Results: ACH-3422 alone showed potency up to 7-fold


Results: ACH-3422 alone showed potency up to 7-fold

greater than the sofosbuvir comparator against genotype-1 through genotype-4 replicons. Short-term combination studies of ACH-3422 with RBV, ACH-3102, sovaprevir, or ACH-2684 showed additive to synergistic effects on antiviral potency. In long-term colony-formation studies, ACH-3422 treatment alone at a concentration approximately 8-fold above its EC50 value led to nearly complete blockage of resistance emergence. The same effect was achieved at lower ACH-3422 concentrations when combined with either ACH-3102 or an NS3 protease inhibitor (sovaprevir or ACH-2684). Further reduction of ACH-3422 concentrations to as low as its EC50 of 60 nM also completely blocked resistance emergence when selleck compound combined with both ACH-3102 and an NS3/4A protease inhibitor (either sovaprevir or ACH-2684). Conclusions: Combinations of ACH-3422 with an NS5A inhibitor, an NS3 protease inhibitor, or both were highly effective in blocking the emergence click here of resistant variants in vitro. These results support clinical investigation of ACH-3422 in combination with ACH-3102 and/or an NS3 protease inhibitor for the treatment of chronic hepatitis C. Disclosures: Jason Wiles – Employment: Achillion

Pharmaceuticals Mingjun Huang – Employment: Achillion Pharmaceuticals Wengang Yang – Employment: Achillion Pharmaceuticals; Stock Shareholder: Mirabegron Achillion Pharmaceuticals The following people have nothing to disclose: Dharaben Patel, Yongsen Zhao, Joanne L. Fabrycki, Guangwei Yang, Steven Podos, Avinash Phadke Purpose: Evaluate the pharmacokinetics and safety of ABT-493 and ABT-530 following 3-day monotherapy in HCV Geno-type-1 infected subjects with or without compensated cirrhosis.

Methods: This study is a randomized, open-label, multicenter dose-ranging study, exploring the safety, pharmacokinetics and antiviral activity of ABT-493 (100, 200 (including cirrhotics), 300, 400, and 700 mg QD) and ABT-530 (15, 40, 120 (including cirrhotics) and 400 mg QD) 3-day monotherapy in HCV genotype-1 infected subjects with or without compensated cirrhosis. Intensive blood samples were collected during the 3-day monotherapy for ABT-493 and ABT 530 pharmacokinetic assessment. Safety and tolerability was assessed throughout the study. Results: A total of 48 subjects received ABT-493 and 40 subjects received ABT-530. Both ABT-493 and ABT-530 showed rapid absorption with Tmax ranging from 2-4 hours. Similar to healthy subjects, increase in ABT-493 exposure was more than dose-proportional; Cmax and AUC24 ranged from 68.3 -12200 ng/mL and 290-71600 ng*h/mL over the 100-700 mg dose range. ABT-493 exposures in HCV infected non-cirrhotic subjects were 2- to 5-fold of healthy subjects, while exposure in subjects with cirrhosis were 6-fold of non-cir-rhotic subjects.

In particular, IL-6 has been put forward as the molecular

In particular, IL-6 has been put forward as the molecular

component released by non-stem cancer cells to allow their conversion to cancer stem cells, and thereby maintain a dynamic equilibrium between these two tumor intrinsic cell types.82 Stat3 upregulates proteins of the Bcl-2 pro-survival family. In epithelial cells, it also induces other proteins that indirectly suppress apoptosis, such as the chaperone protein Hsp70, the C-type lectin-type RegIIIβ, and survivin,83 which are all overexpressed in CRC and IBD. The latter proteins not only suppress apoptosis, but might also promote cell cycle progression through binding to Cdc2. Stat3 also promotes the G1/S phase transition of the cell cycle more directly through the transcriptional Apoptosis inhibitor induction of cyclinB1, cdc2, c-myc, and cyclinD1, Alpelisib order and repression of the cell cycle inhibitor p21.83 As a third tumor-intrinsic property, Stat3 induces expression of the angiogenic factors, VEGF and HIF1α.83 Thus, excessive activation of Stat3 correlates with tumor invasion and metastasis in a variety of cancers. In the absence of epithelial Stat3 expression, the CAC model yields reduced tumor formation. Conversely, excessive Stat3 activation, through epithelial-specific Socs3 ablation or introduction of the Socs3-binding deficient gp130Y757F mutation, results in increased multiplicity and size of these tubular adenomas.84,85 Administration of

