Fig. 4: TLR9 is required for cidal induced increases in inflammatory cytokines.

TNF quantified by ELISA at 6.5 h from WT (A) or Tlr9−/− (B) iBMDM macrophages infected with media alone (black), bacteria without antibiotics (black), KLA (orange), and equivalent concentrations of cidal (blue) and static treated bacteria (red) as indicated. Error bars display SEM. A is reproduced from Fig. 3A for comparison. A Three independent measurements/group, and (B) displays six independent measurements / group (aggregated across two independent replicates). C, D Bacteria were incubated with the indicated antibiotic (or no antibiotic) for 3 h, then spun down. Supernatants were collected, and the pellets of surviving bacteria were lysed. All supernatants (C) and pellets (D) were ethanol precipitated and then run on agarose electrophoresis gels. Representative ladder band sizes are labeled to the left of the gels in kilobases. Total DNA stain intensity in each lane is quantified in the graphs. E Infections were set up as in Fig. 3A in WT macrophages, with an additional positive CpG DNA control, and DNaseI-XT (a nonspecific nuclease) was added to the infections where indicated. The readout is TNF quantified by ELISA at 6.5 h, with three independent measurements per group shown. Error bars display SEM. F TNF quantified by ELISA at 6.5 h from Tlr9−/− iBMDMs (white bars) infected 5xMIC drug treated bacteria across all the antibiotics. – is media only, + is bacteria without antibiotics, KLA is Kdo2-Lipid A. WT infections reproduced for comparison from Fig. 2D (black bars), lethality of tetracycline gradient is indicated below, with three independent measurements per group displayed. Error bars display SEM. A–F are representative of three independent experiments, and *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed t test; statistical comparisons in A, B and E are of cidal drugs (as a group) to static drugs (as a group). Source data and exact p values are provided as a Source Data file.