FIG. 4.

Quantification and kinetics of RANKL mRNA expression by osteoblasts. (A) Expression of RANKL mRNA in osteoblasts infected with viable S. pyogenes JRS4. Mouse primary osteoblasts were cultured in the absence of stimulant (None) or in the presence of PGE2 (2 μM) (PGE₂) for 6 h. Other cultures of osteoblasts were infected with S. pyogenes (GAS) at MOIs of 1, 5, and 10. Total RNA was extracted from osteoblasts and subjected to RT-PCR analysis. (B) Quantification of RANKL by real-time PCR. Relative mRNA levels were expressed as percentages of the mRNA level in osteoblasts stimulated with PGE2 (PGE₂). Cells were not infected (None) or were infected with S. pyogenes JRS4 (GAS) at an MOI of 1, 5, or 10. (C) Time course of RANKL expression in osteoblasts infected with S. pyogenes JRS4. To determine the kinetics of RANKL mRNA expression, osteoblasts were infected with S. pyogenes (MOI, 10) for 1, 3, or 6 h. The relative mRNA levels were expressed as fold increases compared with the RANKL mRNA level in the unstimulated control osteoblasts. (D) Quantification of RANKL mRNA expression in osteoblasts treated with heat-inactivated S. pyogenes. Osteoblasts were cultured in the presence of heat-inactivated S. pyogenes (heated GAS) at an MOI of 10, 50, or 100 for 6 h, and then total RNA was extracted. Relative mRNA levels were determined by real-time PCR. The transcription levels were expressed as percentages of the RANKL mRNA level in osteoblasts stimulated with PGE2 (2 μM) (PGE₂). The values are the means ± standard deviations for triplicate assays. An asterisk indicates that the P value was <0.05 for a comparison with the RANKL mRNA level in cells cultured in the absence of the stimulant (None).