Hcell 100 medium

Vero E6 cells were transfected with mock (pcDNA3.1), TMPRSS2 (pcDNA3.1/TMPRSS2 Uniprot: O15393-1) or TMPRSS2(S441A) (pcDNA3.1/TMPRSS2-S441A) using Lipofectamine 3000 (Thermo Fisher Scientific) in 12-well plates. For the mouse TMPRSS2 assay, empty vector (pCMV6-Entry, Origene PS100001) and TMPRSS2-Myc-DDK (Origene MR207852) were used. After 24 h transfection, cells were washed with PBS and the medium replaced with HCell-100 medium (Wisent) containing 200 µM Boc-QAR-AMC (R&D Systems) and either vehicle (0.01% DMSO) or compounds at the indicated concentration for 24 h. To measure proteolytic activity, 90 µl of cell medium was transferred to a black 96-well plate, and fluorescence was measured at room temperature (excitation: 360 nm, emission: 460 nm) using an FLx800 TBE microplate reader (Bio-Tek Instruments). Background-subtracted (mock-transfected cells) proteolytic activities are presented as the percentage of activity relative to vehicle-treated cells (screen at 10 nM). IC₅₀ values were determined after generating a nonlinear regression analysis from a log([Compound]) versus a proteolytic activity plot using GraphPad Prism software (v.9.0.1). GraphPad Prism was used to identify and eliminate outliers (Q = 1) and assess the goodness of the fits. IC₅₀ values presented are the mean ± s.d. of at least three independent experiments.

Cleavage assays in cellulo using purified recombinant matriptase were performed as described previously^{38 (link)}. Briefly, culture medium of HEK293T was removed and replaced with 2 mL of serum-free HCELL-100 medium (Wisent, St-Bruno, QC, Canada) containing 5 nM of recombinant soluble human WT matriptase or mutant S805A. The conditioned medium was collected after 36 h incubation and concentrated with Amicon Ultra Centrifugal filters 3,000 NMWL (Merck Millipore Ltd., T., C., Co Cork IRL). Both cell lysates and concentrated conditioned medium were then analyzed by SDS-PAGE and immunoblotting.