hyper-IL-6 (a fusion protein between IL-6 and soluble IL-6Rα), but not of enough IL-6, also increased tumor burden in CAC-challenged mice,85 suggesting that the extent of membrane-bound IL-6Rα, rather than gp130, limit the tumor-promoting

response. Consistent with these observations, we found functional redundancy between IL-6 and IL-11, and that both cytokines conferred Stat3-dependent, epithelial resistance to apoptosis and colitis.84 Genetic deficiency for the ligand binding IL-11Rα subunit in the CAC model significantly abrogates colonic tumor formation in gp130Y757F mice, while systemic reduction of Stat3 expression in gp130Y757FStat3+/− mice also reduced their susceptibility to colon tumorigenesis in the CAC model (Ernst et al., unpubl. observ., 2011). Furthermore, intestinal tumor burden is reduced in ApcMin mice lacking IL-6, and in ApcMin mice that are also haplo-insufficient for IL-11Rα or Stat3. However, IL-11 administration protected against radiation-induced mucositis, suggesting that IL-11 signaling might play a physiological role in the maintenance of intestinal epithelial integrity. Notwithstanding the central role played by excessive epithelial Stat3 signaling for the promotion of intestinal tumorigenesis, it has been recently suggested that this might also be part of an epigenetic switch mechanism that initiates tumor formation from non-transformed cells, rather than solely-expanding neoplastic cells that have arisen after exposure to mutagens.

Indeed, on the basis of the similar

way in which sperm ar

Indeed, on the basis of the similar

way in which sperm are stored and utilized in all birds, it seems likely that passive sperm loss is ubiquitous in this taxon. It is important to recognize that second or last male sperm precedence is not the rule in birds, especially in the wild. The experimental studies demonstrating the existence of last male sperm precedence in birds were conducted under very restrictive conditions, notably with equal numbers of equally competitive sperm in two inseminations. This is an unlikely scenario in the wild. Moreover, it is now known that both sperm numbers and sperm quality, which RO4929097 research buy can vary substantially between males, have a marked

influence on the outcome of sperm competition (Birkhead et al., 1999). In some species, males can allocate sperm number strategically (Cornwallis & Birkhead, 2006). It is also known that females can influence the uptake of a male’s sperm, and so the outcome of sperm competition in the wild is likely to be a due to a combination of factors that can obscure or override the influence of insemination order. Sperm competition mechanisms in mammals seem to be simpler than in birds or insects, probably because in most species, there is little or no sperm storage by the female and as a result, the interval between insemination and fertilization is usually much shorter, and sometimes just a few hours. An early, prescient model LEE011 mw of sperm competition Ergoloid in mammals by Ginsberg & Huck (1989) proposed that the timing of insemination relative to sperm capacitation and that in turn relative to when the female ovulated would be crucial for the outcome of sperm competition. There is now good

evidence for this and that the timing of capacitation varies between species (Gomendio et al., 2006). A particularly striking adaptation to sperm competition in rodents is ‘sperm trains’– groups of sperm operating as a unit. The woodmouse Apodemus sylvaticus, for example, is a species with relatively large testes and high levels of multiple paternity (Baker, Makova & Chesser, 1999) in which sperm trains are typical. Moore et al. (2002) found that the curiously extended hook on the sperm head allowed sperm to grasp each others’ flagella and swim as a ‘train’. They also showed that trains swan faster than individual sperm, because their flagella beat in unison, and speculated that this sperm cooperation was an adaptation to sperm competition, allowing sperm to rapidly traverse the hostile vagina and enter the cervix, before moving individually to the site of fertilization. Later, in a comparative study, Immler et al.

