Fig. 4: UNI418 delays the maturation of cathepsins and cleavage of the SARS-CoV-2 spike protein.

a Representative Western blot image depicting pro- and mature cathepsin D after treatment with the indicated concentrations of UNC3230, apilimod and UNI418. GAPDH served as a loading control. b, c Evaluation of cathepsin D and LAMP1 colocalization in Vero cells after treatment with UNC3230, apilimod or UNI418 for 4 h. Immunofluorescence staining (b) and Pearson’s correlation coefficients (c) between LAMP1 and cathepsin D distribution. Scale bar, 15 µm. Images of individual cells, comprising at least 45 cells per sample, were randomly cropped and analyzed for colocalization using JACoP in ImageJ. The error bars indicate the SDs. Statistical significance was determined using Student’s t-test, where ***p < 0.001 and n.s., not significant. d, e Cathepsin L maturation in Vero cells was determined by Western blotting. d Representative Western blot images showing pro- and mature cathepsin L levels after treatment with UNI418 at the indicated concentrations. GAPDH was used as a loading control. e The ratio of cathepsin L to pro-cathepsin L was quantified and is presented as the mean ± SEM, with a sample size of n = 3. Statistical significance was determined by Student’s t test, where *p < 0.05 and **p < 0.01. f Cleavage of the SARS-CoV-2 spike protein was determined with or without UNI418 treatment at specific time points. HEK293T cells stably expressing ACE2 (293T-ACE2) were treated with SARS-CoV-2 spike protein (Spike-HexaPro) in the absence or presence of 0.5 μM UNI418 and subjected to Western blotting for the SARS-CoV-2 spike protein, ACE2 and GAPDH. g The ratios of processed S2/GAPDH were quantified and are presented as the means ± SEMs, with n = 4. Statistical analysis was performed using two-way ANOVA; *p < 0.05.