Fig. 3

Rapamycin promotes Staphylococcus aureus-induced pyroptosis in macrophages. Macrophages were pretreated with rapamycin (100 nM) for 6 h and then infected with S. aureus for 3 h. The pyroptotic characteristics were determined, including pyroptosic protein markers, inflammatory cytokine release, and morphology. (a Western blot analysis of S. aureus-induced NLRP3 and caspase1-p20 expression. b Immunofluorescence assay of S. aureus-induced caspase 1 expression. Representative confocal microscopy images of caspase 1 (green) in cells that were co-stained with DAPI (blue). Scale bars represent 10 μm. c GSDMD-NT expression by western blot. d GSDMD-NT expression by immunofluorescence assay. Representative confocal microscopy images of GSDMD-NT (green) in cells that were co-stained with DAPI (blue). Scale bars represent 10 μm. e Macrophages were pretreated with rapamycin for 6 h and then infected with S. aureus for 3 h. The levels of IL-18 and IL-1β in cell culture medium were determined by ELISA. f GSDMD-NT pores on plasma membrane by scanning electron microscopy. Red arrow indicates GSDMD-NT pore. Scale bar, 500 nm. g Large bubbles by light microscopy, as indicated by red arrows in pyroptotic cells. Scale bars represent 1 μm. The resolved bands were quantified using Gel-Pro Analyzer 4.0 (Media Cybernetics, Inc., Rockville, MD, USA). Fluorescence intensity of the immunofluorescent was measured by imaging analysis software (NIS-Elements Viewer, Nikon Instruments Inc. Shanghai, China). *p < 0.05; **p < 0.01. n = 3 independent experiments. Error bar indicates SD