95% of respondents stated that they would use a liver from an HCV

95% of respondents stated that they would use a liver from an HCV-positive donor for an HCV-positive recipient, and 93% would re-transplant a patient with graft failure due to hepatitis C, although most respondents would not re-transplant a patient less than one year after initial GSI-IX transplant. 62% used an alternative immunosuppression strategy in HCV-infected patients, most commonly a smaller post-operative steroid bolus and a rapid steroid taper. 64% preferred

tacrolimus to cyclosporine for immunosuppression in patients with hepatitis C. 87% performed protocol biopsies at specific time points after transplantation. The trigger to treat HCV was > stage 2 (Metavir) fibrosis (97%), cholestatic hepatitis C (81%), and increased liver enzymes (19%). Treatment included antiviral therapy (97%), discontinuing or decreasing the prednisone and mycophenolate doses (35 and 32% respectively), and conversion from tacrolimus to cyclosporine (30%). Post transplant anti-viral therapies included a protease inhibitor/peg/riba (72%) and peg/riba (28%). Regarding use of sofosbuvir (sof) or simeprevir (sim) pre-transplantation, 62% of respondents would use sof/riba, 32% sof/peg/riba and 8% sim/peg/riba pre-transplant in a patient with genotype-1 infection. Related to all-oral HCV treatment regimens; 78% would use such a regimen; 51% pre-transplant,

selleck compound 19% immediately post-transplant and 30% in patients with clinically significant HCV recurrence in the graft. Conclusions: Pre- and post-transplantation management of hepatitis C varies significantly. Respondents appear willing to use both recently FDA-approved and future interferon-free

therapies to reduce HCV replication pre-and post-transplantation. Disclosures: Paul J. Gaglio – Advisory Committees or Review Panels: Merck, Vertex, Salix, BI, BMS, Janssen; Grant/Research Support: Merck, Gilead, Vertex, Otsuka, Genentech, MCE BI; Speaking and Teaching: Merck, Gilead, Vertex, Salix, Otsuka, Janssen The following people have nothing to disclose: Carly E. Glick, John F. Reinus BACKGROUND: End-stage liver disease caused by hepatitis C virus (HCV) is a leading indication (40%) for liver transplantation (LT) in Western Countries. Viral recurrence in the graft is considered “universal” and represents a major cause of mortality and morbidity after LT. METHODS: We retrospectively analyzed data from the last 14 years regarding patients with HCV-related liver disease who underwent LT in our Centre showing undetectable HCV-RNA at blood tests. We looked for pre-LT predictors of HCV recurrences (both histological and virological) and we conducted grafts and patients follow-up until April 2014. RESULTS: 50 patients were included in the study, 22/50 (44%) being HBV-HCV co-infected. We observed HCV-RNA recurrence in 8/50 (16%) patients, and histological recurrence at liver biopsy in 6/50 (12%).

95% of respondents stated that they would use a liver from an HCV

95% of respondents stated that they would use a liver from an HCV-positive donor for an HCV-positive recipient, and 93% would re-transplant a patient with graft failure due to hepatitis C, although most respondents would not re-transplant a patient less than one year after initial U0126 nmr transplant. 62% used an alternative immunosuppression strategy in HCV-infected patients, most commonly a smaller post-operative steroid bolus and a rapid steroid taper. 64% preferred

tacrolimus to cyclosporine for immunosuppression in patients with hepatitis C. 87% performed protocol biopsies at specific time points after transplantation. The trigger to treat HCV was > stage 2 (Metavir) fibrosis (97%), cholestatic hepatitis C (81%), and increased liver enzymes (19%). Treatment included antiviral therapy (97%), discontinuing or decreasing the prednisone and mycophenolate doses (35 and 32% respectively), and conversion from tacrolimus to cyclosporine (30%). Post transplant anti-viral therapies included a protease inhibitor/peg/riba (72%) and peg/riba (28%). Regarding use of sofosbuvir (sof) or simeprevir (sim) pre-transplantation, 62% of respondents would use sof/riba, 32% sof/peg/riba and 8% sim/peg/riba pre-transplant in a patient with genotype-1 infection. Related to all-oral HCV treatment regimens; 78% would use such a regimen; 51% pre-transplant,

