milliunits heparinase III (Amsbio) for 1 h at 37°C, washed, and used in subsequent experiments.

Techniques: Incubation, Isolation, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, CRISPR

Journal: The EMBO Journal

Article Title: Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

doi: 10.15252/embj.2020106765

Figure Lengend Snippet: A Huh7.5 cells were exposed to SARS‐CoV‐2 pseudovirus directly or after pre‐treatment with different concentrations (0.0001, 0.001, 0.1, 0.5, 1, 5, 50, 100, 250 IU/ml) of UF heparin or LMWH enoxaparin for 30 min at 37°C. Infection was determined by luciferase reporter activity after 5 days. B SARS‐CoV‐2 pseudovirus was pre‐incubated for 30 min at 37°C with UF heparin (250 IU/ml) or LMWH tinzaparin (250 IU/ml) or dalteparin (250 IU/ml) or enoxaparin (250 IU/ml) or nadroparin (250 IU/ml). Huh7.5 was exposed to the SARS‐CoV‐2 pseudovirus, alone or treated with different LMWH for 4 h at 4°C, washed, lysed and binding was determined by ELISA. C SARS‐CoV‐2 pseudovirus was pre‐incubated for 30 min at 37°C with UF heparin (250 IU/ml) or LMWH tinzaparin (250 IU/ml) or dalteparin (250 IU/ml) or enoxaparin (250 IU/ml) or nadroparin (250 IU/ml). Huh7.5 cells were infected with SARS‐CoV‐2 pseudovirus in presence or absence of different LMWH and infection was determined after 5 days by luciferase reporter activity. D 293T cells expressing ACE2 were infected with SARS‐CoV‐2 pseudovirus in presence or absence of antibodies against ACE2, UF heparin (250 IU/ml), or LMWH enoxaparin (250 IU/ml), and infection was determined after 3 days by luciferase reporter activity. E VeroE6 cells were infected with SARS‐CoV‐2 isolate (hCoV‐19/Italy; 100 TCID/ml) previously treated with serial dilutions of LMWH enoxaparin. Cell viability was determined using an MTT assay ( n = 3 donors measured in triplicate). Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) ordinary one‐way ANOVA with Dunnett’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 ( n = 3 donors measured in triplicate). (B) Ordinary one‐way ANOVA with Tukey’s multiple‐comparison test ( n = 3 donors measured in monoplo), (C) ordinary one‐way ANOVA with Dunnett’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 ( n = 3 donors measured in triplicate), (D) ordinary one‐way ANOVA with Dunnett’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01 ( n = 3 measured in triplicate). RLU: relative light units. Source data are available online for this figure.

Article Snippet: HuH7.5 cells were treated in D‐PBS/0.25% BSA with 46 milliunits heparinase III (Amsbio) for 1 h at 37°C, washed, and used in subsequent experiments.

Techniques: Infection, Luciferase, Activity Assay, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, MTT Assay

Journal: The EMBO Journal

Article Title: Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

doi: 10.15252/embj.2020106765

Figure Lengend Snippet: A Syndecan 1 and Syndecan 4 expression by Namalwa cell lines, 293T cells, and Huh7.5 cells was detected by quantitative real‐time PCR. Representative data for an experiment repeated more than three times with similar results. B, C Different Namalwa Syndecan cell lines expressing either Syndecan 1 (B) or Syndecan 4 (C), determined by quantitative real‐time PCR. D Huh7.5 cells and Namalwa cells ectopically expressing Syndecan 1 were exposed to SARS‐CoV‐2 pseudovirus or control pseudovirus lacking S protein, in presence or absence of LMWH enoxaparin (250 IU/ml). Binding was measured after 4 h at 4°C by ELISA. E DCs were stained with antibodies against the surface markers CD209, CD14, and CD11b and analyzed by flow cytometry. F LCs were stained with antibodies against CD207 and CD1a and analyzed by flow cytometry. The histogram shows the cell surface expression of the receptor. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (D) 2‐way ANOVA with Tukey’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 ( n = 2 in triplicate). DCs: Dendritic cells, LCs: Langerhans cells, ND: not determined, FI: fluorescent intensity.

