Staurosporine

Human IFN-β1a was provided by Biogen Inc. Stock solutions for in vitro experiments were prepared in 20 mM sodium acetate, pH 8.4, containing 150 mM arginine hydrochloride. TG02 was provided by Adastra. Staurosporine was purchased from AppliChem, acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl coumarin (ac-DEVD-amc) and benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (zVAD-fmk) were obtained from Bachem. ON-TARGET plus human siRNA SMART pool targeting human X-linked inhibitor of apoptosis protein (XIAP)-associated factor (XAF)-1 or caspase 3 was purchased from Dharmacon.

The apoptosis measurements were carried out according to Volling et al. (2007) with minor modifications [22] (link): 2 × 10⁵ MH-S cells were cultivated on 24-well plates and incubated overnight at 37 °C under 5% (v/v) CO₂ incubation chamber for adherence. 10⁶ resting, heat-killed, and UV-treated spores from LCV and LCA strains of L. corymbifera were co-incubated with MH-S for 3 h at 37 °C at 5% (v/v) CO₂. MH-S cells were washed three times to remove any excess of adherent spores and incubated with 1.5 µM staurosporine (AppliChem, Darmstadt, Germany, A7626) for 16 h to induce apoptosis. The media were removed and the remaining MH-S cells were co-incubated with FITC annexin-V (BioLegend, 640906) and with fixable viability Dye eFluor™ (Thermofisher, 780 65-0865-14) for 15 min in the dark at 4 °C to determine the percentages of apoptotic and necrotic macrophage cells, respectively by flow cytometry (Becton Dickinson LSRII). Three independent biological replicates were conducted.