selleck compound library 19% immediately post-transplant and 30% in patients with clinically significant HCV recurrence in the graft. Conclusions: Pre- and post-transplantation management of hepatitis C varies significantly. Respondents appear willing to use both recently FDA-approved and future interferon-free

therapies to reduce HCV replication pre-and post-transplantation. Disclosures: Paul J. Gaglio – Advisory Committees or Review Panels: Merck, Vertex, Salix, BI, BMS, Janssen; Grant/Research Support: Merck, Gilead, Vertex, Otsuka, Genentech, MCE BI; Speaking and Teaching: Merck, Gilead, Vertex, Salix, Otsuka, Janssen The following people have nothing to disclose: Carly E. Glick, John F. Reinus BACKGROUND: End-stage liver disease caused by hepatitis C virus (HCV) is a leading indication (40%) for liver transplantation (LT) in Western Countries. Viral recurrence in the graft is considered “universal” and represents a major cause of mortality and morbidity after LT. METHODS: We retrospectively analyzed data from the last 14 years regarding patients with HCV-related liver disease who underwent LT in our Centre showing undetectable HCV-RNA at blood tests. We looked for pre-LT predictors of HCV recurrences (both histological and virological) and we conducted grafts and patients follow-up until April 2014. RESULTS: 50 patients were included in the study, 22/50 (44%) being HBV-HCV co-infected. We observed HCV-RNA recurrence in 8/50 (16%) patients, and histological recurrence at liver biopsy in 6/50 (12%).

95% of respondents stated that they would use a liver from an HCV

95% of respondents stated that they would use a liver from an HCV-positive donor for an HCV-positive recipient, and 93% would re-transplant a patient with graft failure due to hepatitis C, although most respondents would not re-transplant a patient less than one year after initial click here transplant. 62% used an alternative immunosuppression strategy in HCV-infected patients, most commonly a smaller post-operative steroid bolus and a rapid steroid taper. 64% preferred

tacrolimus to cyclosporine for immunosuppression in patients with hepatitis C. 87% performed protocol biopsies at specific time points after transplantation. The trigger to treat HCV was > stage 2 (Metavir) fibrosis (97%), cholestatic hepatitis C (81%), and increased liver enzymes (19%). Treatment included antiviral therapy (97%), discontinuing or decreasing the prednisone and mycophenolate doses (35 and 32% respectively), and conversion from tacrolimus to cyclosporine (30%). Post transplant anti-viral therapies included a protease inhibitor/peg/riba (72%) and peg/riba (28%). Regarding use of sofosbuvir (sof) or simeprevir (sim) pre-transplantation, 62% of respondents would use sof/riba, 32% sof/peg/riba and 8% sim/peg/riba pre-transplant in a patient with genotype-1 infection. Related to all-oral HCV treatment regimens; 78% would use such a regimen; 51% pre-transplant,

this website 19% immediately post-transplant and 30% in patients with clinically significant HCV recurrence in the graft. Conclusions: Pre- and post-transplantation management of hepatitis C varies significantly. Respondents appear willing to use both recently FDA-approved and future interferon-free

therapies to reduce HCV replication pre-and post-transplantation. Disclosures: Paul J. Gaglio – Advisory Committees or Review Panels: Merck, Vertex, Salix, BI, BMS, Janssen; Grant/Research Support: Merck, Gilead, Vertex, Otsuka, Genentech, 上海皓元 BI; Speaking and Teaching: Merck, Gilead, Vertex, Salix, Otsuka, Janssen The following people have nothing to disclose: Carly E. Glick, John F. Reinus BACKGROUND: End-stage liver disease caused by hepatitis C virus (HCV) is a leading indication (40%) for liver transplantation (LT) in Western Countries. Viral recurrence in the graft is considered “universal” and represents a major cause of mortality and morbidity after LT. METHODS: We retrospectively analyzed data from the last 14 years regarding patients with HCV-related liver disease who underwent LT in our Centre showing undetectable HCV-RNA at blood tests. We looked for pre-LT predictors of HCV recurrences (both histological and virological) and we conducted grafts and patients follow-up until April 2014. RESULTS: 50 patients were included in the study, 22/50 (44%) being HBV-HCV co-infected. We observed HCV-RNA recurrence in 8/50 (16%) patients, and histological recurrence at liver biopsy in 6/50 (12%).

