Chemosensitivity to 5-FU was evaluated by both cell proliferation assay and apoptosis

assay. Results: Dramatically inhibited cell proliferation and increased apoptosis were indentified in FU, CUR and FU+CUR compared with Ctrl, but FU+CUR showed the most significant changes. This result suggested that curcumin enhanced chemosensitivity of 5-FU against gastric cancer cells. Mechanically, FU+CUR exhibited the most activation status of PERK signaling pathway than FU and CUR, even in which PERK signaling pathway was also activated. This result indicated that activation of PERK signaling pathway was one of the possible mechanisms in curcumin- enhanced chmosensitivity to 5-FU against gastric cancer cells. Conclusion: Curcumin exerts anticancer activity and chemosensitivity to 5-FU by activating PERK signaling pathway. BMS354825 Key Word(s): 1. PERK; 2. Curcumin; 3. Chemosensitivity; Presenting Author: XIN XU Additional Authors: ZHONGWEI LIU, KUNLUN CHEN, YING LIU, JINKAI XU, JIE LI, JIE WU, YI YANG, JIANGYI CAI Corresponding

FDA-approved Drug Library chemical structure Author: XIN XU Affiliations: The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University; Xi’an Aerospace General Hospital Objective: Original studies have shown that trastuzumab improved overall survival rate in patients with advanced gastric cancer. However, a relatively high trastuzumab resistance for rate was reported in several studies. This study was aimed to elucidate the possible mechanism of trastuzumab resistance in gastric cancer cells. Methods: Trastuzumab

was used to incubate gastric carcinoma cell line MKN45 and NCI-N87. Western blotting was applied to evaluate the phosphorylation of PERK (protein kinase RNA – like ER kinase) in each cell line. Then a PERK gene specific siRNA was transfected into gastric cancer cells. RNA interference was examined by RT-PCR. Drug resistance against trastuzumab was assessed by MTT assay. Results: Gastric cancer cell line MKN45 was found resistant to trastuzumab rather than NCI-N87. The expression of phosphorylated PERK, which is the active form of PERK, increased dramatically in NCI-N87 cells than in MKN45 cells. After knockdown PERK gene by RNAi, NCI-87 exhibited drug resistant behavior against trastuzumab. These results indicated that trastuzumab’s anti- proliferation signaling transduction relied on activation of PERK. Low expression or loss of PERK made gastric cells resistant to trastuzumab. Conclusion: Deficiency of PERK plays an important role in trastuzumab resistance in gastric cancer. Key Word(s): 1. PERK; 2. Trastuzumab; 3.

19 HSC cotransplantation markedly enhanced expression of CD62L on infiltrated CD11b+ cells, but not others (Supporting Fig. 1B). The antigen stimulatory activity of these purified CD11b+ cells was examined in a one-way mixed leukocyte reaction (MLR) culture

where carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled B6 spleen T cells were stimulated by CD11b+ cells pulsed with BALB/c spleen cell lysate (without pulsing served as controls). CD11b+ cells from islet/HSC grafts elicited weaker proliferative response in both CD4 and CD8 T cells with less IFN-γ production, but generated more CD4+Foxp3+ cells compared to the islet-alone group (Fig. 2B). To determine their immune regulatory activity, the isolated CD11b+ cells were added to the culture of CFSE-labeled T cells at a ratio of 1:10. T-cell proliferation was elicited by anti-CD3 mAb. Addition of CD11b+ cells from islet/HSC, but not from selleckchem islet alone grafts, markedly suppressed the proliferative response and IFN-γ production in both CD4+ and CD8+ T cells. This was associated with markedly RXDX-106 nmr reduced T-cells numbers (Fig. 2C, right panels, P < 0.05, islet versus islet/HSC) due to enhanced T-cell apoptosis as determined by annexin V staining (Fig. 2C). Taken together, these data on CD11b+ cells in islet/HSC grafts demonstrated many characteristics of MDSC: CD11clow,

