[View Larger Version of this Image]

Figure S1.  mRNA expression of TLR4 and 9 is increased in tlr3^−/− mice after CLP. (A) TLR4 and 9 expression in tlr3^−/− mice. Purified RNA was isolated with TRIzol from whole spleens from WT and tlr3^−/− mice and reverse transcribed into cDNA using TaqMan reverse transcription reagents (Applied Biosystems). TLR4 and 9 chemokine gene expression was determined, and relative expression was determined after calibration of the gene of interest with 18sRNA and normalization to relevant naive control gene expression. (B) TLR4 activation via LPS significantly increased KC production in tlr3^−/− peritoneal neutrophils compared with WT peritoneal neutrophils. Neutrophils were obtained from thioglycollate-challenged WT and tlr3^−/− mice and stimulated by Pam3Cys, LPS, or CpG-DNA for 5 h. KC and CCL3 were quantified using ELISA or Bio-Plex. Data means ± SEM of two experiments in triplicate. *, P < 0.05 compared with tlr3^−/−.