FIGURE 5.

Role of TMPRSS2 in sEV proviral effect on SARS‐CoV‐2 infection of Calu‐3 cells. (A) Effect of the TMPRSS2 inhibitor camostat mesylate (10 μM) on Calu‐3 cell infection by SARS‐CoV‐2 viral particles preincubated or not with 10⁹ VCaP‐sEVs. Intracellular SARS‐CoV‐2 RNA was extracted 48 h post‐infection and quantified by RT‐qPCR. Results were normalized to 18S rRNA, then to control (black bar). They are expressed as mean ± SEM of two independent experiments. *p < 0.05, **p < 0.01. (B) Time‐course analysis of the effect of VCaP‐sEVs on prefusion Spike protein priming and its inhibition by camostat mesylate. Western blot analyses of Spike (anti‐S1 antibodies) and CD9 proteins were performed on SARS‐CoV‐2 viral particles (10 μl of viral inoculum, ∼2.04 × 10⁵ TCID₅₀/ml), on VCaP‐sEVs (10¹⁰), and on mixtures of both, in the absence or presence of camostat mesylate (100 μM) at three time points: T1 (5 min à 37°C), T2 (1 h at 37°C) and T3 (overnight at 4°C). Tri‐S, Di‐S and Mono‐S: uncleaved prefusion Spike trimer, dimer, and monomer, respectively. S1: cleaved prefusion Spike fragment at the S1/S2 boundary