Thymidylate synthase immature phenotype, expressing high iNOS and arginase1, immune inhibitory activity,16, 20 suggesting that cotransplanted HSC are potent inducers of MDSC. MDSC have

been shown to mediate development of Treg cells.18 To study the correlation of MDSC and Treg cells induced by HSC cotransplantation, CD11b+CD11c− cells and CD4+Foxp3+ cells were kinetically analyzed by immunohistochemistry and flow cytometry in the grafts, draining LN, and spleen following transplant. CD11b+CD11c− cells were remarkably increased in islet/HSC grafts, peaking on POD 7, compared to islet alone, gradually declined thereafter, and hardly found in long-term survival grafts (Fig. 3A). An increase in CD11b+CD11c− cells was also seen in dLN and spleen, and remained high there in the recipients with long-term survival grafts (Fig. 3B,C). The changes of CD11b+CD11c− cells (MDSC) were well correlated with that of CD4+Foxp3+ Treg cells, suggesting a close relationship of the two cell populations. Induction of MDSC has been shown to require inflammatory stimulation.21, 22 We hypothesized that HSC might lose their ability to induce MDSC when IFN-γ stimulation was blocked. This was tested by using HSC from IFN-γR1−/− mice for islet cotransplantation. Following transplantation, the graft CD11b+ and CD4+ cells were evaluated by both immunohistochemistry and flow cytometry for expression of CD11c and Foxp3, respectively. As shown in Fig.

The intensity increases to its maximum

slowly, but may be maintained at a lower level with a single push of the button. Two pushes turns the device off. It is approved for patients 18 years and older, and is contraindicated in those with other implanted electrical devices such as pacemakers. In Europe and Canada, the device made by the same manufacturer has 3 stimulation settings: program 1 for acute “crisis treatment,” program 2 with 60 Hz for migraine prevention, and program 3 used for stress reduction and relaxation. FDA approval was based upon 2 studies conducted Selleck JNK inhibitor in Europe. The first was performed in Belgium, using 67 individuals who had at least 2 migraine attacks per month and had not taken any migraine preventive medications for 3 months prior to the study. Patients who were overusing medications, had failed 3 other sound preventive trials, had frequent tension-type headaches, as well as patients who had severe neurologic or psychiatric disorders, were all excluded from the trial. As a double blind trial, patients with similar characteristics were randomly divided into 2 groups. Both groups were given the device, and everyone received some degree of electrical stimulation, but

only half of them got the same degree and type of stimulation offered by the Cefaly NeuroStimulation (tSNS) device. A 30-day baseline record was obtained during which no preventive treatment was used, and headache data were gathered, followed by 3 months in which the actual device or the Selleck Apoptosis Compound Library sham OSBPL9 device was used depending upon the assigned group. The 2 primary outcomes compared baseline with the third treated month, looking for: A change in monthly migraine days. The percentage of subjects who had at least a 50% reduction in monthly migraine days. The first outcome measure, change in the number of migraine days, showed improvement, but not sufficient change to be considered significant. The second outcome, that is the percentage of those having at least a 50% reduction, was positive, meaning that if individuals did respond to the device,

they had a significant decrease in the number of headache days, seen in about 26% of subjects. Almost a third of subjects who did respond experienced a 25% reduction in headache days. The investigators also studied attack frequency, mean headache severity per migraine day, monthly intake of acute, as-needed medications, occurrence of migraine-associated symptoms per migraine day (nausea, light, and noise sensitivity), and percentage of very or moderately satisfied patients. There was a slight decrease in mean headache severity if individuals followed the protocol, and a highly significant almost 75% decrease in monthly acute migraine medications taken. Patient satisfaction was rated as 70% with the actual device compared with 40% with the sham device. There was no change in nausea, light, and noise sensitivity.

