Fig. 6: nCoV400 antibody suppresses N protein-driven aggregation in vitro and in cells. | Communications Biology

Fig. 6: nCoV400 antibody suppresses N protein-driven aggregation in vitro and in cells.

From: Structural basis of a human antibody targeting SARS-CoV-2 nucleocapsid protein dimerization domain and interfering with RNA-binding

Fig. 6

a Representative fluorescence images of AF488-labeled N-CTD protein (20 μM) mixed with vRNA157 (1 μM) to form condensate liquid droplets in vitro, in the absence or presence of nCoV400Fab at an equimolar ratio to N-CTD. Lanes 1 and 2 with the addition of 10% PEG8000 while lane 3 and 4 contained 20% PEG8000. Scale bars, 50 μm. b Fluorescence microscopy images showing condensate droplets formed by incubation of AF488-labeled full-length N-CTD (green, 20 μM) or AF488-labeled truncated N-CTD (Δ264–364) (green, 20 μM) with Cy5-labeled 55-nt vRNA (red, 20 μM) in the presence or absence of different inhibitors. Conditions included full-length N-CTD with vRNA (lane 1), truncated N-CTD (Δ264–364) with vRNA (lane 2), and N-CTD with vRNA incubated separately with nCoV400Fab (lane 3), L-chicoric acid (lane 4), ampicillin (lane 5), or the non-N-CTD-specific antibody nCoV396Fab (lane 6). Merged images (yellow) illustrate colocalization of N-CTD and RNA. Scale bar, 10 μm. c Representative line profile analysis demonstrating the colocalization pattern between AF488-N-CTD and Cy5-55-nt vRNA signals. d Quantitative analysis of condensate formation, including integrated fluorescence intensity, droplet counts, and area occupied by droplets, calculated from four randomly selected fields for each condition. Data represent mean ± SD. Statistical significance was analyzed by one-way ANOVA (Exact p values are indicated; ns, not significant). e Confocal fluorescence microscopy images of HEK293T cells under four conditions, corresponding to lanes 1–4 from left to right: Lane 1: transfected with N-FL-mCherry alone; Lane 2: transfected with N-FL-mCherry and treated with poly(I:C); Lane 3: co-transfected with N-FL-mCherry and plasmids encoding nCoV400-GFP, then treated with poly(I:C); Lane 4: transfected with nCoV400-GFP alone. N-FL-mCherry is shown in red, nCoV400-GFP in green, and merged images (yellow) indicate colocalization. White arrows indicate representative N protein condensates. Scale bar, 10 μm. f Representative fluorescence intensity profiles showing colocalization of N-FL-mCherry (red) and nCoV400-GFP (green) signals. g Quantification of N protein condensates per cell under each condition. Data were collected from three independent experiments, with 17 randomly selected cells analyzed per group. Data represent mean ± SD. Statistical significance was determined by one-way ANOVA (Exact p values are indicated; ns not significant).

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