Fig 1: Characterization of NPC and endosome-to-Golgi trafficking proteins in ORF3a-expressed cells(A) The indicated HeLa-Flp-In cells were quantified for free cholesterol by high-content imaging.(B and C) The indicated cells were transfected with NPC2-mCherry and immunostained with the indicated antibodies. Colocalization of NPC2-mCherry with LAMP1 was assessed by confocal microscopy quantified.(D and E) Vero E6 cells were transfected with NPC2-mcherry and then infected with SARS-CoV-2, fixed, and immunostained 24 h post infection. Colocalization of NPC2-mCherry with LAMP1 was assessed by confocal microscopy and quantified with FIJI.(F and G) Equal volumes of serum-free media was placed for the Flp-In cell culture and subjected to immunoblotting using the antibodies indicated. Cell input was controlled using calnexin in corresponding lysates. Quantification was based on three independent experiments.(H–J) The indicated cells were analyzed for protein localization and colocalization by confocal microscopy (H) and high-content imaging (I). Periphery was defined as a ring area shrunk from cell boundary with a gap distance 15 pixels. Center was defined as a dilated circle from nuclear boundary with a distance of 20 pixels. Middle was defined as the area between periphery and center (I). Pearson correlation coefficient of CI-MPR LAMP2 was quantified with high-content imaging in the indicated cells (J).(K and L) Secretion and cellular levels of indicated proteins were analyzed in wild-type and VPS29 knockout HeLa cells.(M) Vesicular filipin was measured by high-content imaging and normalized to lysosomes in the indicated cells.(N and O) Pearson correlation coefficient was measured with high-content imaging in the indicated cells.Bar graphs are presented as mean ± SD. p values were determined using t test or one-way ANOVA test. *p < 0.05, **p < 0.01 (vs. control), ##p < 0.01 (CoV-2 vs. W193A), ***p < 0.001, ****p < 0.0001 (vs. control), ####p < 0.0001 (CoV-2 vs. W193A); n.s., not significant. Scale bars, 5 μm. Control (ctrl) and linker peptides; CoV, SARS-CoV ORF3a; CoV-2, SARS-CoV-2 ORF3a; and W193A, SARS-CoV-2 ORF3a-W193A mutant.