Fig 1: PD blocks SARS-CoV-2 entry by preventing cholesterol-dependent membrane fusion.a Schematic illustration showing that ACE2+ H1299 cells become GFP-positive when the GFP-conjugated SARS-CoV-2 spike receptor binding domain (RBD) (GFP-S (RBD)) are bound to ACE2 on host cells. b, c ACE2+ cells were incubated in culture medium containing 10% FBS or 10% lipoprotein-free FBS for 24 h prior to the pSARS-CoV-2 entry assay (b) or staining with filipin-III (c). d–f ACE2+ cells were infected with lentiviral particles expressing a nonspecific (NS) shRNA or an shRNA targeting NPC1 or NPC2 for 4 days. The protein levels of NPC1 and NPC2 were assessed by western blot. GAPDH was used as a loading control (d). The cells were used for the pSARS-CoV-2 entry assay (e) or filipin-III staining (f). g ACE2+ and ACE2/TMPRSS2+ cells were pretreated with 10 μM U18666A for 12 h prior to the pSARS-CoV-2 entry assay. h, i ACE2/TMPRSS2+ cells were pretreated with 10 μM U18666A or 5 μM PD for the indicated amount of time, followed by the pSARS-CoV-2 entry assay (h) or staining with filipin-III (i). j, k ACE2+ cells were treated with 5 μM PD in the presence or absence of 100 μM MβCD for 1 h, followed by the pSARS-CoV-2 entry assay (j) or staining with filipin-III (k). The data from the pSARS-CoV2 entry assay are representative of two or three independent experiments with triplicate samples. The error bars indicate the SEM. P values were determined by the unpaired, two-tailed Student’s t-test (b, e, g) or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (j). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS not significant. The error bars are not visible when they are within the symbols (h).