Disclosures: The following people have nothing to

disclose: Huquan Yin, Xiaomei Liang, Joanne M. Ajmo, Brian Finck, Min You BACKGROUND: Alcohol (Al) and weight gain (WG) independently contribute to significant liver disease burden. Concurrent presence of Al and WG can substantially impact liver disease phenotype conceivably by biologically active lipid metabolites. AIMS: To characterize the hepatic lipidome in a mouse model of regular (RAl) and binge (B) alcohol consumption that gains weight BGB324 purchase on western diet (WD) and potential interactions of WG, RAl and binge drinking on disease phenotype. METHODS: Male C57Bl/6 mice received 8 wk chow and WD with and without Al. Another group of WD+RAl fed mice received a weekly alcohol binge while keeping constant the total Al consumed. Molecular lipid species including eicosanoids, ceramides, and sphingolipids were identified by LC/MS lipidomic techniques. Inter-group comparisons were performed using AN〇VA followed by Tukey’s post-hoc tests with adjustment for multiple testing as appropriate. RESULTS: Five groups (Chow, Chow+RAl, WD, WD+RAl and WD+RAl+B) were studied. Mice gained weight on WD (+21. 5%), WD+RAl (+7. 8%) and WD+RAl+B (+10.3%). RAl resulted in mononuclear cell inflammation, perisinusoidal and pericellular fibrosis

while weekly binge induced intense neutrophilic infiltration and 2-fold increase in AST/ALT ratio in WD+RAl+B mice simulating severe alcoholic hepatitis in humans. Lipidomic this website changes: 141/268 (52%) measured lipids were significantly changed in one or more intergroup comparison. Pro-inflammatory lipoxygenase (L〇X) metabolites, 5- and 8-hydroxyeicosatetraenoic (HETE) acids and lipid peroxidation products 9- and 13- hydroxy-octadecadienoic (H〇DE) acid were increased. Interaction effect of RAl:

RAl in WD fed mice with WG dramatically increased hepatic cholesteryl ester (CE) content while a striking increase in diacylglycerol (DAG) species was noted independent of WG. Both 5- and 8-HETE were Decitabine cell line increased in mice fed WD with WG and RAl intake. Interaction effect of RAl+B: Interestingly, RAl or WD alone had only minor effects on hepatic eicosanoids. However, concurrent RAl+B in WD fed mice with WG revealed a consistent trend of increased non-enzymatic eicosanoids (9- & 13-H〇DEs) and a striking significant increase in prostaglandin E2 with RAl (+150%) and RAl+B (+650%). In addition, ceramides were significantly decreased in the RAl+B group and downstream lactoylceramides and other related metabolites showed a consistent increasing trend. CONCLUSION: Concurrent regular alcohol with binge exposure and weight gain on western diet leads to profound pro-inflammatory and oxidative injury compared to weight gain alone. This is mediated via ceramides and related pathways and by eicosanoid metabolites of nonenzymatic and LOX pathways. Disclosures: Puneet Puri – Advisory Committees or Review Panels: Health Diagnostic Laboratory Inc.; Consulting: NPS Pharmaceuticals Inc.

Disclosures: The following people have nothing to

disclose: Huquan Yin, Xiaomei Liang, Joanne M. Ajmo, Brian Finck, Min You BACKGROUND: Alcohol (Al) and weight gain (WG) independently contribute to significant liver disease burden. Concurrent presence of Al and WG can substantially impact liver disease phenotype conceivably by biologically active lipid metabolites. AIMS: To characterize the hepatic lipidome in a mouse model of regular (RAl) and binge (B) alcohol consumption that gains weight GPCR Compound Library high throughput on western diet (WD) and potential interactions of WG, RAl and binge drinking on disease phenotype. METHODS: Male C57Bl/6 mice received 8 wk chow and WD with and without Al. Another group of WD+RAl fed mice received a weekly alcohol binge while keeping constant the total Al consumed. Molecular lipid species including eicosanoids, ceramides, and sphingolipids were identified by LC/MS lipidomic techniques. Inter-group comparisons were performed using AN〇VA followed by Tukey’s post-hoc tests with adjustment for multiple testing as appropriate. RESULTS: Five groups (Chow, Chow+RAl, WD, WD+RAl and WD+RAl+B) were studied. Mice gained weight on WD (+21. 5%), WD+RAl (+7. 8%) and WD+RAl+B (+10.3%). RAl resulted in mononuclear cell inflammation, perisinusoidal and pericellular fibrosis

while weekly binge induced intense neutrophilic infiltration and 2-fold increase in AST/ALT ratio in WD+RAl+B mice simulating severe alcoholic hepatitis in humans. Lipidomic selleck inhibitor changes: 141/268 (52%) measured lipids were significantly changed in one or more intergroup comparison. Pro-inflammatory lipoxygenase (L〇X) metabolites, 5- and 8-hydroxyeicosatetraenoic (HETE) acids and lipid peroxidation products 9- and 13- hydroxy-octadecadienoic (H〇DE) acid were increased. Interaction effect of RAl:

