Figure 2.

Selective suppression of MIF enhances the sensitivity of HPAEC to LPS-induced apoptosis. (A) Quantitative RT-PCR measurements of MIF mRNA levels in untreated HPAEC and in response to LPS. Results are normalized to GAPDH mRNA expression. LPS caused a 2.6-fold increase in MIF mRNA that was unaffected by control (GFP) siRNA but markedly suppressed to less than 10% of control levels by MIF-selective siRNA. (B) Protein expression in HPAEC transfected for 8 hours with control/GFP- or MIF siRNA and then exposed to 100 ng/ml LPS for an additional 16 hours. LPS induced a 3.4-fold increase in MIF protein levels. Both basal and LPS-induced levels were suppressed approximately 20% of unstimulated basal levels by MIF siRNA. (C). HPAEC were transfected with GFP siRNA (solid bars) or MIF siRNA (shaded bars) at a final concentration of 100 nM. Six hours after transfection, cells were challenged with PBS, 100 ng/ml LPS in PBS, 100 ng/ml LPS, and 10 ng/ml recombinant human MIF protein (rHMIF), or rHMIF alone and incubated for an additional 18 hours before quantification of apoptotic cell death by fluorescent microscopy as described in Materials and Methods. **P > 0.005.