Although l-menthol has been reported to be an effective agent for reducing gastric peristalsis, the contributions during endoscopy are not well understood. Our aims were to evaluate and compare the anti-peristaltic effects of those agents during upper gastrointestinal endoscopy. Methods: This study selleck compound was performed as a non-randomized prospective study. A total of 260 patients scheduled to undergo upper gastrointestinal endoscopy were enrolled. Written informed consents were obtained from all patients

before enrollment. HB and GL were injected intramuscularly three minutes ahead of procedure. L-menthol was sprayed on the gastric mucosa through endoscopy within one minute from insertion of endoscopy. Patients <70 years old without contraindications to HB were classified into two groups; administrated HB intramuscularly (group A) or administrated l-menthol on the gastric mucosa

(group B). Patients ≧ 70 years old, or with contraindications to HB were also classified into two groups; administrated this website GL intramuscularly (group C) or administrated l-menthol on the gastric mucosa (group D). The peristaltic score (1–5, 1 represents no peristalsis, 5 represents markedly vigorous peristalsis) was defined according to the degree of peristalsis of the antrum after two minutes from insertion and at the end of procedure. Blood pressure, heart rate, procedure time, and severity of gastric atrophy were also assessed. Statistical analysis was performed by use of GraphPad Prism version5.0 (GraphPad Software, San Diego, CA). Patient characteristics were compared among the groups using Fisher’s exact test. For MCE公司 the peristaltic score, Mann-Whitney U test was used to assess the significance of difference.

P value < 0.05 was considered statistically significant. Results: As four cases were excluded due to the exclusion criteria, 256 cases were analyzed (group A; B; C; D = 61; 58; 71; 66). No significant differences were observed in age, sex, procedure time and severity of gastric atrophy between group A and B as well as C and D. Although the peristaltic score was lower in group B than in group A (1.47 ± 0.81 vs.1.73 ± 0.94) at the end of procedure, there was no significant difference (p = 0.097). The peristaltic score in Group D was significantly lower than the one in group C (after two minutes 1.17 ± 0.51 vs. 1.48 ± 0.78, at the end of procedure 1.33 ± 0.70 vs. 1.83 ± 1.01) in both of periods (p < 0.05).

Collectively, Bortezomib ic50 these results indicate that mTOR activation could suppress LHBs expression through a negative feedback loop that repressed pre-S1 promoter activity. To determine the critical elements responsible for pre-S1 promoter repression by mTOR signal, we constructed three 5′-deletion mutants of pre-S1 promoter reporter plasmids (Fig. 5Aa,b,c). As shown in Fig. 5Ba,b,c, luciferase activities of all these deletion mutants consistently showed that only approximately 50% of the activities were detected in pre-S2 mutant-expressed cells, compared with those in control cells, indicating that nucleotide 2789-2845 of the pre-S1 promoter was the minimal

region for mediating mTOR signal-induced transcriptional repression. A computer search further revealed that this region contains three putative transcription factor binding sites at nucleotide PD0325901 chemical structure 2794-2801 (site 1), 2812-2816 (site 2), and 2820-2825 (site 3) of the pre-S1 promoter. We, therefore, generated pre-S1 promoter constructs with mutations at each one of these three sites (Fig. 5Ad,e,f) for further assays. As shown in Fig. 5Bd,f, activities of pre-S1 promoter-carrying mutations at site 1 or site 3 still showed mTOR activation-dependent repression, indicating that these

two sites were not responsive elements for the mTOR signal. Interestingly and unexpectedly, the mutation at site 2 resulted in a relatively low or insignificant luciferase activity of the pre-S1 promoter (Fig. 5Be), suggesting that site 2 was transcriptionally necessary for pre-S1 promoter activity. To further evaluate the role of site 2 in pre-S1 promoter repression by mTOR, we created