11, 12 Elastin is an insoluble nonpolar protein, formed by polyme

11, 12 Elastin is an insoluble nonpolar protein, formed by polymerization of the soluble monomer tropoelastin.13 The tropoelastin molecule is rich in alanine and lysine residues, which are principal sites for crosslinking reactions. Such reactions are potentially catalyzed by either lysyl-oxidase

(LOX) or tissue transglutaminase (tTG)14, 15; in addition, in mature scars a nonenzymatic reaction EGFR inhibitor is possible.11 Thus, intermolecular crosslinks increase the insolubility of the elastic fibers and render matrix resistant to degradation, in turn limiting the reversibility of fibrosis. Elastic fibers are present in the normal liver in the capsule and portal tracts and their number increases in fibrosis and cirrhosis.16, 17 Furthermore, the ratio between elastin and collagen

increases as liver fibrosis check details progresses.18 In parallel, an increase in crosslinking is observed.19 Despite this clear contribution of elastin to liver fibrosis and progression of liver disease, the regulation of elastin secretion and turnover has not been investigated in liver fibrosis. Two main cell types are responsible for elastin degradation, neutrophils, secreting neutrophil elastase (NE), and macrophages through macrophage metalloelastase (MMP-12).20 Like other MMPs, MMP-12 is transcriptionally regulated,21 secreted as a proenzyme, and subsequently activated by (self)-cleavage in selleck screening library the extracellular space. Macrophage depletion during spontaneous recovery from fibrosis leads to a failure of matrix degradation, associated with an increase in scar elastin relative to control22 (see below). This suggests that macrophages serve a discrete function mediating degradation of elastin. Furthermore, Fallowfield et al.23 have shown expression of MMP-13 (collagenase 3) by scar-associated macrophages, suggesting that these cells may be critically important in mediating matrix remodeling during fibrosis. We therefore deployed targeted gene mutation and conditional macrophage depletion studies to define

the role of macrophages and MMP-12 in mediating elastin turnover during progressive fibrosis. Our data provide evidence that elastin is regulated at the level of degradation during experimental liver fibrosis. Specifically, macrophage-derived MMP-12 appears to be critical for regulating elastin degradation in progressive experimental liver fibrosis. CCl4, carbon tetrachloride; HSC, hepatic stellate cell; LOX, lysyl-oxidase; MMP, macrophage metalloelastase; NE, neutrophil elastase; TAA, thioacetamide; TIMP, tissue inhibitors of metalloproteinase; tTG, tissue transglutaminase; WT, wildtype. Animals were housed in standard sterile conditions with free access to chow and water. All procedures were undertaken in accordance with the local ethical committee.

Therefore, we now know that under certain situations recombinant

Therefore, we now know that under certain situations recombinant viruses can be oncogenic if they insert into the genome in the proximity of a gene that regulates cellular growth. As a consequence of this

serious issue, clinical studies are now using gene-transfer systems based on lentiviral vectors. Lentiviral vectors, such as those derived from HIV-1, have Dasatinib cell line multiple advantages compared to γ-retroviruses. Recent evidence shows that the use of advanced generation, self-inactivating recombinant lentiviral vectors for HSC gene transfer is safer than γ-retroviruses. It now is well documented that lentiviral vectors, unlike γ-retroviruses, do not integrate with high frequency near the promoters of proto-oncogenes and genes that control cell proliferation, and recent studies showed that

they have a much BGB324 ic50 lower oncogenic potential than other retroviruses. In addition, lentiviral vectors transduce HSCs as efficiently or, under some conditions, more efficiently than γ-retrovirus vectors. The use of haematopoietic stem cells (HSCs) as the target cell population for lentiviral-mediated gene therapy applications is the most advanced application of this technology, and the use of lentiviral vectors for the treatment of haemophilia A has benefited from clinical trials that targeted HSCs for other genetic diseases. Because lentiviral-based gene transfer results in the genetic modification of the transduced nearly cell’s genome, the transduction process permanently

