Fig. 6: Activation of both caspase-1 and caspase-4 is required for Ef.LTA/NaB-induced inflammasome activation.

A PMA-differentiated THP-1 cells were pre-treated with the indicated concentrations of the caspase-1 inhibitor (Z-YVAD-FMK), the caspase-4 inhibitor (Z-LEVD-FMK), for 1 h and subsequently co-stimulated with Ef.LTA (10 μg/ml) and NaB (10 mM) for an additional 6 h. IL-1β expression in the culture supernatants was measured by ELISA. B Pro- or active caspase-1 and IL-1β in the culture supernatants (Sup) and pro-IL-1β and β-actin in the cell lysates (Cell) were detected by immunoblotting. Pam3CSK4 was used as a positive control. C The cells were co-stimulated with Ef.LTA (10 μg/ml) and NaB (10 mM) for 6 h. Then, the culture supernatants were collected and the level of cleaved GSDMD was determined by immunoblotting. D The cells were co-stimulated with Ef.LTA (10 μg/ml) and NaB (10 mM) for 24 h. The LDH in culture supernatants was measured using the LDH-cytotoxicity colorimetric assay kit. E The cells were co-stimulated with Ef.LTA (10 μg/ml) and NaB (10 mM) for 6 h and stained with PI. The images were captured under a microscope and PI-positive cells were counted by ImageJ software. VC denotes vehicle control (DMSO). The results shown are representative of triplicate experiments. All results are expressed as mean ± SD of triplicate samples. *p < 0.05.