one additional mutant construct, in which site 2 remains intact, but site 1 and site 3 are mutated (Fig. 5Ag). We observed that this mutant construct could negatively respond to mTOR activation (Fig. 5Bg). Therefore, the 2812-2816 site appeared to be not only transcriptionally important for pre-S1 promoter activity, but also capable of mediating a suppressive effect under the status of mTOR activation. Because sequence analysis revealed that the 2812-2816 site of pre-S1 promoter is the putative binding site for transcription factor YY1, we performed EMSA and DAPA to confirm MCE公司 the binding of YY1 to this site. In the EMSA experiment, two major shifted bands (a and b) were detected using WT probes containing the 2812-2816 site, one (a band) of which was not detected using the Mut probes, which destroyed the 2812-2816 site (Fig. 6A). Coincubation with competitors abrogated the formation of these two bands. The data suggest that the slower migrating band (a), but not the faster migrating band (b), represented the YY1-probe complex. To further confirm this speculation, we additionally added YY1 antibody to the reaction mixtures and observed specifically reduced intensity of the slower migrating band (a). This data further verified the interaction of YY1 with the 2812-2816 site.

This theoretical background CYC202 nmr is subsequently used to explain why current interventions for hepatic I/R injury have not been very successful. Moreover, novel therapeutic modalities are addressed, including MitoSNO and nilotinib, and metalloporphyrins

on the basis of the updated paradigm of hepatic I/R injury. Liver resection or transplantation often constitutes the only curative treatment option for patients who suffer from a hepatic malignancy or end-stage liver disease. Irrespective of the underlying disease, intraoperative cessation of hepatic blood supply (i.e. ischemia) is sometimes necessary during resection or is inherent to the transplantation procedure, and causes further impairment of organ function. Following resection or transplantation, ischemia is alleviated by the restoration of blood flow to the organ (i.e. reperfusion), which triggers a cascade of molecular events that entails oxidative stress, induction of an immune response, and inflammation.[1, 2] The damaging effects that result from these events, which are highlighted in this paper, are known as Navitoclax datasheet ischemia and reperfusion (I/R) injury and collectively determine the postoperative outcome. The need for major liver surgery is likely to increase as the number of surgical patients in which a malignancy coexists with parenchymal liver disease is expected to rise.[3, 4] Given

that the severity of I/R injury correlates positively with the extent of preexisting liver disease, for example non-alcoholic fatty liver disease,[5] this trend will not only result in an increased number of hepatic resections, but also in an increased percentage of high-risk procedures. The same applies to liver transplantation, where the gap between organ supply and demand has been partially filled by the utilization of marginal (e.g. steatotic) grafts.[6] The use of suboptimal organs, however, remains associated

with dissatisfying outcomes, while the already insufficient donor pool suffers from a deteriorating quality of grafts.[7] As a result, hepatic I/R injury will most likely become even more prevalent, necessitating the development of effective treatment strategies. In spite of these cues, few options are currently available to 上海皓元 prevent or treat hepatic I/R injury in patients. The modalities that have been clinically evaluated can roughly be divided into surgical and pharmacological interventions.[8] Surgical approaches have mainly focused on local and remote ischemic preconditioning and the fine-tuning of portal triad clamping regimens, which has yielded modest improvements at best.[9, 10] Pharmaceutical approaches have primarily addressed the use of antioxidants (e.g. vitamin E), vasodilators (e.g. prostaglandin E1), and volatile anesthetics (e.g. sevoflurane).