modifies the DNA of the targeted cell. Bone marrow transplant studies in children have shown that transplanted HSCs survive for the lifetime of the recipient and that genetically engineered HSCs can both self replicate and/or differentiate into all cells of the haematopoietic system. In theory, transduction and transplantation of a single genetically modified HSC can result in the complete repopulation of the haematopoietic compartment, whereby all cells would be genetically modified. In the clinical setting, many diseases have already been treated using lentiviral-modified HSCs, including adrenoleukodystrophy, metachromatic leukodystrophy, Wiskott-Aldrich syndrome, chronic granulomatous disease, SCID-X1, HIV and thalassemia [65-71]. Based on encouraging clinical results using lentiviral vectors, preclinical studies using genetically engineered HSCs to treat haemophilia A are advancing towards clinical trials. Platelet-specific promoters have been used to treat both murine and canine models of haemophilia A. It is thought that this technology can be most useful in the setting of patients with pre-existing FVIII inhibitors. Lentiviral designs using promoters with more ubiquitous expression patterns have advanced to the stage of US FDA review.

We next explored the methylation status of the PAX5 promoter by u

We next explored the methylation status of the PAX5 promoter by using MSP (Fig. 2A). Full or partial methylation was detected in HCC cell lines (Hep3B, huH4, huH6, Mahlavu, SNU398, and PLC5), which showed silenced or down-regulated PAX5 expression, whereas methylation was not detected in the cell lines with PAX5 expression (hUH7, SNU475, Selleck Vemurafenib and SNU449) (Fig. 2A). The methylation density within the PAX5

promoter region was then characterized and validated by BGS (Fig. 2B). The BGS results were consistent with those of MSP in which dense methylation was found in methylated cell lines by MSP, but not in normal liver tissues (Fig. 2B). To confirm whether the promoter methylation is involved

in the silencing of PAX5, five cell lines with silenced PAX5 expression including Hep3B, HepG2, SNU387, SNU398, and PLC-5 were treated with 5-Aza combined with or without trichostatin A. This treatment resulted in the restoration of PAX5 expression in all cell lines examined (Fig. Erlotinib in vivo 2C), further implicating that the transcriptional silence of PAX5 was mediated by promoter methylation. The frequent inactivation of PAX5 in HCC cell lines but not in normal liver tissue suggested that PAX5 may play a role in tumor growth. We thus examined the growth-suppressive effect through ectopic expression of PAX5 in HepG2 and Hep3B, which showed no PAX5 expression. Reexpression of PAX5 in the stable transfected HepG2 and Hep3B cells was confirmed by RT-PCR (Fig. 3A1) and western blot (Fig. 3A2). Ectopic expression of PAX5 in these HCC cell lines caused a significant decrease in cell viability (Fig. 3B). The inhibitory effect on HCC cell growth was further confirmed by colony formation assay. The colonies formed in PAX5-transfected cells were significantly fewer in number and smaller in size than in empty vector-transfected

cells (down to 44%-54% of vector control, P < 0.01) (Fig. 3C). We examined the contribution of apoptosis to the observed growth inhibition in HCC cells derived by PAX5. The number of HepG2 cells with sub-G1 DNA content after PAX5 transfection was substantially increased compared with the control vector transfection Calpain (24.75% ± 2.09% versus 33.11% ± 2.06%; P < 0.05). Apoptosis was further assessed by immunoblot detection of the active form of caspase-7, caspase-8, caspase-9, and poly (ADP-ribose) polymerase (PARP). As shown in Fig. 3D, overexpression of PAX5 enhanced the levels of active caspase-7, -8, -9, and PARP. The subcutaneous tumor growth curve of Hep3B stably transfected with PAX5 or empty vector in vivo is shown in Fig. 4A. The tumor volume was significantly lower in PAX5-transfected nude mice as compared to the vector control mice (P < 0.0001). At the end of experiments tumors were isolated and weighed.

