Figure 4

Transfection of MKP-1 siRNA into primary human blood monocytes can increase LPS-induced TNF-α level while decrease BCG-induced TNF-α expression. Cells were first transfected with control (ctrl) or MKP-1 siRNA (MKP-1 si, 200 nM) for 24 hours and then treated with different inducers as indicated. (A) After transfection, cells were treated with Mock (M) or BCG (B, MOI = 1 CFU/cell) for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR. (B) Monocytes were treated as in (A) for the indicated time points and MKP-1 proteins were measured by Western blotting. (C) Cells were treated with LPS (50 ng/ml) for the indicated time points. RNA was harvested, and levels of MKP-1 and TNF-α were measured by RT-PCR. For both (B) and (C), the intensities of the PCR/protein bands were determined by using Bio-Rad Quantity One imaging software. The intensities of bands were normalized to the corresponding control. The values in parenthesis are the relative normalized intensities compared to those of the control siRNA-transfected cells without other treatment. (D) BCG (MOI = 1 CFU/cell) was added for 3 hours. RNA was harvested and levels of TNF-α, IL-6 and IL-10 were measured by QPCR. Percentage change was defined as the percentage of the fold induction of (MKP-1 si + BCG) over the fold induction of (ctrl + BCG). Results of (ctrl + BCG) were set as 100%. For (A)-(C), independent experiments were done on monocytes from 3 different donors and one representative set of results is shown. For (D), experiments were performed on 4 different donors and the results are shown as mean ± SD. * = p < 0.05.