2B, RBP4 treatment markedly decreased cytosolic SREBP-1 but elevated nuclear SREBP-1 levels. We further determined the messenger RNA (mRNA) amounts of SREBP-1a, SREBP-1c, and SREBP-2 by real-time polymerase chain reaction (PCR). Moreover, the mRNA levels of SREBP-1c but not SREBP-1a were significantly increased in a dose-dependent manner in response to RBP4 treatment (Fig. 2C), despite the fact that the SREBP-1c to SREBP-1a ratio (1:2) of human HepG2 cells was much less than that of mouse hepatocytes (9:1).[27] However, RBP4 did not significantly

affect the degree of SREBP-2 nuclear form (Fig. S3A) and its mRNA expression (Fig. S3B). To determine the functional effects of increased nuclear SREBP-1 translocation by RBP4, the gene expression of key target enzymes of SREBP-1 in the HepG2 cells was evaluated by quantitative reverse-transcription (RT)-PCR. As expected in Luminespib molecular weight the case of dynamically altered nuclear SREBP-1c, RBP4 dose-dependently increased the expression of endogenous lipogenic genes, including FAS, ACC1, and DGAT2, involved in fatty acid and TAG synthesis in HepG2 cells. Similar to the lack of an effect on nuclear SREBP-2, the expression of mRNAs encoding two key enzymes of cholesterol biosynthesis, 3′-hydroxylmethyl glutaryl coenzyme A reductase (HMGCR) Apoptosis Compound Library purchase and low-density lipoprotein

receptor (LDLR), was not altered in RBP4-treated HepG2 cells (Fig. 3A). MCE公司 In contrast, RBP4 exerted less effect on the nuclear SREBP-2-mediated transcriptional activation of SRE-containing

target genes, including the 4×SRE-Lucand LDLR-Luc reporter genes (Fig. 3B). We next mapped the human SREBP-1c promoter and identified the element responsible for RBP4 action. Transcriptional activation of the wildtype SREBP-1c promoter was markedly induced by RBP4 in HepG2 cells. Disruption of the LXRE and SRE motif in the same promoter diminished the level of basal transcription and prevented the further induction caused by RBP4 (Fig. 3C). These data show that the LXRE and SRE motifs are necessary for the RBP4-dependent induction of SREBP-1c transcription. PGC-1β is a recently identified transcriptional coactivator closely related to lipid metabolism.[28] To evaluate the effects of RBP4 on PGC-1β expression, we exposed HepG2 cells to recombinant RBP4 for different time periods and examined the effects on PGC-1β expression. RBP4 treatment was found to cause a time-dependent increase in the expression of Ppargc1b mRNA as determined by northern blot (Fig. 4A) and quantitative RT-PCR (Fig. 4B). The levels of Ppargc1b mRNA, over the control at 8 hours, increased as early as 2 hours after the addition of RBP4 to the cells, increased as early as 2 hours after RBP4 treatment. Moreover, the PGC-1β transcript levels remained high throughout the 24-hour treatment period. The effects of RBP4 were further found to be dose-dependent (Fig. 4C).

2%, 11.8%, and 42.4% of patients, respectively.

Mild CD patients within initial bowel damage showed a statistical significant rising tendency compared to those without, in need of surgery (Log Rank test, P < 0.001). Patients with a baseline elevated CRP level were more likely to experience surgical resection (P = 0.016). Conclusion: The clinically mild CD patients who have bowel damage at baseline would rather intensive therapy, such as biological agent, than immunomodulator therapy. Patients with a baseline elevated CRP level always should be cautious for the future risk of surgical resection. Key Word(s): 1. Crohn's disease; 2. azathioprine; 3. immunomodulator Presenting Author: MAKI MIYAKAWA Additional Authors: RYOSUKE SAKEMI, MASANAO NASUNO, HIROKI TANAKA, Inhibitor Library SATOSHI MOTOYA, AKIMICHI IMAMURA Corresponding Author: HIROKI TANAKA Affiliations: Sapporo Kosei General Hospital, Sapporo Kosei General Hospital, Sapporo Kosei General Hospital, Sapporo Kosei General Hospital, Sapporo Kosei General Hospital Objective: It is not clear whether a resultant Mayo endoscopic subscore of 0 is 5-Fluoracil ic50 associated