RASA1 protein expression in HCT116 with upregulated miR-21 was si

RASA1 protein expression in HCT116 with upregulated miR-21 was significantly lower than that in those with downregulated miR-21. The ability of proliferation in HCT116, RKO with upregulated miR-21 was enhanced over time and vice versa. Conclusion: RASA1 can be a promising molecular target for therapeutic intervention in patients with colon cancer. Key Word(s): 1. miR-21; 2. RASA1; 3. colon cancer; 4. KRAS; Presenting Author: PING DU Additional Authors: NINGNING CONG,

FAJUAN SHEN, QINGYU ZHANG, CHUNSHENG KANG Corresponding Author: QINGYU BMS-777607 mw ZHANG, CHUNSHENG KANG Affiliations: Department of Gastroenterology, General Hospital of Tianjin Medical University; Laboratory of Neuro-oncology, Tian jin Neurological Institute Objective: Wnt/β-catenin signaling pathway is widely studied in many tumors including gastric cancer, which is a leading cause of death in China. Gastric adenocarcinoma is the most common type of gastric cancer. When Wnt/β-catenin signaling pathway is activated, the combination of β-catenin and T-cell factor / Lymphoid enhancer-binding factor (TCF/LEF) is necessary for the expression downstream factors

such as c-Myc and cyclin-D1. We use β-catenin/Tcf inhibitor FH535 to observe its effect on proliferation and invasion of gastric adenocarcinoma cell line SGC-7901 in vivo and in vitro. Methods: Human gastric adenocarcinoma SGC-7901 cells and human glioblastoma LN229 cells were treated with 20 μmol/L FH535 and solvent DMSO for 48 h respectively. HM781-36B purchase The cell cycle and apoptosis of treated cells were analyzed by flow cytometry. The invasive ability of SGC-7901 and LN-229 cells was determined by Transwell assay. We also used wound healing test to evaluate the cell migrating ability. Western-blot was used to find the malignancy related protein alteration. Subcutaneous tumor xenograft model was adopted to detect the influence of FH535 on SGC-7901 cell in vivo. 0.3 mg FH535 was delivered to each mice via intraperitoneal

injection every two days. FH535 was totally injected 6 times. Curve of tumor growth was plotted to describe the proliferation of SGC-7901 cell in vivo. Immunohistochemistry analysis and TUNEL Tolmetin staining were carried out to evaluate the apoptosis and proliferation state of SGC-7901 cells in vivo. Results: The experiments shows that the group treated with FH535 had significantly higher percentage of cells blocked at G0/G1 phase, lower percentage of cell cloning, less trans-membrane cell numbers, lower migrating rate after 48 h than the other groups. Protein levels of cyclin-D1 and c-Myc decreased in the FH535 group. While there was no significant difference on the percentage of apoptotic cells between the FH535 group and the others. The tumor from FH535 group grew slower than the other two groups. Immunohistochemistry analysis showed higher level of caspase-3 and lower level of Proliferating Cell Nuclear Antigen (PCNA) in the tumor tissue from FH535 group.

PJC improved the diagnostic utility of EUS-FNA for pancreatic tum

PJC improved the diagnostic utility of EUS-FNA for pancreatic tumor. Endoscopic ultrasonography (EUS) is a widely accepted modality for detecting pancreatobiliary diseases, determining the depth of gastrointestinal malignancies, and, often, for visualizing lesions more precisely than other imaging modalities. Endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNA) has enhanced the diagnostic

capabilities of EUS by providing additional pathological findings.[1] More than 20 years have passed since the use of EUS-FNA was demonstrated Trichostatin A cost for pancreatic disease,[2] and now, this technique is popular worldwide. However, EUS cannot detect minimally invasive carcinoma, and EUS-FNA cannot be performed for intraductal papillary mucinous carcinoma (IPMC) because of concerns about needle tract seeding.[3, 4] Since the introduction of endoscopic retrograde cholangiopancreatography (ERCP), pancreatic juice cytology (PJC) has yielded sensitivities for pancreatic cancer that have ranged from 33% to 67%.[5, RXDX-106 mw 6] Recently, Uehara et al. have shown the usefulness of PJC for pancreatic cancer.[7] However, whether PJC strengthens the diagnostic power of EUS-FNA for pancreatic masses remains unclear. In the present study, the

