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. Author manuscript; available in PMC: 2016 Nov 11.
Published in final edited form as: Cell Host Microbe. 2015 Nov 11;18(5):604–612. doi: 10.1016/j.chom.2015.10.011

Figure 2. PGN made by PBP2A requires intracellular degradation to activate the inflammasome.

Figure 2

PGN from MRSA grown overnight in the absence or presence of cefoxitin was used in macrophage stimulation assays. (A) IL-1β mRNA expression (relative to β-actin mRNA) by BMDM challenged with PGN (40 μg/ml) for 6 h (n=3). (B) IL-1β release by LPS-primed BMDM incubated with PGN (40 μg/ml) (rep. data of 3 exp.). (C) Detection of cleaved caspase-1 in supernatants of LPS-primed PGN-stimulated BMDM (rep. data of 4 exp.). (D) IL-1β release by WT, Nlrp3−/−, and Casp1−/− BMDM after overnight stimulation with PGN (80 μg/ml) (rep. data of 4 exp.). (E) Effect of lysostaphin degraded PGN (40 μg/ml) from cefoxitin-challenged or unchallenged MRSA on IL-1β release by BMDM (rep. data of 3 exp.). (F–H) IL-1β release by BMDM treated with cytochalasin D (F), bafilomycin A1 (G), or E64d (H) and stimulated with PGN (40 μg/ml) (rep. data of 3 exp.). (I) Degradation of PGN by purified lysosomal extract after 24 h (n=4). (J) Effect of cefoxitin on MRSA survival within BMDM. The assay was repeated with ampicillin-treated MRSA (Fig. S2G). (K) Growth curve of MRSA initially cultured with or without cefoxitin, and subsequently grown in antibiotic-free media. See also Fig. S2.