with improved long-term outcomes compared with a resultant subscore of 1. We analyzed the relationship between long-term remission rates and Mayo endoscopic subscores in UC patients. Methods: We retrospectively analyzed the medical records of patients with UC who underwent endoscopy from January 2009 to December 2010. The inclusion criteria were as follows: 1) maintenance of clinical remission for at least 1 year before the day of endoscopy; 2) no change in maintenance therapy before the day of endoscopy; 3) a Mayo endoscopic subscore of 0–2; and 4) patients not receiving maintenance treatment. Clinical remission and recurrence were defined as Lichtiger’s clinical activity index MCE (CAI) scores of ≤4 and ≥5, respectively. The cumulative remission rate since the day of endoscopy was estimated for each Mayo

endoscopic subscore using the Kaplan–Meier method. Results: A total of 166 patients were included in the present study. The 1-year cumulative remission rates were 86% for a Mayo endoscopic subscore of 0; 77% for a Mayo endoscopic subscore of 1; and 55% for a Mayo endoscopic subscore of 2. The cumulative remission rates for a Mayo endoscopic subscore of 0 were higher than those for a Mayo endoscopic subscore of 1, although the differences were not statistically significant. Conclusion: In UC patients in clinical remission, a Mayo endoscopic subscore of 0 may be associated with a reduced risk of recurrence compared with a Mayo endoscopic subscore of 1. Key Word(s): 1. Ulcerative colitis; 2.

13 The vast majority of these factors activate STAT3, underscoring STAT3 as an important transcription factor in MDSC differentiation. Indeed, ablation of STAT3 using conditional knockout mice reduced the expansion of MDSCs and improved T-cell responses selleck kinase inhibitor in tumor-bearing mice.14 MDSCs have been shown to suppress T-cell responses by way of numerous mechanisms including expression of inhibitory cell surface molecules, production of regulatory cytokines, the metabolism of arginine through activation of arginase-1, production of nitric oxide, and the up-regulation of reactive oxygen species (ROS).9 Arginase-1 inhibits

T-cell responses through depletion of nonessential amino acid, L-arginine, resulting in down-regulation of CD3-ζ and inhibition of T-cell proliferation.15, 16 Nitric oxide (NO) production in MDSCs is induced through up-regulation of inducible nitric oxide synthase (iNOS), NO down-regulates MHC class II in APCs and leads to T-cell apoptosis.17, 18 In leukocytes, ROS is primarily generated through NADPH oxidase. The oxidase is a multicomponent enzyme Selleck p38 MAPK inhibitor consisting of two membrane proteins, gp91 and p22, and at least four cytosolic components:

p47phox, p67phox, p40phox, and a small G protein Rac.19 In MDSCs a number of these components have been shown to be up-regulated, including p47phox and gp91.20 Notably, the regulation of these proteins was shown to be dependent on STAT3 activation, which provides further evidence for the importance of this transcription factor.20 Here we show that HCV induces the accumulation of MDSC through extracellular core protein. Human CD33+ cells cocultured with HCV-infected hepatocytes, or treated with HCV core,

suppress the activation of autologous T cells. Additionally, the suppression of T cells by HCV core-treated MDSCs is ROS-dependent. Core-treated CD33+ cells were CD14+CD11blow/+ and HLADR−/low. Further, HCV core treatment up-regulated NOX2 component, p47phox. Lastly, CD33+ cells from chronically infected patients were CD11b+CD14+ and HLADR−/low; these cells also up-regulated p47phox compared with healthy donors. These data provide evidence that HCV core induces the accumulation of ROS producing MDSCs, thereby inhibiting host T-cell responses. Therefore, this study describes a novel MCE mechanism for HCV-mediated immune regulation, and suggests that regulation of the MDSC population may be an attractive target for future HCV therapies. APC, antigen-presenting cell; DC, dendritic cell; IFN-α, interferon-alpha; IL, interleukin; HCV, chronic hepatitis C virus; MDSC, myeloid-derived suppressor cell; PBMC, peripheral blood mononuclear cell; PMN, polymorphonuclear; ROS, reactive oxygen species; STAT3, signal transducer and activator of transcription 3; TLR, Toll-like receptor. The human hepatoma cell line Huh 7.5.

