FIG 3 .

SpyA stimulates a caspase-1-dependent inflammatory response and IL-1β production in macrophages. (A) BMDMs were infected with GAS (AP) at an MOI of ~10. At the end of the assay, cells were washed and stained for 1 h with FAM YVAD-FMK to visualize activated caspase-1 (green) and for 5 min with Hoechst 33342 to visualize DNA (blue) under an epifluorescent microscope. Scale bar, 100 µm. (B) Quantification of FMK YVAD-FMK-positive cells (per 100 cells) from multiple random fields of view (n = 10) per sample. (C) Western blot illustrating the abundance of pro-caspase-1 and its p10 subunit in GAS-infected BMDM cell lysates and caspase-1 p10 subunit released in the supernatants (Sup). Data represent densitometry quantifications illustrating average levels of active caspase-1 p10 expression in lysates and supernatants relative to pro-caspase 1 expression. Error bars, SEM. *, P < 0.05 (n = 3). Experiments were performed in duplicate using BMDMs isolated from four different C57BL/6 mice. (D and E) Real-time qPCR (D) and ELISA (E) quantification of IL-1β and TNF-α transcripts and proteins produced by BMDMs infected with GAS 2 h after total killing (n > 3). (F) IL-1β released by BMDMs after 4 h infection with S. aureus RN4220 expressing pSpyA or control vector (n = 4). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s two-tailed unpaired t test). UI, uninfected.