Article Snippet: HuH7.5 cells were treated in D‐PBS/0.25% BSA with 46 milliunits heparinase III (Amsbio) for 1 h at 37°C, washed, and used in subsequent experiments.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

Journal: The EMBO Journal

Article Title: Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

doi: 10.15252/embj.2020106765

Figure Lengend Snippet: A, B SARS‐CoV‐2 binding to monocyte‐derived DCs (A) or primary LCs (B) in absence or presence of UF heparin (250 IU/ml) or LMWH enoxaparin (250 IU/ml). C DCs and LCs were infected with SARS‐CoV‐2 pseudovirus and infection was determined after 5 days by measuring luciferase reporter activity. As positive controls, Huh7.5 cells were infected. D ACE2 cell surface expression on DCs, LCs, and Huh7.5 cells. Representative data for an experiment repeated more than three times with similar results ( n = 3 in duplicate). E Graphical overview of the cell‐to‐cell viral transmission assay. F, G DCs (F) and LCs (G) were pre‐incubated with SARS‐CoV‐2 pseudovirus for 4 h at 37°C in presence or absence of UF heparin (250 IU/ml) or LMWH enoxaparin (250 IU/ml), extensively washed, and co‐cultured with Huh7.5 cells. Transmission by DCs or LCs to Huh7.5 cells was determined by luciferase reporter activity. H, I SARS‐CoV‐2 isolate (hCoV‐19/Italy) was pre‐treated with LMWH enoxaparin (250 IU/ml) for 30 min at 37°C. DCs (H) and LCs (I) were exposed to either the untreated or pre‐treated SARS‐CoV‐2 isolate (100 TCID/ml) for 24 h, washed thoroughly, and co‐cultured with Huh7.5 cells. Quantification of viral RNA was measured by quantitative real‐time PCR. Data information: Data show the mean values and error bars are the SEM. (A, B) ordinary one‐way ANOVA with Tukey’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (A) ( n = 4), (B) ( n = 4), (C) ordinary one‐way ANOVA with Tukey’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 ( n = 4 measured in triplicate), (F) ordinary one‐way ANOVA with Dunnett’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01 ( n = 4 measured in triplicate), (G) ordinary one‐way ANOVA with Tukey’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 ( n = 3 measured in triplicate). (H, I) ordinary one‐way ANOVA with Tukey’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (H) ( n = 3 in duplicate), (I) ( n = 4 in duplicate). DCs: Dendritic cells, LCs: Langerhans cells, RLU: relative light units, ND: not determined. Source data are available online for this figure.

Article Snippet: HuH7.5 cells were treated in D‐PBS/0.25% BSA with 46 milliunits heparinase III (Amsbio) for 1 h at 37°C, washed, and used in subsequent experiments.

Techniques: Binding Assay, Derivative Assay, Infection, Luciferase, Activity Assay, Expressing, Transmission Assay, Incubation, Cell Culture, Real-time Polymerase Chain Reaction

Journal: The EMBO Journal

Article Title: Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

doi: 10.15252/embj.2020106765

Figure Lengend Snippet: A, B SARS‐CoV‐2 isolate (hCoV‐19/Italy, 100 TCID/ml) was either pre‐treated with LMWH enoxaparin (250 IU/ml) for 30 min at 37°C. DCs ( n = 3 independent donors) (A) and LCs (B) were exposed to untreated or pre‐treated SARS‐CoV‐2 (isolated for 24 h, washed thoroughly, and subsequently co‐cultured with Huh7.5 cells for another 24 h). Quantification of viral RNA was measured by quantitative real‐time PCR of Huh7.5 cells after removal of DCs or LCs.

Article Snippet: HuH7.5 cells were treated in D‐PBS/0.25% BSA with 46 milliunits heparinase III (Amsbio) for 1 h at 37°C, washed, and used in subsequent experiments.

Techniques: Isolation, Cell Culture, Real-time Polymerase Chain Reaction