The Mbd1 protein was localized mainly in the hepatocyte nuclei and demonstrated stronger staining in hepatocytes from B6 Mdr2-KO mice versus Mdr2+/− mice; at the age of 2 months, the difference between mutant and control livers was even larger (left panels, Fig. 5B,C). Mbd1 staining was much weaker in the FVB strain versus the B6 strain, whereas the patterns of expression were similar and showed prominent localization in the hepatocyte nuclei (right panels, Fig. 5B,C). When we tested the expression levels of these proteins, Lipin-1 and Mbd1, in liver tumors from old Mdr2-KO mice of both strains, we found variable expression patterns for both proteins. They evidenced higher expression in some

tumors or nodules and lower expression in others in comparison with the noncancerous surrounding tissue (Supporting Fig. 5). Although genome-scale gene expression profiling

did not reveal aberrant expression of the Mat1a www.selleckchem.com/products/dinaciclib-sch727965.html transcript in the livers of Mdr2-KO mice of both strains, we compared Mat1a protein levels in total liver extracts from mutant and control mice at the age of 3 months. We found a significant decrease in Mat1a at the protein level in livers of Mdr2-KO/FVB mice but not in livers of Mdr2-KO/B6 mice (Fig. 6A,B). Both WT (Fig. 6A) and Mdr2+/− males (not shown) of the RAD001 ic50 FVB and B6 strains were used as controls, and they showed similar expression of Mat1a. Because of the paradoxical anti-inflammatory and protumorigenic effects of Gal-1 and its significant up-regulation in Mdr2-KO/FVB mice, we further validated the functional relevance of this endogenous lectin in both strains. MCE公司 We observed up-regulation of the Lgals1 transcript encoding the Gal-1 protein in the livers of 3-month-old Mdr2-KO/B6 mice by RT-PCR (Fig. 4C). We confirmed the increased expression of the Gal-1 protein in both mutant

Mdr2-KO livers (Figs. 5D and 6C,D) starting from the first month of age. The Gal-1 protein was localized mainly in the cytoplasm of nonhepatocyte cells, including immune cells and cholangiocytes, and rarely in the cytoplasm of hepatocytes. Remarkably, in the bile ducts, Gal-1 was detected only in proliferating cholangiocytes (ductular reaction). Expression of the Gal-1 protein increased between 1 and 3 months of age in both Mdr2-KO strains. When the strains were compared, the level of Gal-1 was higher in Mdr2-KO/B6 livers at the age of 1 month, but the levels were similar in the two strains at the age of 3 months (Fig. 6D). Previous studies showed that the administration of exogenous recombinant Gal-1 protected mice against autoimmune hepatitis induced by ConA.14 Here we demonstrate that B6 Gal-1–KO mice had an increased sensitivity to ConA-induced hepatitis (Fig. 7A,B), and this highlights the relevance of endogenous Gal-1. Reflecting hepatocyte injury, ALT blood levels were significantly higher in the B6 Gal-1–KO mice versus the control WT mice at every time tested (Fig.

Immunohistochemistry using the antibody reacting with the C terminus of Foxp3 detected only mononuclear cells (Treg cells), but the antibody reacting with the N terminus highlighted cholangiocarcinoma cells as well as Treg cells (Fig. 4A). The cytoplasm

