Figure 3.

ANO6 is responsible for phosphatidylserine externalization evoked by pseudotyped SARS-CoV-2 S virus (SARS2-PsV)

HeLa cells expressing ACE2 (HeLa-ACE2) were incubated with a lentivirus-based SARS2-PsV (100 ng p24/mL; 20 MOI) or an authentic SARS-CoV-2 (10 MOI) for 15 min and then with Lact-C2-mCherry for 45 min.

(A and B) Chelation of cytosolic Ca²⁺ (BAPTA-AM; 10 μM; 1 h) inhibits SARS2-PsV-induced PS externalization. Representative images are shown in (A), and the quantification results of multiple experiments are summarized in (B) (n = 3, each from 3 to 5 fields per experimental condition).

(C and D) Control experiments in ACE2-negative HeLa cells. PS externalization was induced by ionomycin (10 μM; 10 min). Representative images are shown in (C), and the quantification results of multiple experiments are summarized in (D) (n = 3). ns, not significant.

(E and F) Silencing of ANO6 (siRNAs against ANO6; 100 nM; 24 h) inhibits the phosphatidylserine (PS) externalization evoked by SARS2-PsV. Cell nuclei were stained with DAPI. Representative images are shown in (E), and the quantification results of multiple experiments are summarized in (F) (n = 3, each from 3 to 5 fields per experimental condition).

(G and H) ANO6 inhibitors (A6-001 and A6-004, each 10 μM, 1 h) inhibit SARS2-PsV-induced PS externalization. Representative images are shown in (G), and the quantification results of multiple experiments are summarized in (H) (n = 3–4).

(I and J) Authentic SARS-CoV-2 virus evokes Ca²⁺- and ANO6-dependent PS externalization. The HeLa-ACE2 cells were pretreated with compounds (10 μM) for 1 h and infected with SARS-CoV-2 for 15 min. Representative images are shown in (I), and the quantification results of multiple experiments are summarized in (J) (n = 3).

Bar graph data are shown as the mean ± SEM. ^∗p < 0.05 and ^∗∗p < 0.01. Data were analyzed using one-way analysis of variance, followed by Tukey’s multiple comparison test. Scale bars: 50 μm. See also Figure S3.