diagnostic ability of EUS-FNA and/or PJC in pancreatic disease was examined. A total of 161 patients (103 men, 58 women; age range, 24–86 years; mean age, 67.0 years) with pancreatic disease was enrolled (Table 1). Of these, 90 patients (54 men, 36 women; age range, 24–86 years; mean age, 65.1 years) had malignant disease, and 71 patients (49 men, 22 women; age range, 28–82 years; mean age, 69.5 years) had benign disease. All patients who underwent EUS-FNA and/or PJC between April 2009 and March 2012 were reviewed. Ten patients were repeated during follow up in some patients

(Table 1). The true number of patients who underwent EUS-FNA, PJC, and both of them are 124, 89, and 36. Patients were referred for EUS-FNA and/or PJC based check details on the need to evaluate them for malignancy. Cytodiagnosis of the specimen was performed by Papanicolaou’s method; rapid cytopathologic diagnosis was not used. Informed consent was obtained from all patients. Eight patients were excluded from EUS-FNA, but not from PJC, if they had thrombocytopenia or uncontrolled coagulopathy. EUS-FNA was not performed for cases of intraductal papillary mucinous neoplasm (IPMN) and IPMC, as it is contraindicated in Japan in such cases. EUS-FNA and PJC were performed in an inpatient endoscopy suite as previously described.[7-11] EUS-FNA was performed using a 7.5-MHz, convex, linear array echoendoscope (GF-UCT240; Olympus Optical Co. Ltd, Tokyo, Japan), a 22-G needle (NA-200H-8022, Olympus), and a 25-G needle (ECHO-25 Cook Medical Inc, Winston-Salem, NC, USA, M00550020; Boston Scientific Corporation, Natick, MA, USA). PJC was performed using a lateral-viewing endoscope (JF260V; Olympus), a cannula (M00535700; Boston Scientific Corporation), and a 0.

The selective detection of IFNα biological activity on cells expr

The selective detection of IFNα biological activity on cells expressing the cognate HBV peptide/HLA-complex is thus the direct consequence of the exclusive targeting of IFNα to cells expressing the cognate HBV-peptide/HLA-complexes, whereas IFNα biological activity on cells not expressing the cognate complexes is dramatically reduced by the covalent attachment of IFNα to the antibodies. The therapeutic efficacy of IFNα treatment is linked to its antiviral and immunomodulatory effects. We tested whether our TCR-L/IFNα selleck kinase inhibitor could specifically induce antiviral and immunomodulatory

effects. The antiviral effector function of TCR-L/IFNα was measured using a system in which HepG2 cells were transfected with the entire HBV genome. These HBV-transfected HepG2 cells secrete HBsAg that is quantifiable in the supernatants of the cells Nutlin-3 mw 4-5 days after transfection. In the experiment shown in Fig. 6A, HepG2 cells were treated with the indicated concentrations of either cTCR-L/IFNα ± HBc18-27 peptide, or IFNα 6 hours after transfection with the HBV construct. On day 4 supernatants were collected and the respective concentrations of HBsAg were quantified by enzyme-linked immunosorbent assay (ELISA). Although IFNα significantly reduced the amount of HBsAg secreted into the cell culture medium, the addition of cTCR-L/IFNα fusion protein did not have any effect

on HBsAg production by the transfected cells. However, if the same cells were pulsed with HBc18-27 peptide at the time of cTCR-L/IFNα treatment, significant inhibition of HBsAg secretion was observed, similar to the levels detected