as well as nucleus of tumor cells was positive in several cases. However, because Afatinib datasheet Foxp3 is a transcription factor, the nuclear pattern was evaluated as functional expression. Consequently, 21 of 54 (39%) cholangiocarcinomas tested positive for Foxp3 by the antibody reacting with the N terminus. The relation between the IgG4 reaction and Foxp3 expression in cholangiocarcinoma cells is shown in Fig. 5. In cases of positivity for Fopx3, the number of IgG4-positive cells was significantly higher than in cases of negativity for Foxp3. RT-PCR analysis demonstrated that a cholangiocarcinoma cell line, HuCCT1, expressed the mRNA of Foxp3, but close examination using four sets of primers corresponding to exons 1, 3, 10-12, and 12 revealed a lack of exon 3 (Fig. 6), suggesting the presence of a splicing variant of Foxp3 in cholangiocarcinoma cells. Moreover, RT-PCR and ELISA revealed that HuCCT1 cells expressed IL-10 mRNA (Fig. 6) and protein in the culture medium at 7.8-15.6 pg/mL. IgG4 is important to the pathogenesis of IgG4-related diseases. selleck inhibitor However, patients with pancreatic

adenocarcinomas accompanying IgG4 reactions and/or elevated serum IgG4 levels4, 18-20 and with pancreatic and biliary cancers arising from IgG4-related diseases20-22 have been reported, though a cause-and-effect

relationship between IgG4 reactions Celecoxib and cancers has yet to be demonstrated. Moreover, in IgG4-nonrelated diseases, including primary sclerosing cholangitis, IgG4 reactions were found to various degrees.23, 24 Therefore, the presence of IgG4-positive cells is not a histological hallmark of IgG4-related diseases, and IgG4 reactions are speculated to be nonspecific in several pathological conditions, including cholangiocarcinomas. The present study also demonstrated the presence of extrahepatic cholangiocarcinoma cases with abundant IgG4 reaction, though there was no significant difference in IgG4-positive cell counts among anatomical locations of extrahepatic cholangiocarcinomas (common bile ducts, gallbladder, and the Papilla of Vater). The significance and mechanisms of IgG4 reactions in cancers as well as IgG4-related diseases are still unknown, but we speculated that cancer cells directly participate in the histogenesis of IgG4 reactions. Because the regulatory cytokine IL-10 is known to induce the differentiation of IgG4-positive plasma cells or favor B cell switching to IgG4 in the presence of IL-4,5, 6 we noted the IL-10–related regulatory cytokine network around cholangiocarcinoma tissue in this study.

37 HLA typing was carried out using a Luminex multianalyzer profiling system (Luminex, Austin, TX) with a LAB type SSO One Lambda typing kit (One Lambda, Inc., Canoga Park, CA), which is based on polymerase

chain reaction sequence-specific oligonucleotide probes. HLA genotypes were determined by sequence-based typing. Peptide sequences of all HLA-DRB1 alleles in the IMGT/HLA database release 3.4.0 (April 2011) were aligned. Phenotype frequencies were estimated by direct counting for each HLA allele. The significance of an association was evaluated by determining the standard P values after chi-squared analysis or Fisher’s exact test. A P value of less than 0.05 was considered statistically significant. FK866 ic50 Association strength was estimated by calculating

the odds ratio (OR) and 95% confidence interval (CI). Among HLA class I alleles, the frequencies of A*02:01 and C*03:03 were significantly increased in patients with PBC, compared with healthy subjects (16% versus 11%, P = 0.0029, and 18% versus 13%, P = 0.012, respectively) (Table 2). In contrast, patients had significantly lower frequencies of A*02:06 (6% versus 9%; P = 0.038), A*33:03 (4% versus 8%; P = 0.0025), B*44:03 (2% versus 7%; P = 0.0011), C*08:01 (5% versus 10%; P = 0.005), C*14:03 (3% versus 7%; P = 0.0018), and C*15:02 (2% versus 4%; P Dabrafenib order = 0.03) alleles, compared with controls (Table 2). No other HLA A, B, or C alleles differed significantly between the groups. Among DRB1 alleles, DRB1*04:05 and DRB1*08:03 were significantly associated with PBC, compared with healthy subjects (17% versus 13%, P = 0.044, and 13% versus 6%, P = 0.000025, respectively) (Table 2). Patients with PBC had a significantly lower frequency of DRB1*11:01

