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(A) Schematic of the ACE2 and ACE2-GPI constructs used in this study. For ACE2-GPI, the transmembrane domain and cytoplasmic tail of ACE2 were replaced with Thy1, a GPI-anchored protein, to localize ACE2 to lipid rafts. (B) Surface expression of ACE2 and ACE2-GPI by flow cytometry analysis by ACE2 surface staining with anti-ACE2 VHH-B07-Fc. A serial dilution of ACE2-expression vector was used to transfect 293T cells before surface staining. Shown is the total percentage of ACE2-positive cells (left) and representative contour plots taken from one independent repeat (right). Data are means ±s.d. of three independent experiments. (C) 293T cells expressing ACE2 or ACE2-GPI were incubated with recombinant biotinylayed-SARS-CoV-2 RBD (Wuhan variant). Cell surface staining with streptavidin AF-488 was carried out at indicated timepoints. Data were normalized to 0h post-RBD staining. Data are means ±s.d. of three independent experiments. (D) Treatment of 293T cells with phospholipase C at indicated concentrations for 2 hours at 37°C. ACE2 surface levels were assessed by flow cytometry. Data were normalized to NT conditions. Data are means ±s.d. of three independent experiments. (E) Histogram plots from flow cytometry analysis of ACE2 cell surface staining of 293T cells (left) and MRC-5 cells (right) stably expressing ACE2 or ACE2-GPI. Results are representative of at least three independent experiments. (F) Cell lysates of 293T cells (above) and MRC-5 cells (below) stably expressing ACE2 or ACE2-GPI were ultracentrifuged in an <t>iodixanol</t> gradient to isolate membranes on their lipid content. * = Lipid raft associated protein, flotillin-1, was used as a control for lipid raft localization. For B, C, and D, Mann-Whitney tests were performed to compare ACE2 and ACE2-GPI conditions, * P < 0.05, ** P < 0.001, *** P < 0.0001, ns = not significant.
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(A) Schematic of the ACE2 and ACE2-GPI constructs used in this study. For ACE2-GPI, the transmembrane domain and cytoplasmic tail of ACE2 were replaced with Thy1, a GPI-anchored protein, to localize ACE2 to lipid rafts. (B) Surface expression of ACE2 and ACE2-GPI by flow cytometry analysis by ACE2 surface staining with anti-ACE2 VHH-B07-Fc. A serial dilution of ACE2-expression vector was used to transfect 293T cells before surface staining. Shown is the total percentage of ACE2-positive cells (left) and representative contour plots taken from one independent repeat (right). Data are means ±s.d. of three independent experiments. (C) 293T cells expressing ACE2 or ACE2-GPI were incubated with recombinant biotinylayed-SARS-CoV-2 RBD (Wuhan variant). Cell surface staining with streptavidin AF-488 was carried out at indicated timepoints. Data were normalized to 0h post-RBD staining. Data are means ±s.d. of three independent experiments. (D) Treatment of 293T cells with phospholipase C at indicated concentrations for 2 hours at 37°C. ACE2 surface levels were assessed by flow cytometry. Data were normalized to NT conditions. Data are means ±s.d. of three independent experiments. (E) Histogram plots from flow cytometry analysis of ACE2 cell surface staining of 293T cells (left) and MRC-5 cells (right) stably expressing ACE2 or ACE2-GPI. Results are representative of at least three independent experiments. (F) Cell lysates of 293T cells (above) and MRC-5 cells (below) stably expressing ACE2 or ACE2-GPI were ultracentrifuged in an <t>iodixanol</t> gradient to isolate membranes on their lipid content. * = Lipid raft associated protein, flotillin-1, was used as a control for lipid raft localization. For B, C, and D, Mann-Whitney tests were performed to compare ACE2 and ACE2-GPI conditions, * P < 0.05, ** P < 0.001, *** P < 0.0001, ns = not significant.
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(A) Schematic of the ACE2 and ACE2-GPI constructs used in this study. For ACE2-GPI, the transmembrane domain and cytoplasmic tail of ACE2 were replaced with Thy1, a GPI-anchored protein, to localize ACE2 to lipid rafts. (B) Surface expression of ACE2 and ACE2-GPI by flow cytometry analysis by ACE2 surface staining with anti-ACE2 VHH-B07-Fc. A serial dilution of ACE2-expression vector was used to transfect 293T cells before surface staining. Shown is the total percentage of ACE2-positive cells (left) and representative contour plots taken from one independent repeat (right). Data are means ±s.d. of three independent experiments. (C) 293T cells expressing ACE2 or ACE2-GPI were incubated with recombinant biotinylayed-SARS-CoV-2 RBD (Wuhan variant). Cell surface staining with streptavidin AF-488 was carried out at indicated timepoints. Data were normalized to 0h post-RBD staining. Data are means ±s.d. of three independent experiments. (D) Treatment of 293T cells with phospholipase C at indicated concentrations for 2 hours at 37°C. ACE2 surface levels were assessed by flow cytometry. Data were normalized to NT conditions. Data are means ±s.d. of three independent experiments. (E) Histogram plots from flow cytometry analysis of ACE2 cell surface staining of 293T cells (left) and MRC-5 cells (right) stably expressing ACE2 or ACE2-GPI. Results are representative of at least three independent experiments. (F) Cell lysates of 293T cells (above) and MRC-5 cells (below) stably expressing ACE2 or ACE2-GPI were ultracentrifuged in an iodixanol gradient to isolate membranes on their lipid content. * = Lipid raft associated protein, flotillin-1, was used as a control for lipid raft localization. For B, C, and D, Mann-Whitney tests were performed to compare ACE2 and ACE2-GPI conditions, * P < 0.05, ** P < 0.001, *** P < 0.0001, ns = not significant.

