Journal: Research
Article Title: The Selective DHCR24 Blocker SH42 Inhibits ACE2 Binding and Cellular Entry of SARS-CoV-2 Spike Proteins More Efficiently Than Atorvastatin
doi: 10.34133/research.1280
Figure Lengend Snippet: SH42 reduces cholesterol abundance in the plasma membrane in general and lipid rafts in particular, and decreases lipid raft area more efficiently than atorvastatin (ATO). Control HEK293T (A) and Calu-3 (B) cells and those treated for 96 h with 10 nM or 1 μM ATO or SH42 were labeled with cholesterol-binding mCherry-conjugated D4H*, the D434S mutant of domain 4 (D4) of C. perfringens theta-toxin. Fluorescence intensities correlating with plasma membrane cholesterol levels of at least 10,000 individual cells per sample were subsequently measured using a flow cytometer. The average intensity values obtained in n = 9 independent biological replicates, and their average values (± SEM) are plotted in the figure. (C) To examine changes in the cholesterol content of raft and non-raft microdomains of the plasma membrane, control HEK/ACE2 + TMPRSS2 cells and those treated as above were labeled with Alexa Fluor 647-conjugated cholera toxin subunit B (CTX-AF647), a lipid raft marker, and F66. F66 is a fluorescent indicator with spectral properties depending on the cholesterol-dependent local molecular order (dipole potential) of the membrane; therefore, this dye, combined with CTX-AF647, can provide information about the extent of cholesterol reduction separately in raft and non-raft membrane regions. Representative confocal microscopic images taken from the flat, bottom membrane region adjacent to the coverglass show F66 intensities detected in 2 wavelength ranges of emission (“F66 N*” and “F66 T*”), their ratio (“F66 emission ratio” calculated as T*/N* pixel by pixel), and CTX-AF647 intensities. Cell “membrane masks” selected manually in CTX images were segmented using the maxentropy algorithm to CTX-high “rafts” and CTX-low “non-rafts” corresponding to high- and low-intensity regions, respectively, as shown by the representative images. Violin plots were generated from median F66 emission ratio values determined separately for the CTX-high “raft” (D) and CTX-low “non-raft” (E) masks of n = 54 to 73 individual cells, which also display median values with quartiles. (F) Pixelwise distributions of the F66 emission ratio in CTX-high “rafts” and CTX-low “non-rafts” of control cells are displayed. For the quantification of the relative area of lipid rafts, as an alternative definition for raft regions, a threshold value of the F66 emission ratio was determined (green dashed line) and membrane pixels were considered as “F66 raft” and “F66 non-raft” regions when being above and below the threshold, respectively. (G) Violin plots were generated from the relative fraction of F66 raft pixels (“F66 raft area”) of individual cells, which also display median values with quartiles. (H) Representative images show changes in the lateral distribution of the F66 emission ratio on a color-scale image and reduction in the relative F66 raft area induced by 1 μM SH42. Throughout the figure, asterisks indicate significant differences compared to control samples (* P < 0.05, **** P < 0.0001), while hashes show that between samples treated with ATO and SH42 at identical concentrations ( # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001), which were determined by Tukey’s HSD test carried out after significant differences were obtained for between-group effects in ANOVA.
Article Snippet: The human embryonic kidney HEK293T cell line that stably expresses ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (HEK/ACE2 + TMPRSS2) was obtained from GeneCopoeia (Rockville, MD; SL222), while the Calu-3 lung adenocarcinoma cell line with an endogenous expression of ACE2 and TMPRSS2, and the original HEK293T cell line lacking considerable ACE2 and TMPRSS2 were purchased from the American Type Culture Collection (Manassas, VA; HTB-55 and CRL-3216, respectively).
Techniques: Clinical Proteomics, Membrane, Control, Labeling, Binding Assay, Mutagenesis, Fluorescence, Flow Cytometry, Marker, Generated
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