Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition (Sim et al)
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Original datasets and Supplemental Videos : Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition
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Figure 2D. The effects of ANO6 inhibitors on the surface exposure of phosphatidylserine (PS) were assayed in FRT-ANO6 cells. 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. Figure 4. Mechanisms involved in the SARS-CoV-2 Spike-mediated PS-scrambling and membrane fusion. Figure 6. The single-round infection of pseudotyped SARS-CoV-2 S virus (SARS2-PsV) is Ca2+- and ANO6-dependent. The single-round infection of SARS2-PsV was performed using a lentivirus-based SARS2-PsV encoding GFP. The cell viability was assessed using the mCherry fluorescence of HEK 293T-ACE2-TMPRSS2 cells. Figure 7F. Viral replication of SARS-CoV-2 (1 MOI) was assayed in HNE cells using the plaque assay. Figure S1A. Immunoblotting of ANO6 in FRT and Jurkat cells with exogenous ANO6 expression. Figure S2G. Effects of ANO6 inhibitors on cytoskeletal structure and on the nuclei were analyzed using F-actin and DAPI staining. Figure S2H. The dose responses of A6-001 on ANO6 PS scramblase inhibition were analyzed. PS externalization was assessed via the fluorescence intensity of Lact-C2-GFP. Figure S3C. Effects of camostat on ionomycin (10 M, 10 min)-induced PS scrambling. Figure S3E. Immunoblot analysis of the SARS-CoV-2 Spike glycoprotein. The cell lysates of wild-type and R682S/R685G SARS2-PsV infected HEK 293T cells were blotted with primary antibodies against the S2 domain of Spike and aldolase A. Figure S5A. Viral replication of SARS-CoV-2 in Vero cells with an infection dose of 0.01 MOI. In the light microscopic analysis, A6-001 reduced the virus-induced cytolysis. Supplemental Videos Video S1. Time-lapse imaging of HEK 293T-ACE2-TMPRSS2 (Red)/CHO-Control (Green) co-cultures, Related to Figures 4G and 4H Video S2. Time-lapse imaging of HEK 293T-ACE2-TMPRSS2 (Red)/CHO-Spike (Green) co-cultures, Related to Figures 4G and 4H Video S3. Time-lapse imaging of HEK 293T-ACE2-TMPRSS2 (Red)/CHO-Spike (Green) co-cultures, A6-001 (10 μM) treated, Related to Figures 4G and 4H Video S4. [Ca2+]i measurement in HEK 293T-ACE2-TMPRSS2 cells: mock vehicle, Related to Figures 5A and 5B Video S5. [Ca2+]i measurement in HEK 293T-ACE2-TMPRSS2 cells: SARS2-PsV alone, Related to Figures 5C and 5D Video S6. [Ca2+]i measurement in HEK 293T-ACE2-TMPRSS2 cells: BAPTA pretreatment (BAPTA-AM, 3 μM, 20 min) plus SARS2-PsV, Related to Figures 5E and 5F Video S7. [Ca2+]i measurement in HEK 293T-ACE2-TMPRSS2 cells: camostat pretreatment (10 μM) plus SARS2-PsV, Related to Figures 5G and 5H Video S8. [Ca2+]i measurement in HEK 293T-ACE2-TMPRSS2 cells: A6-001 pretreatment (10 μM) plus SARS2-PsV, Related to Figures 5I and 5J