Extended Data Fig. 9: MRSA g-hemolysin B (HlgB) binds and promotes AMFR-mediated NF-kB activation.
(a) qRT-PCR analysis of Tnf, Il1b, and Il6 mRNA expression in BMDMs isolated from Amfrfl/fl (Amfr+/+) and LysM-Cre Amfrfl/fl (Amfr−/−) mice and treated with heat-killed MRSA43300 (HKSA) (MOI = 8). (b) qRT-PCR analysis of Tnf, Il1b, and Il6 mRNA expression in BMDMs isolated from Amfrfl/fl (Amfr+/+) and LysM-Cre Amfrfl/fl (Amfr−/−) mice and treated with LPS, or E.coli. (c) qRT-PCR analysis of Tnf, Il1b, and Il6 mRNA expression in BMDMs isolated from Amfrfl/fl (Amfr+/+) and LysM-Cre Amfrfl/fl (Amfr−/−) mice and treated with Poly(I:C). The results are shown as the relative levels of gene transcripts, with that of unstimulated cells set as 1. (d) Luciferase assay of NF-kB activation in HEK293T cells transfected with TAB3 and together with different MRSA leucotoxins expression vector (FLAG-tagged). The expression of each protein was shown below. (e) Densitometric analysis of immunoblot analysis of phosphorylated (p-) p65, p38, JNK, and ERK in Fig. 5c. The samples derive from the same experiment and that gels/blots were processed in parallel. (f) Coimmunoprecipitation and immunoblot analysis of HEK293T cells co-transfected with MYC-AMFR plus FLAG-Hld, or FLAG-HlgC, followed by IP with an anti-MYC Ab. (g) qRT-PCR analysis of Tnf, Il1b, and Il6 mRNA expression in Amfr+/+ and Amfr−/− BMDMs and infected with 43300∆hld. Data are representative of three independent experiments (f), means ± SEM in a-d and g, or means ± SD in e, one-way ANOVA (Tukey’s test), two-way ANOVA (Sidak’s test), or unpaired two-tailed Student’s t test.