Next, we investigated whether ectopic Lcn2 expression by SH-J1 ce

Next, we investigated whether ectopic Lcn2 expression by SH-J1 cells significantly inhibited wound closure (Fig. 4A, right panels). Cells harboring stable transfectants were significantly less likely to migrate to the wounded area compared with parent or vector control cells. In a modified Boyden chamber assay, stable transfectants penetrated the matrix and colonized the bottom surface of the Matrigel-coated membrane to DNA Synthesis inhibitor a lesser extent than vector control cells; this was true for both SH-J1 (Fig. 4B, left panels) and SH-J1-luc stable transfectants (Fig. 4B, right panels).

Lcn2 expression therefore appears to inhibit the migration and invasiveness of cells in vitro. We also evaluated the effects of EGF and TGF-β1 treatment on the ability of Lcn2-positive or -negative cell lines to close wounds (Supporting Fig. S11). EGF and TGF-β1 treatment MG-132 datasheet significantly enhanced the wound closure ability of HCC cells endogenously

expressing Lcn2, but not that of HCC cells ectopically expressing Lcn2. These results suggested that Lcn2 functions downstream of EGF and TGF-β1 signaling. Next, to determine the functional role of Lcn2 in HCC cell metastasis, we used SH-J1-luc cells expressing luciferase and established Lcn2-expressing SH-J1-luc cells (Lcn2-luc) (Fig. 4C, left panels). The metastatic phenotype of the Lcn2-luc cells was examined by injection of the cells into the tail veins (200 μL of 5 × 105 cells) of nude mice, followed by detection of multiple metastatic nodules in the lungs (Fig. 4C, right panel). Vector control cells first colonized and then continued growing in the lungs with many metastatic nodules, whereas Lcn2-expressing cells formed far fewer metastatic nodules in the lungs. Sufficient bioluminescence data were obtained 40 days postinjection (Fig. 4D). These results suggest that Lcn2

plays a critical role in inhibiting metastasis and invasion in HCC. Twist expression has been shown to promote migration and invasion in HCC.[31] MCE We found that Twist1 protein expression was significantly down-regulated in stable transfectants expressing Lcn2 (Fig. 5A) and in cells transduced with Lcn2-expressing adenovirus (Fig. 5B; Supporting Fig. S12). In contrast, Twist2, Slug, and Snail expression did not change (Supporting Fig. S13). Next, to investigate whether Twist1 down-regulation is dependent on transcriptional regulation, we examined Lcn2-mediated Twist1 mRNA expression by real-time PCR analysis in HEK293T (Supporting Fig. S14, left panel) and SH-J1 cells (Fig. 5C, left panel). We found that Twist1 down-regulation was associated with a decrease in transcript levels of this gene. Furthermore, a promoter assay revealed that Lcn2 effectively decreased Twist1 promoter activity in HEK293T (Supporting Fig. S14, right panel) and SH-J1 cells transfected with a Twist1-luc construct containing the human Twist1 promoter linked to a luciferase reporter gene (Fig. 5C, right panel).

Next, we investigated whether ectopic Lcn2 expression by SH-J1 ce

Next, we investigated whether ectopic Lcn2 expression by SH-J1 cells significantly inhibited wound closure (Fig. 4A, right panels). Cells harboring stable transfectants were significantly less likely to migrate to the wounded area compared with parent or vector control cells. In a modified Boyden chamber assay, stable transfectants penetrated the matrix and colonized the bottom surface of the Matrigel-coated membrane to Selleck FK506 a lesser extent than vector control cells; this was true for both SH-J1 (Fig. 4B, left panels) and SH-J1-luc stable transfectants (Fig. 4B, right panels).