RAl in WD fed mice with WG dramatically increased hepatic cholesteryl ester (CE) content while a striking increase in diacylglycerol (DAG) species was noted independent of WG. Both 5- and 8-HETE were Loperamide increased in mice fed WD with WG and RAl intake. Interaction effect of RAl+B: Interestingly, RAl or WD alone had only minor effects on hepatic eicosanoids. However, concurrent RAl+B in WD fed mice with WG revealed a consistent trend of increased non-enzymatic eicosanoids (9- & 13-H〇DEs) and a striking significant increase in prostaglandin E2 with RAl (+150%) and RAl+B (+650%). In addition, ceramides were significantly decreased in the RAl+B group and downstream lactoylceramides and other related metabolites showed a consistent increasing trend. CONCLUSION: Concurrent regular alcohol with binge exposure and weight gain on western diet leads to profound pro-inflammatory and oxidative injury compared to weight gain alone. This is mediated via ceramides and related pathways and by eicosanoid metabolites of nonenzymatic and LOX pathways. Disclosures: Puneet Puri – Advisory Committees or Review Panels: Health Diagnostic Laboratory Inc.; Consulting: NPS Pharmaceuticals Inc.

5B). Morphologically, tumorigenic EpCAM+ cells showed this website an epithelial cell shape, whereas CD90+ cells showed a mesenchymal VEC shape (Fig. 5C and Supporting Fig. 3C). FACS analysis indicated that P12 HCC cells

showed abundant expression of vascular endothelial growth factor receptor (VEGFR) 1 and a vascular endothelial marker endoglin (CD105) (Fig. 5D). By contrast, P4 and P7 HCC cells did not express these vascular endothelial markers (data not shown). Lung metastasis was detected in NOD/SCID mice transplanted with P12 HCC cells, but not in mice transplanted with P4 and P7 HCC cells (Fig. 5E,F). Taken together, these results suggest that the tumorigenic and metastatic capability of primary HCC may depend on the presence of distinct EpCAM+ or CD90+ CSCs. EpCAM+ cells were associated with a high tumorigenic capacity with hepatic epithelial stem cell features, whereas CD90+ cells were related to the metastatic propensity with VEC PI3K Inhibitor Library features. We previously demonstrated that Wnt/β-catenin signaling

inhibitors could successfully attenuate the tumorigenic capacity of EpCAM+ CSCs in HCC.8, 10 To explore the potential molecular targets activated in CD90+ CSCs, we investigated the expression of the known VEC markers, CD105, VEGFR1 (encoded by FLT1), and c-Kit (encoded by KIT), in cell lines and showed that they were abundantly expressed in CD90+ cell lines, but not EpCAM+ cell lines (Fig. 6A). No expression of VEGFR2 was detected in this set of cell lines, suggesting that molecular reagents specifically targeting VEGFR2 may have no effects on CD90+ CSCs. CD44, a stem cell marker that functionally regulates redox status and is a potential target of CD90+ CSCs, was also abundantly expressed in CD90+ cell lines (Supporting Fig. 4A), consistent with previous data.5, 6-phosphogluconolactonase 13 No significant difference was detected in the expression of the hematopoietic marker, CD34, or ABCG2 between EpCAM+ and CD90+ cell lines (Supporting Fig. 4A). Among these molecular

targets, we focused on the characterization of c-Kit because the c-Kit tyrosine kinase inhibitor, imatinib mesylate, is readily available, is widely used for the treatment of gastrointestinal stromal tumor with activation of c-Kit, and may have potential antitumor activity against a subset of HCC.14 We explored the effect of imatinib mesylate on HCC cell lines and found that treatment with 10 μM reduced cell proliferation and spheroid formation in CD90+ cell lines, but had no effect on EpCAM+ cell lines (Supporting Fig. S4B,C). We further explored the effect of imatinib mesylate in vivo. Because EpCAM+ and CD90+ cells reside in the primary HCC, but not in established cell lines, we SC injected HuH7 and HLF cell lines to generate tumors organized by EpCAM+ and CD90+ CSCs.

