FIGURE 3.

Role of MyD88-dependent signals in regulating iNOS expression. A, MyD88 KO and WT microglia were seeded at 2 × 10⁶ cells/ well in 6-well plates and incubated overnight. The following day, cells were stimulated with 10⁷ heat-inactivated S. aureus, 10 µg/ml PGN, or 100 ng/ml LPS for 6 or 24 h, whereupon total RNA was isolated and examined for iNOS expression by qRT-PCR as described in Materials and Methods. Gene expression levels were calculated after normalizing iNOS signals against GAPDH and are presented in relative mRNA expression units (mean + SEM of three independent experiments). Significant differences in iNOS mRNA expression between unstimulated (data not shown) vs PAMP-treated microglia are denoted with asterisks (*, p < 0.05), whereas significant differences between MyD88 KO vs WT microglia are denoted with a hatched sign (#, p < 0.05). B, MyD88 KO and WT primary microglia were stimulated with either 10⁷ heat-inactivated S. aureus, 10 µg/ml PGN, or 100 ng/ml LPS for 24 h, whereupon protein extracts from whole cell lysates (40 µg/sample) were evaluated for iNOS expression by Western blotting as described in Materials and Methods. Following analysis, blots were stripped and reprobed with an Ab specific for β-actin to verify uniformity in gel loading. Results are representative of three independent experiments.