Lcn2 expression therefore appears to inhibit the migration and invasiveness of cells in vitro. We also evaluated the effects of EGF and TGF-β1 treatment on the ability of Lcn2-positive or -negative cell lines to close wounds (Supporting Fig. S11). EGF and TGF-β1 treatment GPCR & G Protein inhibitor significantly enhanced the wound closure ability of HCC cells endogenously

expressing Lcn2, but not that of HCC cells ectopically expressing Lcn2. These results suggested that Lcn2 functions downstream of EGF and TGF-β1 signaling. Next, to determine the functional role of Lcn2 in HCC cell metastasis, we used SH-J1-luc cells expressing luciferase and established Lcn2-expressing SH-J1-luc cells (Lcn2-luc) (Fig. 4C, left panels). The metastatic phenotype of the Lcn2-luc cells was examined by injection of the cells into the tail veins (200 μL of 5 × 105 cells) of nude mice, followed by detection of multiple metastatic nodules in the lungs (Fig. 4C, right panel). Vector control cells first colonized and then continued growing in the lungs with many metastatic nodules, whereas Lcn2-expressing cells formed far fewer metastatic nodules in the lungs. Sufficient bioluminescence data were obtained 40 days postinjection (Fig. 4D). These results suggest that Lcn2

plays a critical role in inhibiting metastasis and invasion in HCC. Twist expression has been shown to promote migration and invasion in HCC.[31] 上海皓元 We found that Twist1 protein expression was significantly down-regulated in stable transfectants expressing Lcn2 (Fig. 5A) and in cells transduced with Lcn2-expressing adenovirus (Fig. 5B; Supporting Fig. S12). In contrast, Twist2, Slug, and Snail expression did not change (Supporting Fig. S13). Next, to investigate whether Twist1 down-regulation is dependent on transcriptional regulation, we examined Lcn2-mediated Twist1 mRNA expression by real-time PCR analysis in HEK293T (Supporting Fig. S14, left panel) and SH-J1 cells (Fig. 5C, left panel). We found that Twist1 down-regulation was associated with a decrease in transcript levels of this gene. Furthermore, a promoter assay revealed that Lcn2 effectively decreased Twist1 promoter activity in HEK293T (Supporting Fig. S14, right panel) and SH-J1 cells transfected with a Twist1-luc construct containing the human Twist1 promoter linked to a luciferase reporter gene (Fig. 5C, right panel).

Proteins that constitute acute phase response to tissue injury/in

Proteins that constitute acute phase response to tissue injury/infection and the complement cascade have also been explored as candidates involved in the inflammatory state present in fatty liver disease. In agreement with a previous report,36 we found that some serum acute phase proteins were significantly elevated in NASH compared to controls,

but found no changes in the expression levels of others. The same was observed with several proteins that comprise the complement system, which have been identified in previous proteomic studies as important diagnostic biomarkers for patients with cirrhosis and hepatocellular carcinoma.37, 38 Coagulation and development of liver fibrosis are tightly coupled and proteins that contribute to inflammation and immunity, production and remodeling of extracellular matrices, and cell proliferation, motility, selleckchem and survival are all involved in this process.39 Serum levels of most proteins involved in platelet aggregation and coagulation were elevated in NAFLD and NASH patients; however, circulating levels of

fibrinogen β chain and fibrinogen γ chain were significantly reduced. Interestingly, in the only other proteomics study using serum from NAFLD patients, Younossi et al.25 provisionally identified fibrinogen γ chain as one of the protein peaks that differed significantly among patient groups and controls. Taken together, these findings highlight the Volasertib importance of coagulation in the pathogenesis of NAFLD. Structural and extracellular matrix proteins also play a critical role in tissue remodeling and fibrosis in the liver,