On the other hand, animals in Lt/HF group decreased energy expenditure with lost BAT compensations and developed more severe lipid and glucose intolerance, hepatic steatosis, inflammation and hepatocyte ballooning. Contrary to our expectations, cBATX caused a further recruitment of iBAT both in St/C and St/HF groups, which could compensate energy expenditure loss by excised tissue, but not in Lt/HF group. Ad-MSCs population in St/HF group was higher (Sca-1: >95%, CD29: >99%, CD44: >60%, CD105: >40%) with abundant differentiation capacity to WAT and iBAT than that in Lt/HF group. Interestingly,

Ad-MSCs Tx significantly improved body weight gain, energy expenditure loss and findings of NAFLD with the appearance of donor cell derived iBAT in Lt/HF group. Conclusion: Impaired metabolic compensation in adipose tissues resulted in progression of NAFLD, which selleckchem was based on deteriorated Ad-MSCs capacity. New approach targeted this pathway as cell transplantation could be a potent strategy for preventing NAFLD. Disclosures: Kohichiroh Yasui – Grant/Research Support: AstraZeneca K.K., CHUGAI Pharmaceutical Co., Ltd., Dainippon Sumitomo Pharma Co., Ltd., Eisai Co., Ltd., FUJIFILM Medical Co., Ltd., Merck Serono, MSD K.K., Otsuka Pharmaceutical Co., Ltd. Yoshito Itoh – Grant/Research Support: MSD KK, Bristol-Meyers

Squibb, Dain-ippon Sumitomo Pharm. Co., Ltd., Otsuka Pharmaceutical Co., Chugai Pharm Co., Ltd, Mitsubish iTanabe Pharm. Co.,Ltd., SAHA HDAC clinical trial Daiichi Sankyo Pharm. Co.,Ltd., Takeda Pharm. Co.,Ltd., AstraZeneca K.K.:, Eisai

Co.,Pharm.Ltd, FUJIFILM Medical Co.,Ltd. The following people have nothing to disclose: Taichiro Nishikawa, Hisakazu Nakajima, Satoru Sugimoto, Ikuyo Itoh, Kazuki Koudou, Tomoki Nakajima, Kanji Yamaguchi, Michihisa Moriguchi, Yoshio Sumida, Hironori Mitsuyoshi, Masahito Minami The pathogenesis of liver injury and inflammation Etofibrate in nonalcoholic steatohepatitis (NASH) is obscure. We have previously reported that toxic free fatty acids induce hepatocyte lipoapoptosis in vitro by activating the TRAIL receptor (TR). The AIMS of this study were to examine the role of TR-mediated signaling in a murine model of NASH. METHODS: TR knockout (TR−/−) mice, mice with conditional deletion of caspase-8 in hepatocytes (Casp8Δhepa) and wild-type (WT) littermates were fed a diet high in saturated fat, cholesterol and fructose (FFC) or chow for 6 or 3 months, respectively. RESULTS: FFC-fed WT and TR−/− mice had comparable caloric intake and activity. However, the FFC-fed TR−/− mice had reduced liver triglyceride content, lower serum ALT values, and hepatocyte apoptosis by TUNEL assay as compared to WT mice. Moreover, TR−/− mice displayed attenuation of liver fibrosis by mRNA levels of collagen-1a and Sirius red staining.

