Z ietd fmk

5-Aminolevulinic acid (ALA) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St Louis, MO, USA). Necrostatin-1 (Nec-1), z-DEVD-fmk (ZDEVD) and z-IETD-fmk (ZIETD) were from Santa Cruz Biotechnology (CA). Cu-oxidised LDL (ox-LDL) and red fluorescent marked DiI-ox-LDL were both purchased from Peking Union-Biology Co. Ltd (Beijing, China). Hoechst-33342, DAPI, the ATP assay kit, the lactate dehydrogenase (LDH) release assay, and the caspase-3 and caspase-8 activity assay kit were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). The ApoAlert Annexin V-FITC kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Other drugs and chemicals used in this study were obtained from Sigma-Aldrich. EGS (ethylene glycol bis(succinimidyl succinate)) was obtained from Thermo Scientific.

Bone marrow-derived macrophages derived from wild-type (WT) or knockout mice were infected with F. pedrosoi for 6 (RT-qPCR analysis) and 24 h (other assays) at a multiplicity of infection (MOI) of 3 for conidial infection (except for fungicidal assays, performed with a MOI of 1) and of 1 for hyphal. For inhibition assays, BMDMs received 2 h prior to the infection a Myd88 inhibitor peptide (50 µM) (InvivoGen), Syk inhibitor R406 (5 µM) (InvivoGen), NF-kB inhibitor celastrol (5 µM) (InvivoGen), caspase-1 inhibitor AC-YVAD-CHO (50 µM) (Santa Cruz Biotechnology), caspase-8 inhibitor Z-IETD-FMK (50 µM) (Santa Cruz Biotechnology), intracellular potassium efflux inhibitors glyburide (150 µM) (InvivoGen) and KCl (50 mM) (Sigma-Aldrich), ROS inhibitor DPI (diphenyleneiodonium chloride) (20 µM) (Sigma-Aldrich), endosomal acidification inhibitor bafilomycin A (250 nM) (InvivoGen) or cathepsin B inhibitor CA-074 Me (50 µM) (Sigma-Aldrich). In some experiments, BMDMs and BMDCs were treated 2 h previously to the fungal infection with 500 ng/mL of LPS (Sigma-Aldrich) and/or 20 µM of nigericin (InvivoGen) during the last 40 min of incubation (1 h for fungicidal assays). Cells stimulated with both LPS and nigericin served as a positive control for NLRP3-mediated inflammasome activation.

The following reagents were used: Doxorubicin (Sigma), Etoposide (Sigma), Z-IETD-FMK (Santa Cruz), LCL161 (Selleckchem), anti-PAR-4 (sc-1807, Santa Cruz), anti-PAR-4 (ab5787, Abcam), anti-GAPDH (sc-32233, Santa Cruz), anti-caspase-8 (#9746, Cell Signaling), anti-cleaved caspase-8 (#9496, Cell Signaling), anti-PARP-1 (#9542, Cell Signaling), anti-cIAP1 (#7065, Cell Signaling), anti-cIAP2 (#3130, Cell Signaling), anti-XIAP (#2045, Cell Signaling), anti-phospho-p65 Ser536 (#3033, Cell Signaling), anti-IκBα (#9242, Cell Signaling), anti-GFP (Rockland), anti-rabbit-HRP (P0448, DAKO), anti-mouse-HRP (P0447, DAKO), anti-rabbit-Alexa-Fluor-555 (A-31572, Thermo Fisher) and anti-rabbit-Alexa-Fluor-488 (A-21206, Thermo Fisher).