intact cos cells expressing high levels of rat recombinant pth receptors on the cell surface  (Charles River Laboratories)

Journal: eBioMedicine

Article Title: Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2

doi: 10.1016/j.ebiom.2024.105281

Figure Lengend Snippet: BTN3A2 inhibited SARS-CoV-2 entry and interacted with Spike protein via its RBD . a : Stable expression of BTN3A2 suppressed SARS-CoV-2 attachment to the cell. Huh7 cells overexpressing BTN3A2-L (BTN3A2-L Huh7) or BTN3A2-S (BTN3A2-S Huh7) and the control (Vector Huh7) cells were treated with DOX (1 μg/mL) for 24 h, then incubated with SARS-CoV-2 (MOI = 1) on ice at 4 °C for 1 h. Cells were washed by PBS three times and harvested to measure viral RNA copies based on SARS-CoV-2 N gene level. b : Representative immunofluorescence images of cell–cell fusion assay showing no apparent effect of BTN3A2 overexpression (BTN3A2-L and BTN3A2-S) on syncytia formation mediated by Spike of Delta. The HEK293T cells were transfected with Spike of Delta expression vector, and the Huh7 cells were transfected with px-300 for 16 h. The transfected Huh7 and HEK293T cells were co-cultured for 20 h and harvested for immunofluorescence assays. Scale bar, 10 μm. c : BTN3A2 interacted with the SARS-CoV-2 Spike protein. HEK293T cells (1 × 10 7 ) were co-transfected with expression vector of BTN3A2 isoform (BTN3A2-L-His or BTN3A2-S-His, each 5 μg) and Spike protein (5 μg) for 48 h. Whole-cell lysates were immunoprecipitated (IP) using anti-His antibody. Immunoblotting (IB) for His (BTN3A2-L-His or BTN3A2-S-His), Spike, and Tubulin was performed using anti-His, anti-Spike, and anti-Tubulin antibodies, respectively. d : BTN3A2-L interacted with the S1 subunit but not the S2 subunit of Spike protein. Spike S1 and S2 subunits were Flag-tagged. e : Endogenous BTN3A2 interacted with the Spike S1 protein. Huh7 cells (1 × 10 8 ) were transfected with expression vector for Spike S1 protein (S1-Flag, 20 μg) or empty vector (Vector) for 36 h, then cells were treated with or without IFN-α (450 U) for 12 h. Whole-cell lysates were immunoprecipitated (IP) using anti-Flag antibody. Immunoblotting (IB) for BTN3A2, Flag (S1-Flag), and Tubulin was performed using anti- BTN3A2, anti-Flag, and anti-Tubulin antibodies, respectively. f-g : Biolayer interferometry analyses of Spike S1 protein binding to immobilized BTN3A2-L-His ( f ) and BTN3A2-S-His ( g ). Different concentrations (1 000, 500, 250, and 125 nM, corresponding to kinetic curves from top to bottom) of Spike S1 protein were used for binding to BTN3A2-L-His and BTN3A2-S-His (lacked 125 nM), respectively. h : BTN3A2-S interacted with the Flag-tagged S1 subunit of the Spike protein. i : BTN3A2 interacted with Spike RBDs of different SARS-CoV-2 variants. Procedures for IP and IB in ( d ), ( h ) and ( i ) are the same as those in ( c ). Data are representative of three independent experiments with similar results ( b – i ). ( a ) Values were presented as mean ± SD, and differences between groups were determined by comparing to the Dox-group; ANOVA with Dunnett's multiple comparisons, ∗∗∗, P < 0.001.

Article Snippet: To measure the cell surface expression level of ACE2, Huh7 cells and Calu-3 cells with BTN3A2 overexpression (BTN3A2-L Huh7 cells, BTN3A2-S Huh7 cells, BTN3A2-L Calu-3 cells and BTN3A2-S Calu-3 cells) or knockout (sgBTN3A2-1 Huh7 cells, sgBTN3A2-3 Huh7 cells, sgBTN3A2-1 Calu-3 cells, and sgBTN3A2-3 Calu-3 cells), and corresponding control cells (Vector Huh7, Vector Calu-3, sgNC Huh7, and sgNC Calu-3) were washed with PBS and stained with mouse anti-ACE2 (66699-1-Ig, Proteintech, China) at a 1:50 dilution for 30 min at 4 °C.

