cells through conditional HNF4a knockout experiments [4], another group reported that the expression level of Kir6.2 in HNF4a knockout mice was unchanged as compared with control mice [3]. Our findings partially support the latter observation, although we cannot rule out the possibility that MED25 involvement is for only a subset of endogenous HNF4a target genes and another Mediator subunit might be involved in HNF4a-mediated Kir6.2 transactivation. The involvement of MED25 in HNF4a-mediated transcription and insulin secretion was further tested using the MED25 LXXLL-motif mutant (MED25 NR) and the knockdown of MED25 using shRNA (Figure S2 for validation of MED25 knockdown by shRNA). In keeping with the results from siRNA treatments for the luciferase assays, the expression of all three target genes in ß-cells (PPARa, L-PK, and GLUT2) were markedly reduced by both the MED25 NR mutant and MED25 shRNA treatment (Figure 3C). As a result, the overall insulin HNF4a-MED25 Interactions in Beta-Cells secretion from MIN6 cells was also reduced significantly by both treatments (Figure 3D). Taken together, these preliminary data indicate that MED25 is a critical Mediator component involved in HNF4a-mediated transcription that promotes glucose-stimulated insulin secretion in pancreatic ß-cells. MED25 Lacks Synergistic Activation with MED1, Another Mediator Component Known for HNF4a MED1, the best-studied Mediator subunit, is also known to be involved in activation of HNF4a-mediated transcription [26,34]. Thus, we tested for a potential synergistic activation by MED25 and MED1 towards HNF4a by co-transfection of both protein- producing vectors. As shown in Fig. 4, they appear to act individually on HNF4a, and lack synergistic activation. These effects are quite consistent throughout the transcription assays, target gene expression, and insulin secretion assays (Fig. 4A–D), Figure 3. Effects of MED25 on HNF4a target gene expression implicated in insulin secretion from pancreatic ß-cells. (A) The RNA amounts were quantified by real-time PCR with the primers against each HNF4a target genes following transfection of the indicated expression vectors. Control indicates transfection with an empty vector, and error bars indicate the range of experiments performed in duplicate. Each graph bar corresponds to each target gene as indicated in the inset box. PPARa displayed the highest response (12-fold increase) followed by L-PK and GLU2 (both 8-fold increases), while Kir6.2 showed no response. (B) Q-PCR results of the same RNA quantifications. The experimental results are shown in triplicate while actin, a house-keeping gene product, was used as a loading control in the bottom panel. These results are in good agreement with the real time PCR data shown in (A). Data represent the mean6SE (n = 3). Compared with HNF4a alone, *p,0.01. (C) Effects of the MED25 NR mutant and shMED25 on HNF4a target gene expression shown by Q-PCR. The experimental results are shown in duplicate and again actin was used as loading control. They all showed the MED25-specific effects. (D) Effects of the MED25 NR mutant and shMED25 on insulin secretion shown by color change (right) and its quantification by absorption at 450 nm (left). Darker colors indicate more insulin secretion as shown by higher bars in their actual numerical value plots. These data show the direct correlation between HNF4a target gene expression and insulin secretion. Data represent the mean6SE (n = 3). Compared with the control, *p,0.01. Compared with MED25 plus HNF4a, **p,0.01. doi:10.1371/journal.pone.0044007.g003 PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e44007