Those who prefer unhealthier food have higher prevalence of GI symptoms. Conclusions:  These data suggest that psycho-behavioral conditions may affect the development of functional GI symptoms regardless of the subtypes of GI symptoms, and may explain the high proportion of overlap in the subtypes. “
“The necroinflammatory reaction plays a central role in hepatitis B virus (HBV) elimination. Cluster of differentiation (CD)8-positive cytotoxic T lymphocytes (CTLs) are thought to be a main player in the elimination of infected cells, and a recent report suggests that natural

killer (NK) Stem Cells inhibitor cells also play an important role. Here, we demonstrate the elimination of HBV-infected hepatocytes by NK cells and dendritic cells (DCs) using urokinase-type plasminogen activator/severe combined immunodeficiency mice, in which the livers were highly repopulated with human hepatocytes. After establishing HBV infection, we injected human peripheral blood mononuclear cells (PBMCs) into the mice and analyzed liver pathology and infiltrating human immune cells with flow cytometry. Severe hepatocyte degeneration was observed only in HBV-infected mice transplanted with human PBMCs. We provide the first direct evidence that massive

liver cell death can be caused by Fas/Fas ligand (FasL) interaction provided by NK cells activated by DCs. Treatment of mice with anti-Fas antibody completely prevented severe hepatocyte degeneration. Furthermore, severe hepatocyte death can be prevented by CDK inhibitor depletion of DCs, whereas depletion of CD8-positive CTLs did not disturb the development

of massive liver cell apoptosis. Conclusion: Our findings provide the first direct evidence that DC-activated NK cells induce Epothilone B (EPO906, Patupilone) massive HBV-infected hepatocyte degeneration through the Fas/FasL system and may indicate new therapeutic implications for acute severe/fulminant hepatitis B. (HEPATOLOGY 2012) Between 4% and 32% of fulminant hepatitis cases, characterized by acute massive hepatocyte degeneration and subsequent development of hepatic encephalopathy and liver failure, are caused by acute hepatitis B virus (HBV) infection.1 Host2 and viral factors3 may influence the development of fulminant hepatitis, but these factors have not been fully elucidated. Innate and adaptive immunity both play a role in the elimination of viral infections. In the innate immune response, cytoplasmic and membrane-bound receptors recognize viruses and induce interferon (IFN)-β production, which, in turn, up-regulates IFN-α and induces an antiviral state in surrounding cells.4 In the adaptive immune response, viruses are recognized by dendritic cells (DCs), which activate cluster of differentiation (CD)8-positive T cells to reduce viral replication through cytolytic5 and noncytolytic mechanisms.

Rat IEC-6 intestinal epithelial cells and human Caco-2 colon epithelial cells (American Type Culture Collection, Manassas, VA) were maintained in Ham’s F-12 medium containing 10% fetal calf serum. The anti-Hu antigen R (HuR), anti-tristetraprolin (TTP), anti-Auf-1, anti-KSRP antibodies, HuR and TTP small interfering RNA (siRNA) and control Tanespimycin solubility dmso siRNA (siScr) were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). The plasmid construct pcDNA3.1/mHuRcoding-3′ untranslated region (UTR)/Flag contains a wildtype (wt) HuR gene and was a generous gift

from Dr. Beth S. Lee (Ohio State University, Columbus, OH).12 The antihuman ASBT antibody was a generous gift from Dr. Paul Dawson (Wake Forest University School of Medicine, Winston Salem, NC).13 The anti-β-actin antibody was purchased from Sigma (St. Louis, MO). Recombinant plasmids were prepared using standard techniques with details provided in the Supporting Methods. The full-length rat and human ASBT 3′UTR were subcloned into pCRII-TOPO (pCRII-rASBT3′/3.1 and pCRII-hASBT3′/2.1 kb 3′UTR, respectively). Three fragments EGFR inhibitor drugs of the rat ASBT 3′UTR 1155-4270 (3.1 kb, the full 3′UTR region), 2434-4128 (1.7 kb), and 2114-2434 (0.3 kb) (GenBank NM_017222) (Fig. 1) were subcloned into

either a pGL3 vector for luciferase assays or into a β-globin reporter for mRNA half-life determinations. IEC-6 or Caco-2 cells (5 × 105/well) were transfected with 3 μg of the rASBT3′-luciferase construct of interest and 0.1 μg of a quantification

