1 mM EDTA, 0.5% NP-40, 5% glycerol) supplemented with a protease inhibitor cocktail mix (Roche). Cell lysates were pre- cleared by pre-incubation with protein A Sepharose 4 Fast Flow beads (GE Healthcare) for 15 min, and incubated for 2 hrs with the beads and a 1:200 dilution of HNF4a and anti-rabbit IgG antibodies. The beads were then washed once with IP lysis buffer and twice with PBS, and the immune complexes were released from the beads by boiling in sample buffer for 5 min. Following electrophoresis on 10% SDS-PAGE, immunoprecipitates were transferred onto PVDF membrane, and immunoblotted with a specific MED25 antibody (Abgent). Proteins were visualized using the enhanced chemiluminescence (ECL) detection system (GE Healthcare). Western Blotting Immunodetection of expression of MED 25 in MIN6 cells was performed using whole cell lysates, rabbit anti- MED 25 antibody (Abgent), mouse anti-Actin antibody (Santa Cruz), and goat anti-rabbit antibody conjugated with HRP (Cell Signalling Technology). Briefly, transfected cells were lysed with cell lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.5% TritonX-100, 0.5% NP-40, and 1 mM EDTA) with protease inhibitors. Cell lysates were centrifuged and the protein concentration of each cell lysate was determined using the Bio-Rad protein assay reagent. The same amounts of cell lysates were loaded onto a 10% SDS-PAGE, and the proteins were fractionated by electrophoresis and transferred to a PVDF membrane. The blotted membrane was incubated with aforementioned antibod- ies and signals were detected using the ECL-based method (GE Healthcare). Gene Knock-down (siRNA or shRNA) for Cellular Studies The sequence of MED25-siRNA used for initial transcription assays was described elsewhere [21]. A negative control scrambled siRNA (Ambion) was used to demonstrate that transfection did not induce nonspecific effects on gene expression. One microgram of each siRNA oligo for every 16106 cells was electroporated into HeLa cells using the Amaxa nucleofactor and cell line nucleofactor kit V according to manufacturer’s protocols. Cells were then incubated in DMEM containing 5 mM glucose and 10% FBS. After 12 hr incubation, cells were switched to DMEM containing 5 mM glucose, 10% FBS, and antibiotic/antimycotic solution for an additional 36–40 hrs. Cells were then processed for luciferase assays. We also used short hairpin RNA (shRNA) for MED25 interference in the later real time PCR/Q-PCR and insulin secretion assays. MED25-shRNA was purchased from Origene with the following sequences: 59-GAC CAG AGC GGC TTC GTC AAT GGC ATC CG-39. Cells grown to 50% confluence were transfected using Metafectene Pro Transfection reagent (Biontex) with MED25 or scrambled shRNA according to the manufacturer’s instructions. Quantitative Real Time PCR MIN6 cells were transfected with MED25 (wt or the NR mutant), HNF4a (wt or the MODY mutant) plasmids. 24 hours after transfection, total RNA was extracted using Tri reagent (Sigma Aldrich) and 0.5 mg of RNA was reverse transcribed using Onestep RT-PCR kit (Qiagen) and amplified by PCR whose product formation was monitored continuously during PCR using Sequence Detection System software (ver. 1.7; Applied Biosys- tems). Accumulated PCR products were detected directly by monitoring the increase of the reporter dye (SYBR). The expression levels of PPARa, L-pyruvate kinase (PK), Kir6.2, and HNF4a-MED25 Interactions in Beta-Cells GLUT2 in the exposed cells were compared to those in control cells at each time point using the comparative cycle threshold (Ct)- method. The quantity of each transcript was calculated as described in the instrument manual and normalized to the amount of actin, a housekeeping gene. Semi-Quantitative Polymerase Chain Reaction (Q-PCR) MIN6 cells were transiently transfected with MED25 (wt or the NR mutant) and/or HNF4a (wt or the MODY mutant) and MED25 shRNA using Metafectene pro transfection reagent (Biontex) for 24 hrs. Total RNA from the treated cells was prepared with the Tri reagent (Sigma Aldrich) according to the manufacturer’s protocol. cDNA synthesis and semi-quantitative PCR for PPARa, L-PK, Kir6.2, and GLUT2 mRNA were performed, and the PCR reactions were electrophoresed using a 2% agarose gel. The band intensities of the amplified DNA products were visualized using the SYBR Green I DNA gel stain kit (Invitrogen). Insulin Secretion Assays This functional study was performed with the MIN6 cell line which exhibits the characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets [22]. To quantify the amount of insulin secreted, MIN6 cells were grown on a 6-well dish (about 16106 cells) and transfected with various vectors harboring MED25, HNF4a, MED25 NR mutant, MED25 shRNA, or HNF4a MODY mutants and incubated for 24 hrs. After the incubation period, the cells were washed three times with KRB buffer (119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 10 mM Hepes pH 7.4, and 0.1 g BSA), and further incubated in 1 ml pre-warmed KRB buffer containing 1 mM glucose for 1 hr at 37uC. The cell culture media (total of 1 ml) was collected and used to measure the level of insulin release with the insulin ELISA kit (Mercodia) by means of an enzyme immunoassay followed by an optical density reading at 450 nm. The amount of stimulation (fold increase) refers to insulin secretion after various treatments relative to insulin secretion in 1 mM glucose treated cells, which was set as 1-fold. Values are expressed as means 6 SD of data obtained from three independent experiments (n = 3), each performed in duplicate. Statistical Analysis Presented data are expressed as mean 6 standard error of the mean (SEM) of at least three independent groups. Statistical significance was determined by one-way ANOVA followed by Student-Newman-Keuls method using Sigma Stat 3.1 software (Systat Software, San Jose, CA). A probability value p,0.05 was considered statistically significant. Results Initial Identification of HNF4a/MED25 Molecular Interactions The current model of eukaryotic gene regulation is best described by the combinatorial recruitment involving multiple transcriptional regulators [23,24]; however, the full extent of tissue-specific and protein-specific recruitment has not been well characterized. Thus, to elucidate the detailed molecular interac- tion network of HNF4a-mediated transactivation in pancreatic ß- cells, we performed a yeast two-hybrid screen and identified MED25 as one of the binding partners of HNF4a. The bait vectors containing various constructs of HNF4a were constructed and screened against the pre-transformed mouse pancreatic PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e44007