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. 2022 Jul 11;150(4):796–805. doi: 10.1016/j.jaci.2022.05.029

Fig 4.

Fig 4

ORF3a-dependent ASC speck formation in THP1-cell–derived macrophages. A, Representative confocal images of ASC speck formation (in yellow) in the different experimental conditions (empty plasmid, ORF3a, LPS/ATP). Images are a result of a 3-D reconstruction of Z stack. Cells were stained with anti-ASC antibody (yellow), anti-NLRP3 (green), and DAPI (blue). Transfected cells PCMV6-AC-rfp-ORF3a and PCMV6-AC-rfp are in red. Scale bar is 5 μm. B, Representative confocal images of ASC specks (yellow) that colocalized with ORF3a protein (red). Images are a result of a 3-D reconstruction of Z stack. Cells were stained with anti-ASC antibody (yellow) and DAPI (blue). Transfected cells PCMV6-AC-rfp-ORF3a are in red. Scale bar is 5 μm. C, Percentage of THP1-macrophage cells forming ASC specks quantified from n = 100 cells per condition in quadruplicate (n = 4). THP1 macrophage cells were stimulated with LPS (100 ng/mL) for 3 hours followed by ATP (5 mM) for 30 minutes or transfected with PCMV6-AC-rfp-ORF3a and PCMV6-AC-rfp. 3-D, Three-dimensional; DAPI, 4'-6-diamidino-2-phenylindole, dihydrochloride; IQR, interquartile range. Data are expressed as median ± IQR (Fig 4, B and C). ∗P < .05 and ∗∗P < .01 as assessed by Mann-Whitney test.