and we observed significant changes in several of these proteins in NAFLD. Specifically, the expression of lumican, a protein involved in collagen 上海皓元医药股份有限公司 fibril assembly, was significantly elevated in the NASH F3/F4 group. This finding is consistent with the recent proteomics report by Charlton et al.26 in which they also demonstrated increased lumican messenger RNA (mRNA) and protein expression in liver tissue from patients with NAFLD and progressive NASH. The liver is the primary site of synthesis for most apolipoproteins and is responsible for the maintenance of lipoproteins and lipid metabolism.40–42 Serum apolipoprotein C1 and its precursor have been previously identified as potential biomarkers for patients with hepatitis C virus (HCV)-induced cirrhosis that progresses to HCV-induced hepatocellular carcinoma.37, 43 Similarly, we observed changes in the serum lipoprotein profile of patients with NAFLD and NASH. These findings may reflect the common observation of hypercholesterolemia and dyslipidemia in fatty liver disease. Finally, we observed a significant reduction in serum levels of proteins known to possess antiinflammatory and antioxidant capabilities, such as the high-density lipoprotein (HDL) particle-associated paraoxonase 1 and several apolipoproteins, in patients with NAFLD and NASH.

Proteins that constitute acute phase response to tissue injury/in

Proteins that constitute acute phase response to tissue injury/infection and the complement cascade have also been explored as candidates involved in the inflammatory state present in fatty liver disease. In agreement with a previous report,36 we found that some serum acute phase proteins were significantly elevated in NASH compared to controls,

but found no changes in the expression levels of others. The same was observed with several proteins that comprise the complement system, which have been identified in previous proteomic studies as important diagnostic biomarkers for patients with cirrhosis and hepatocellular carcinoma.37, 38 Coagulation and development of liver fibrosis are tightly coupled and proteins that contribute to inflammation and immunity, production and remodeling of extracellular matrices, and cell proliferation, motility, click here and survival are all involved in this process.39 Serum levels of most proteins involved in platelet aggregation and coagulation were elevated in NAFLD and NASH patients; however, circulating levels of

fibrinogen β chain and fibrinogen γ chain were significantly reduced. Interestingly, in the only other proteomics study using serum from NAFLD patients, Younossi et al.25 provisionally identified fibrinogen γ chain as one of the protein peaks that differed significantly among patient groups and controls. Taken together, these findings highlight the Bioactive Compound Library concentration importance of coagulation in the pathogenesis of NAFLD. Structural and extracellular matrix proteins also play a critical role in tissue remodeling and fibrosis in the liver,

and we observed significant changes in several of these proteins in NAFLD. Specifically, the expression of lumican, a protein involved in collagen 上海皓元医药股份有限公司 fibril assembly, was significantly elevated in the NASH F3/F4 group. This finding is consistent with the recent proteomics report by Charlton et al.26 in which they also demonstrated increased lumican messenger RNA (mRNA) and protein expression in liver tissue from patients with NAFLD and progressive NASH. The liver is the primary site of synthesis for most apolipoproteins and is responsible for the maintenance of lipoproteins and lipid metabolism.40–42 Serum apolipoprotein C1 and its precursor have been previously identified as potential biomarkers for patients with hepatitis C virus (HCV)-induced cirrhosis that progresses to HCV-induced hepatocellular carcinoma.37, 43 Similarly, we observed changes in the serum lipoprotein profile of patients with NAFLD and NASH. These findings may reflect the common observation of hypercholesterolemia and dyslipidemia in fatty liver disease. Finally, we observed a significant reduction in serum levels of proteins known to possess antiinflammatory and antioxidant capabilities, such as the high-density lipoprotein (HDL) particle-associated paraoxonase 1 and several apolipoproteins, in patients with NAFLD and NASH.

