Extended Data Fig. 2: Incubation with 1,5-AG did not affect the cell glycolysis and membrane fluidity.
From: A glucose-like metabolite deficient in diabetes inhibits cellular entry of SARS-CoV-2
(a-e) Incubation with 1,5-AG did not affect glycolysis in Vero cells. (a-b) Vero cells incubated with or without 1,5-AG were exploited to measure the O2 consumption rate (OCR) (a) and the extracellular acidification rate (ECAR) (b) upon glycolytic stress (n = 11 vehicle, n = 12 1,5-AG 100 μM, n = 10 1,5-AG 1000 μM). (c-e) Incubation with 1,5-AG did not regulate glycolysis in Vero cells. Each dot represents one cell sample (n = 11 vehicle, n = 12 1,5-AG 100 μM, n = 10 1,5-AG 1000 μM). Glycolysis (following glucose injection) (c), glycolytic capacity (following oligomycin injection) (d) and glycolytic reserve (glycolytic capacity; glycolysis) (e) were calculated from the extracellular acidification rate (ECAR) analysis. (f-h) Incubation with 1,5-AG could not affect the interaction between spike and hACE2 as well as membrane fluidity. (f) The interaction between the full-length SARS-CoV-2 spike protein and human ACE2 was evaluated by an ELISA kit. The human ACE2 antibody served as a positive control (n = 2 biologically independent samples). P < 0.0001 for vehicle versus hACE2 antibody. (g-h) Incubation with 1,5-AG did not alter the membrane fluidity of 293 T cells. The membrane fluidity was measured by a fluorescence recovery after photobleaching (FRAP) assay. Incubation with 1,5-AG did not cause a significant change in 293 T cells fluorescence recovery (g) and Thalf (h) compared to PBS treatment (n = 3 biologically independent samples). (g) The data are presented as the mean. (i-j) Protein purification. The full-length spike protein (i) was expressed and purified in human 293 F cells. The purified protein was detected by an SDS-PAGE gel. (j) The peptides of S2 and S2M were expressed and purified in E. coli. The trimers of S2 and S2M were detected by a nondenaturing electrophoresis. (a-f, h) The data are presented as the means±S.E.M. The data were analyzed using two-tailed Student’s t-test. P values were adjusted using Dunnett’s test to account for multiple comparisons. (a-j) Experiments were performed independently at least three biological replicates with comparable results.