Techniques: Expressing, Control, Plasmid Preparation, Incubation, Immunofluorescence, Cell-Cell Fusion Assay, Over Expression, Transfection, Cell Culture, Immunoprecipitation, Western Blot, Protein Binding, Binding Assay

Journal: eBioMedicine

Article Title: Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2

doi: 10.1016/j.ebiom.2024.105281

Figure Lengend Snippet: BTN3A2 inhibited ACE2 expression . a : Differential expression of BTN3A2 and ACE2 in scRNA-seq dataset of lung tissues from COVID-19 patients and uninfected individuals. Original dataset was reported in Melms et al. Significance was assessed by two-sided Wilcox rank sum test. b : Stable expression of BTN3A2 decreased ACE2 mRNA level in Huh7 cells. RNA was extracted from Huh7 cells overexpressing BTN3A2-L and BTN3A2-S, or control (Vector Huh7) cells (each group 1 × 10 5 cells). BTN3A2 and ACE2 mRNA levels were measured by qRT-PCR, with normalization to GAPDH . c-d : Overexpression of BTN3A2-L ( c ) and BTN3A2-S ( d ) suppressed ACE2 protein expression in HEK293T cells. Cells (5 × 10 5 ) were transfected with indicated expression vector or empty vector (each 1.25 μg) for 48 h, then harvested for western blotting. e : Dose-dependent inhibitory effect of BTN3A2-L on ACE2 protein expression. HEK293T cells (5 × 10 5 ) were transfected with indicated expression vector or empty vector (each 0.5 μg), together with increased BTN3A2-L-His (0.25, 0.50, 0.75, and 1.50 μg, with empty vector to reach a total amount of 2.5 μg of plasmid) for 48 h, then harvested for western blotting. f : Knockout of BTN3A2 increased ACE2 mRNA level in Huh7 cells. The procedure was similar to that in (b). g : Knockout of BTN3A2 promoted ACE2 protein expression in Huh7 ( upper ) and Calu-3 ( lower ) cells. h-i : Flow cytometry was conducted to show surface ACE2 in Calu-3 cells with BTN3A2 overexpression (BTN3A2-L and BTN3A2-S) or knockout (sgBTN3A2-1 and sgBTN3A2-2), together with the control cells (vector and sgNC) and unstained cells. Cell surface expression of ACE2 was measured ( h ), and quantification of mean fluorescence intensity (MFI (%), n = 3) was based on three independent experiments ( i ). j : ACE2 in BTN3A2-tg and WT mice infected with indicated AAV-GFP and AAV-hACE2. Immunoblotting for ACE2, BTN3A2, GFP, and Tubulin was performed using anti-ACE2, anti-BTN3A2, anti-GFP, and anti-Tubulin antibodies, respectively. k : Representative images of immunohistochemical staining of ACE2 and BTN3A2 in lung tissues in ( j ). Scale bar, 100 μm. Data were representative of three independent experiments with similar results ( b-k ). Values were presented as mean ± SD ( b , f , and i ). Significance was determined compared to Vector ( b and i ) or sgNC ( f and i ). ns, not significant; ∗, P < 0.05; ∗∗, P < 0.01; ANOVA with Dunnett's multiple comparisons.

Article Snippet: To measure the cell surface expression level of ACE2, Huh7 cells and Calu-3 cells with BTN3A2 overexpression (BTN3A2-L Huh7 cells, BTN3A2-S Huh7 cells, BTN3A2-L Calu-3 cells and BTN3A2-S Calu-3 cells) or knockout (sgBTN3A2-1 Huh7 cells, sgBTN3A2-3 Huh7 cells, sgBTN3A2-1 Calu-3 cells, and sgBTN3A2-3 Calu-3 cells), and corresponding control cells (Vector Huh7, Vector Calu-3, sgNC Huh7, and sgNC Calu-3) were washed with PBS and stained with mouse anti-ACE2 (66699-1-Ig, Proteintech, China) at a 1:50 dilution for 30 min at 4 °C.

Techniques: Expressing, Control, Plasmid Preparation, Quantitative RT-PCR, Over Expression, Transfection, Western Blot, Knock-Out, Flow Cytometry, Fluorescence, Infection, Immunohistochemical staining, Staining