This could be a methodological issue: patients with more severe b

This could be a methodological issue: patients with more severe bleeding disorders often had a longer duration

of infection, because they were generally treated and thus infected at an earlier age. The gold standard for the diagnosis of cirrhosis is a liver selleck chemicals biopsy, although sampling error is a problem. In haemophilia, there is also the issue of bleeding risk and cost of substitution therapy. Both in persons with haemophilia and others, there is a lot of interest in non-invasive methods to diagnose fibrosis and cirrhosis [10]. Cirrhosis may well be evident on ultrasound (US), if there is irregularity of the liver surface or nodularity of the liver [7], but can be missed. The most well-tested non-invasive options are FibroTest, which is a panel of five biochemical markers and FibroScan which uses transient elastography, an ultrasound-based technique,

to measure liver stiffness. In a meta-analysis of diagnostic accuracy (vs. biopsy as the gold standard), the sensitivity of FibroScan was 64% for F2-4 fibrosis and 86% for cirrhosis. For FibroTest, data were difficult to summarize because different cut-off levels were used. If sensitivity above 80% was required, specificity dropped to 40–60% [11]. FibroTest and FibroScan have been tested in haemophilia, but without comparison with liver biopsy [12–14]. When the two tests were compared with each other in haemophilia, concordance Acalabrutinib concentration was not very good in F2–3 fibrosis, but reasonable (85%) in cirrhosis 上海皓元医药股份有限公司 [12]. Non-invasive tests overcome the issue of bleeding with liver biopsy, but not completely that of costs: FibroTest is only available commercially and cannot be performed in-house and FibroScan requires investment in expensive equipment. The American Association for the Study of Liver Diseases (AASLD) does not recommend the use of

the currently available non-invasive tests instead of liver biopsy in routine clinical practice [4]. With the present options for treatment, the main question in clinical practice is whether there are signs of cirrhosis and this question can be answered by a combination of routine liver tests and a routine ultrasound in most patients. Once patients have developed cirrhosis, they are at risk for liver cancer, i.e. hepatocellular carcinoma (HCC). HCC is the leading cause of death in patients with cirrhosis because of hepatitis C. In the western world, the incidence of HCC is clearly increasing because of chronic hepatitis C. In 2000, 60–70% of HCC in Europe and 50–60% in North America was related to hepatitis C [15]. In other parts of the world, the background incidence of HCC is higher because of chronic hepatitis B and exposure to toxins. In large studies, the rate of development of HCC was 3–6% per year in patients with HCV cirrhosis. In patients with advanced fibrosis, the rate is approximately half of that [16,17].

J CHANG,1,2 M IP,2 M YANG,2 B WONG,2 M ARSHI,3 T PHAN,3 R LEONG1,

J CHANG,1,2 M IP,2 M YANG,2 B WONG,2 M ARSHI,3 T PHAN,3 R LEONG1,2 1Gastroenterology and Liver Services, Bankstown Hospital, South Western Sydney Local Health District, 2The University of New South Wales, 3Garvan Institute of Medical Research, Sydney Background: It

is increasing recognized that patients with inflammatory bowel disease (IBD) with mucosal healing (MH) who continue to have ongoing symptoms, may be suffering from possible irritable bowel syndrome (IBS) overlap. Impairments in small intestinal permeability have previously been demonstrated by confocal laser endomicroscopy (CLE) in patients with IBD and IBS cohorts independently, but little is known in those suffering with both. Aims: This study aims to examine small intestinal permeability using CLE (EC-3870FK, Pentax) in symptomatic selleck chemicals and asymptomatic IBD patients who have complete mucosal healing. Methods: Patients with IBD were prospectively recruited from Bankstown-Lidcombe Hospital for CLE. Confocal images were obtained with fluorescein sodium as an intravenous contrast from 5 separate sites within the terminal ileum. Only patients with MH were included for final analysis. This was defined in Crohn’s Disease (CD) as no endoscopic disease and in Ulcerative colitis (UC) as endoscopic Mayo score of 0 or 1. All patients had histology to demonstrate no active disease. Blinded