Journal: bioRxiv

Article Title: SARS-CoV-2 entry and fusion are independent of ACE2 localization to lipid rafts

doi: 10.1101/2024.07.13.603361

Figure Lengend Snippet: (A) Schematic of the ACE2 and ACE2-GPI constructs used in this study. For ACE2-GPI, the transmembrane domain and cytoplasmic tail of ACE2 were replaced with Thy1, a GPI-anchored protein, to localize ACE2 to lipid rafts. (B) Surface expression of ACE2 and ACE2-GPI by flow cytometry analysis by ACE2 surface staining with anti-ACE2 VHH-B07-Fc. A serial dilution of ACE2-expression vector was used to transfect 293T cells before surface staining. Shown is the total percentage of ACE2-positive cells (left) and representative contour plots taken from one independent repeat (right). Data are means ±s.d. of three independent experiments. (C) 293T cells expressing ACE2 or ACE2-GPI were incubated with recombinant biotinylayed-SARS-CoV-2 RBD (Wuhan variant). Cell surface staining with streptavidin AF-488 was carried out at indicated timepoints. Data were normalized to 0h post-RBD staining. Data are means ±s.d. of three independent experiments. (D) Treatment of 293T cells with phospholipase C at indicated concentrations for 2 hours at 37°C. ACE2 surface levels were assessed by flow cytometry. Data were normalized to NT conditions. Data are means ±s.d. of three independent experiments. (E) Histogram plots from flow cytometry analysis of ACE2 cell surface staining of 293T cells (left) and MRC-5 cells (right) stably expressing ACE2 or ACE2-GPI. Results are representative of at least three independent experiments. (F) Cell lysates of 293T cells (above) and MRC-5 cells (below) stably expressing ACE2 or ACE2-GPI were ultracentrifuged in an iodixanol gradient to isolate membranes on their lipid content. * = Lipid raft associated protein, flotillin-1, was used as a control for lipid raft localization. For B, C, and D, Mann-Whitney tests were performed to compare ACE2 and ACE2-GPI conditions, * P < 0.05, ** P < 0.001, *** P < 0.0001, ns = not significant.

Article Snippet: Lysates were gently mixed with 2 mL of 60% OptiPrep iodixanol density gradient medium (Sigma-Aldrich) within 15 mL ultracentrifuge tubes.

Techniques: Construct, Expressing, Flow Cytometry, Staining, Serial Dilution, Plasmid Preparation, Incubation, Recombinant, Variant Assay, Stable Transfection, Control, MANN-WHITNEY