Fig 2: A. TLR2 and TLR4 localization in WT mouse liver by immunofluorescence microscopy.Representative paraffin sections were stained for TLR2 in HC (top left) and 6 h PI (bottom left) and for TLR4 in HC (top right) and 6 h PI (bottom right). TLR staining is red; nuclear staining with DAPI is blue. B. Blue native PAGE on whole liver extracts from WT mice at 6 h after inoculation with S. aureus. Each blot shows three lanes: Lane 1, NativeMark molecular weight standard; Lane 2, sample in 0.5% DDM with no DTT or heating; lane 3, sample in 4% SDS with 100 mM DTT, boiled at 95°C for 5 min. At the left, Coomassie staining of entire blot showing molecular markers. Western blots were performed with anti-TLR2, TLR4, or TRAM. A complex near 300 kD was identified by all three primary antibodies (arrows) suggesting a possible interaction among the three proteins.

Fig 3: Potential TLR signaling pathways for Ppargc1 metabolic co-activator gene activation after S. aureus infection.Pathway 1 shows the canonical TLR2 MyD88-dependent signaling pathway that activates NF-kB after S. aureus. Pathway 2 shows TLR4 MyD88-dependent signaling to NF-kB and MyD88–independent signaling to TRIF/TRAM. Both MyD88 pathways have been excluded as causes of the Ppargc1a gene expression. Pathway 3 shows a putative TLR2-TLR4 heterodimer interacting with TRIF/TRAM. Pathway 4 indicates TLR2 in the TLR4 null state, as a homodimer or a heterodimer involving a non-TLR4 partner such as TLR1 or 6, interacting with TRIF/TRAM and unmasking the innate immune regulation of Ppargc1a expression. Pathway 5 shows canonical TLR3 endosome signaling also excluded in Ppargc1 gene regulation after S. aureus; however, independent TLR3 activation partially rescues the Ppargc1 phenotype in mice. TIRAP is Toll/interleukin-1 receptor domain-containing adapter protein (MAL); IRAK4 is Interleukin-1 receptor-associated kinase 4; TRAF3 and TRAF6 are TNF receptor-associated factor 3 and 6; TAK1 is TGF-beta-activated kinase 1 and TBK1 is NF-kappa-B-activating kinase.

Fig 4: Nutlin induces TLR2 and TLR5 mRNA expression as well as proteins.(A) Induction of TLR2 and 5 protein expression in stimulated lymphocytes cells from subjects BS25 and BS26 by nutlin (10 µM) after 24 hr post-treatment. Proteins were determined by western blot analysis using antibodies specific to TLR2 and TLR5 in the membrane fractions. Actin protein provided a loading control. (While there is little change in the amount of actin protein detected, there may be differences in the nonspecific bands.) (B) Induction of TLR2 and 5 mRNA expression in stimulated lymphocytes from subjects BS25 and BS26 (data obtained from Figure 1) by nutlin.

Fig 5: a3 integrin levels in TLR2-silenced A549 cells incubated with P. brasiliensis yeasts or PAM3CSK4. A549 cells were transfected with siRNA directed to TLR2 (siRNA TLR2) or negative control siRNA (NC). Next, cells were incubated with P. brasiliensis yeasts (MOI = 2.5) (Pb) (a) or with 5 µg/mL of the TLR2-specific ligand PAM3CSK4 (b). After 24 h, epithelial cells were lysed and aliquots were submitted to SDS–PAGE. a3 integrin levels (a3 int) were analysed by Western blot. ß-actin was used as loading protein control. Relative a3 integrin levels were determined by densitometric analysis. Values represent the ratio of the intensity of integrin band divided by the corresponding intensity of ß-actin band. Similar results were obtained from three independent experiments. Dashed lines in (a,b) represent cropped images. Uncropped images are presented in Supplementary Fig. S5.