Dose-dependent and time course effects of RPR-EA-S1 on the production of IL-10 in BCG-stimulated PBMac. PBMac (5 × 105) were pretreated with 0.05% DMSO or different dose of RPR-EA-S1 overnight and then stimulated with BCG (MOI = 1). (A) Supernatants were collected after 24 hours. The levels of IL-10 in the supernatants were measured by ELISA. (B) Total mRNA was extracted after 3 hours for cDNA synthesis. The levels of mRNA were determined by q-PCR. The mRNA or protein expression levels of DMSO + BCG sample were set as 100%, and the rest were compared to DMSO + BCG to obtain a percentage value. (C) PBMac (5 × 105) were pretreated with 0.01% DMSO or 20 μg/mL of RPR-EA-S1 overnight and then stimulated with BCG (MOI = 1) for 0, 1, 3, 6, 12 and 24 hours. Total mRNA was used to synthesize cDNA. The levels of IL-10 mRNA were determined by q-PCR. Results are shown as mean ± SD from independent experiments on PBMac obtained from three different healthy donors. *P < 0.05, **P < 0.0001 compared to the DMSO + BCG sample (one-way ANOVA, Tukey's test for Figures 5A and B, t-test for Figure 5C).