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. 2024 Feb 2;15(3):e03358-23. doi: 10.1128/mbio.03358-23

Fig 1.

Fig 1

NSP6 inhibited the biogenesis of ACE2-exos. (A) The screening of viral proteins related to exosome formation. HEK293T cells were transfected with 300 ng of control empty vector or GFP-tagged SARS-CoV-2 protein-expressing plasmids, respectively. Cells were collected at 48 h post transfection (hpt). The frequencies of CD63-positive (CD63+) cells were analyzed by flow cytometry, which was first gated on GFP+ cells. (B) A dose-dependent manner of NSP6-mediated CD63 downregulation. HEK293T cells were transfected with various amounts (50 ng, 100 ng, and 200 ng) of GFP-tagged NSP6 (NSP6-GFP) plasmids, and mean fluorescence intensities (MFIs) of CD63 were analyzed by flow cytometry, gating on GFP+ cells. (C) The mRNA level of CD63 after the overexpression of NSP6 in HEK293T cells, normalized to GAPDH mRNA expression. (D) The downregulation of CD63 by NSP6 was validated by Western blot in HEK293T cells. HEK293T cells were transfected with various amounts (0.25 µg, 0.50 µg, 0.75 µg, and 1.00 µg) of NSP6-GFP plasmids, followed by Western blot against calnexin (internal control) and CD63. (E) The downregulation of CD63 by NSP6 was validated by Western blot in A549 cells. A549 cells were transfected with various amounts (0.25 µg, 0.50 µg, 1.00 µg, and 1.50 µg) of NSP6-GFP plasmids, followed by Western blot against calnexin (internal control) and CD63. (F) The downregulation of CD63 by NSP6 was validated by flow cytometry in A549 cells. A549 cells were transfected with control empty vector or NSP6-GFP plasmids, and MFIs of CD63 were analyzed. (G) The downregulation of CD63 by NSP6 was validated by Western blot in Calu-3 cells. Calu-3 cells were transfected with various amounts (0.25 µg, 0.50 µg, 1.00 µg, and 1.50 µg) of NSP6-GFP plasmids, followed by Western blot against calnexin (internal control) and CD63. (H) The downregulation of CD63 by NSP6 was validated by flow cytometry in Calu-3 cells. Calu-3 cells were transfected with control empty vector or NSP6-GFP plasmids, and MFIs of CD63 were analyzed. (I) The size and concentration of purified exosomes, which were derived from CD63-overexpressing (OE CD63) or CD63-knockout (sgCD63) HEK293T cells (OE CD63 and sgCD63), were detected by Nano-FCM. (J) Western blot analysis of purified exosomes derived from HEK293T, A549, and Calu-3 cells which were co-overexpressed with NSP6 (HEK293T-NSP6, A549-NSP6, and Calu-3-NSP6). (K) The size and concentration of purified exosomes, which were derived from NSP6-overexpressing HEK293T cells or wild-type HEK293T cells, were detected by Nano-FCM. (L) Effect of NSP6 on proportions and MFIs of ACE2-exos. The ratio of ACE2-positive (ACE2+) and ACE2-negative (ACE2−) exosomes derived from wild-type HEK293T cells (Mock) or NSP6-overexpressing HEK293T cells (NSP6) and the MFIs of ACE2+ exosomes were analyzed by Nano-FCM. The data were shown as mean ± SD (error bars) in triplicate. P-values were calculated by one-way analysis of variance (ANOVA) tests (A and B) or Student’s t-test (C, F, and H). ****P < 0.0001; ns, not significant.