control plasmid pRL-TK containing a thymidine kinase promoter-driven Renilla luciferase gene (Promega, Madison, WI). Transfection and luciferase activity measurements were performed as described.14 Results are expressed as relative light units (RLU), the ratio of firefly to Renilla luciferase activities.15 IEC-6 cells second (5 × 106) were transfected with 30 μg of rASBT3-β-globin hybrid constructs plus or minus 10 μM siHuR for 48 hours before mRNA half-life assays. In addition to withdrawal of serum from the medium, transcription and translation of logarithmically growing cells were terminated by withdrawal of serum and addition of 10 μg/mL actinomycin D and 10 μg/mL cycloheximide. Total cellular RNA was extracted at different times using TRIzol reagent (Invitrogen, Carlsbad, CA). Twenty micrograms of total cellular RNA were analyzed by northern blotting16 with 32P-labeled β-globin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary DNA (cDNA).16, 17 Signal was quantified using a Phosphoimager Molecular Imager FX (Bio-Rad, Hercules, CA). mRNA half-life was determined by linear regression of a natural log transformation against time. The 32P-labeled RNA probes of the 0.3 kb rASBT 3′UTR were synthesized by in vitro transcription of 0.

Rat IEC-6 intestinal epithelial cells and human Caco-2 colon epithelial cells (American Type Culture Collection, Manassas, VA) were maintained in Ham’s F-12 medium containing 10% fetal calf serum. The anti-Hu antigen R (HuR), anti-tristetraprolin (TTP), anti-Auf-1, anti-KSRP antibodies, HuR and TTP small interfering RNA (siRNA) and control MEK inhibitor siRNA (siScr) were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). The plasmid construct pcDNA3.1/mHuRcoding-3′ untranslated region (UTR)/Flag contains a wildtype (wt) HuR gene and was a generous gift

from Dr. Beth S. Lee (Ohio State University, Columbus, OH).12 The antihuman ASBT antibody was a generous gift from Dr. Paul Dawson (Wake Forest University School of Medicine, Winston Salem, NC).13 The anti-β-actin antibody was purchased from Sigma (St. Louis, MO). Recombinant plasmids were prepared using standard techniques with details provided in the Supporting Methods. The full-length rat and human ASBT 3′UTR were subcloned into pCRII-TOPO (pCRII-rASBT3′/3.1 and pCRII-hASBT3′/2.1 kb 3′UTR, respectively). Three fragments Pirfenidone concentration of the rat ASBT 3′UTR 1155-4270 (3.1 kb, the full 3′UTR region), 2434-4128 (1.7 kb), and 2114-2434 (0.3 kb) (GenBank NM_017222) (Fig. 1) were subcloned into

either a pGL3 vector for luciferase assays or into a β-globin reporter for mRNA half-life determinations. IEC-6 or Caco-2 cells (5 × 105/well) were transfected with 3 μg of the rASBT3′-luciferase construct of interest and 0.1 μg of a quantification

control plasmid pRL-TK containing a thymidine kinase promoter-driven Renilla luciferase gene (Promega, Madison, WI). Transfection and luciferase activity measurements were performed as described.14 Results are expressed as relative light units (RLU), the ratio of firefly to Renilla luciferase activities.15 IEC-6 cells Baf-A1 cell line (5 × 106) were transfected with 30 μg of rASBT3-β-globin hybrid constructs plus or minus 10 μM siHuR for 48 hours before mRNA half-life assays. In addition to withdrawal of serum from the medium, transcription and translation of logarithmically growing cells were terminated by withdrawal of serum and addition of 10 μg/mL actinomycin D and 10 μg/mL cycloheximide. Total cellular RNA was extracted at different times using TRIzol reagent (Invitrogen, Carlsbad, CA). Twenty micrograms of total cellular RNA were analyzed by northern blotting16 with 32P-labeled β-globin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary DNA (cDNA).16, 17 Signal was quantified using a Phosphoimager Molecular Imager FX (Bio-Rad, Hercules, CA). mRNA half-life was determined by linear regression of a natural log transformation against time. The 32P-labeled RNA probes of the 0.3 kb rASBT 3′UTR were synthesized by in vitro transcription of 0.