with the control IFNα molecule (Fig. 6A). Under these experimental conditions (HBV transfection) therefore, the expression of endogenous HBV peptide may have not been sufficient Methocarbamol to allow adequate levels of TCR-L binding. Nevertheless, these results show that the cTCR-L/IFNα fusion molecule possesses an HBV-specific antiviral effect, dependent on the level of HBV peptide/HLA-complexes on the surface of target cells. We also analyzed whether immunomodulatory functions directly mediated by IFNα could be directed toward the specific target cells. First, we tested whether TCR-L/IFNα was able to up-regulate HLA-class I molecules or NK-cell costimulatory molecules MICA and MICB.3 Experiments using HBV-transfected or parental HepG2 cell lines showed only a minimal increase of HLA-class I, MICA and MICB expression, and only in the presence of very high concentrations of TCR-L/IFNα (1 nM, data not shown). Using a different cell line of hepatic origin (PLC), an HBsAg+ hepatocellular carcinoma line that can be recognized by HBV-specific CD8T cells,23 we observed the ability of TCR-L/IFNα to slightly up-regulate HLA-class I expression in an antigen-specific fashion and at low concentrations (50 pM). No changes of MICA and MICB expression were observed in these cell lines at all concentrations tested (Fig. 6B).

All of the described experiments

were performed using mal

All of the described experiments

were performed using male mice aged between 8 and 12 weeks. For quantitative real-time polymerase chain reaction (PCR) messenger RNA (mRNA) was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA) after complementary DNA synthesis expression was determined using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA) (see Supporting Information for details). selleck compound For immunohistochemical analysis, paraffin-embedded tissue slides were stained using a primary anti-Glut2 antibody (1:150) (Abcam, Cambridge, MA) and fluorescence or horseradish peroxidase (HRP)-labeled secondary antibodies (Vectorstain ABC-Kit, Vector Laboratories, Burlingame, CA). Staining was detected using a Nikon light, or fluorescence microscope, respectively (see Supporting Information for details). Proteins were separated by way of sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransfered onto nitrocellulose membranes (Invitrogen, Carlsbad, CA), and protein expression was determined using the indicated primary antibodies (Supporting Table 1). Binding of the antibody was detected using HRP-labeled secondary antibodies (BioRad, Hercules, CA) and the Amersham ECL Plus Western Blotting Detection Reagents Rapamycin chemical structure (GE Healthcare, Baie d’Urfe, Quebec, Canada). Chemiluminescence was determined using a KODAK ImageStation

4000MM (Mandel, Guelph, Ontario, Canada). Animals were fed ad libitum using a western diet (TestDiet, Richmond, IN) containing 16.8% protein, 6.5% fiber, 48% carbohydrates, and 20% fat. After 6 weeks of feeding, wild-type and Slco1b2−/− mice were sacrificed and blood samples were collected. The measurement of cholesterol and Akt inhibitor TSH was performed at Charles River Laboratories (Wilmington, MA). Total and free thyroxine (T4) and triiodothyronine (T3) in plasma were determined using enzyme-linked immunosorbent assay (ELISA) kits from Alpha-Diagnostics (San Antonio, TX). Insulin levels were determined using the UltraSensitive Mouse Insulin ELISA kit (Crystal

Chem Inc., Downers Grove, IL). Total bile acids or 7-α-hydroxy-4-cholesten-3-one were determined using a commercially available colorimetric assay (BioQuant, San Diego, CA) or mass spectrometry, respectively (see Supporting Information for details). Glucose tolerance testing and pyruvate challenge were performed using 2 g/kg glucose or pyruvate. Glucose levels were determined using a glucometer (OneTouch, LifeScan Inc., Milpitas, CA). For thyroid hormone (TH) extraction, tissue was homogenized in methanol. After addition of chloroform (2:1) and centrifugation (15 minutes, 1,900g, 4°C), pellets were re-extracted with a chloroform/methanol (2:1) mixture. Both supernatants were combined and further extracted with chloroform/methanol/water (8:4:3) and 0.05% CaCl2. The mixed solution was centrifuged (10 minutes, 800g, 4°C). Lower apolar phase was re-extracted with chloroform/methanol/water (3:49:48).