(1% versus 4%; P = 0.02) and DRB1*13:02 (3% versus 6%; P = 0.029) allele carriage, compared with controls (Table 2). Among DQB1 alleles, the DQB1*04:01 and DQB1*06:01 alleles were significantly associated with an increased risk of PBC (18% versus 13%, P = 0.02, and 23% versus 15%, P = 0.000091, respectively) (Table 2). Conversely, http://www.selleck.co.jp/products/pembrolizumab.html DQB1*03:01 (6% versus 12%; P = 0.00027), DQB1*06:02 (7% versus 12%; P = 0.019), and DQB1*06:04 (2% versus 5%; P = 0.0041) all conferred a reduced risk of PBC occurrence (Table 2). No other HLA DRB1 or DQB1 alleles were significantly associated with PBC, compared with healthy subjects. We also examined the influence of DRB1 and DQB1 allele homozygosity with PBC susceptibility and protection, but found no significant associations. However, the DRB1*08:03 and DQB1*06:01 alleles were significantly associated with PBC, compared to comparison cases with chronic hepatitis C (13% versus 5%, P = 0.0017, and 23% versus 16%, P = 0.02, respectively) (Supporting Table 1).

S1P2 knockout mice (S1P2−/−) were a gift from Richard Proia (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). They were housed under a reverse light cycle (12:12 hours) for 2 weeks before use. Mice were bred in pathogen-free conditions, under normal lighting, and wild-type and knockout mice were from the same litters. All animals were fed normal rodent chow and water ad libitum. All Midostaurin nmr procedures were approved by the VCU IACUC committee, which is accredited

by the Association for Assessment and Accreditation of Laboratory Animal Care International. Biliary fistulas and intraduodenal cannulas were placed in male Sprague-Dawley rats under brief anesthesia as described.14, 26 After surgery, they were placed in individual metabolic cages with water and normal chow ad libitum. All animals received continuous infusion of glucose-electrolyte replacement

solution. After 48 hours of chronic biliary diversion, TCA was infused at a rate of 1.05 mL/100 g rat/h and at a concentration of 36 μmol/100 g rat/h for 3 hours. JTE-013 was intraperitoneally injected 2 hours before TCA infusion at a dose of 2 mg/kg.27 At the end of the experiment, 0.1 g of liver was harvested to isolate RNA as described,14 and the rest of the liver was flash-frozen in liquid nitrogen in several pieces. One piece was used to make total cell lysates (see western blot analysis below). Animal research was conducted see more in conformity with PHS policy and with approval of the Institutional Animal Care and Use Committee of McGuire Veterans Affairs Medical Center of Richmond. Primary rat and mouse hepatocyte monolayer cultures were prepared

from male Sprague-Dawley rats or wild-type and S1P2−/− mice by the collagenase-perfusion technique of Bissell and Guzelian as described.28 Cells were plated at 2 × 106 cells per collagen-coated 60-mm dish in serum-free Williams E medium containing penicillin, dexamethasone (0.1 μM), and thyroxine (1 μM). In some experiments, PTX (300 ng/mL) was added 4 hours after plating and allowed to incubate for 16 hours before starting experiments. Most experiments were conducted after 24 hours of culture, but shRNA experiments used an incubation period why of 40 hours to allow for lentivirus-mediated gene expression before treatment. Total cell lysates were prepared as described.14 Fifty μg of protein were resolved on 10% Bis-Tris NuPAGE gels (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes. Immunoblots were blocked for 1 hour at room temperature (RT) with 5% nonfat milk in Tris-buffered saline (TBS) buffer and then incubated with antibodies to phosphor (p)-AKT, p-ERK, total-AKT, total-ERK, or actin in 1% bovine serum albumin (BSA) or nonfat milk for 24 hours at 4°C.