post procedure interpretation of images were performed with previously validated CLE features of “fluorescein leak”, “cell junction enhancement” and “cell drop out”. Calculation of a numerical “confocal leak score” (CLS), allowed quantification click here of the degree of small intestinal permeability. Patients were assessed to be symptomatic in CD based on their Crohn’s Disease Activity Index >150 and in UC with a partial mayo MCE公司 >2. A symptom diary of diarrhoea motions/day and severity of abdominal pain was

collected. 20 healthy controls also underwent CLE for assessment of normal range. Statistical analysis was performed using the Mann Whitney U for non-parametric data, chi square for categorical outcomes, spearman rank for correlation and regression analysis to establish the association of symptoms to CLS. Results: A total of 71 consecutive CLE cases (exclusive of 20 controls) were performed with 41 cases fulfilling the condition of MH, (61% F, 22 CD, 19 UC). 33 cases were classified into asymptomatic and 8 symptomatic based on the above criteria. There were no differences in baseline characteristics of median images/case, age, disease duration, C-reactive protein, erythrocyte sedimentary rate, smoking status, and non-steroidal anti-inflammatory use in the two groups (p > 0.05 in all). Median CLS for combined asymptomatic IBD and symptomatic IBD were 8.04 and 18.95 respectively (p = 0.001). Median CLS in controls was 5.94. In CD, median CLS in asymptomatic and symptomatic groups were 8.42 and 17.67 respectively (p = 0.019). In UC, median CLS in asymptomatic and symptomatic groups were 6.87 and 22.

Animals were maintained on a standard diet and housed under a

Animals were maintained on a standard diet and housed under a

12-hour light/dark cycle. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (publication 86-23, revised 1985). SkHep-1 cells plated onto coverslips were fixed with 4% paraformaldehyde. Confocal immunofluorescence (IF) was performed as previously described.[18, 19] SkHep-1 cell and Holtzman rat hepatocyte immunoblottings and separation of nuclear and non-nuclear protein extracts were carried out as previously described.[11] Cell-surface biotinylation and streptavidin pull-down were performed, with modifications, as previously described.[14] Plasmids were generated,[14] and adenoviral constructs were amplified and purified as previously described.[20] Ca2+ signals were detected and measured by time find more lapse confocal microscopy as described.[14, 18, 19] Validated small interfering RNAs (siRNAs) for clathrin heavy chain (cla) and caveolin-1 (cav) were obtained from Ambion (Austin, TX). SkHep-1 cells were transfected with 5 nM of each siRNA using Lipofectamine 2000, according to the

manufacturer’s instructions (Gibco, Grand Island, NY). Cells were used 48 hours after transfection. Cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation using an enzyme-linked immunosorbent assay (Roche Applied Science, Indianapolis, IN), according to the manufacturer’s instructions. Two-thirds (partial) hepatectomy (PH) was performed Tanespimycin order 上海皓元医药股份有限公司 on adult male Holztman rats as previously described.[21] Immunohistochemistry (IHC) was performed following standard methods for microwave antigen retrieval.[22] Glucose content in the blood was measured using an enzymatic colorimetric assay method (Analisa, Belo Horizonte, Brazil), according to the manufacturer’s instructions. Glycogen content from liver samples was determined by a phenol-sulfuric acid method, as described by Dubois

et al.,[23] with modifications. Results are expressed as mean values ± standard deviation (SD). PRISM software (GraphPad, La Jolla, CA) was used for data analysis. Groups of data were compared using the Student t test or one-way analysis of variance (ANOVA; which was used because data sets included only one independent variable), followed by Bonferroni’s post-tests, and P < 0.05 was taken to indicate statistical significance. Detailed and additional methods are available in the Supporting Materials and Methods. Translocation of the IR to the nucleus has been observed in primary rat hepatocytes.[11] To investigate whether the IR translocates to the nucleus in the SkHep-1 human hepatoma cell line as well, cells were analyzed by confocal IF microscopy to monitor localization of the IR before and after insulin stimulation. This liver cell line was used because, as in primary hepatocytes, it contains Ca2+-signaling machinery in both the cytoplasm and the nucleus.