However, despite profound therapeutic implications the prognostic relevance of CSCs and their cellular localization R788 mw within the tumor formation remain controversial. Methods: Expression levels and localization of established CSC markers were assessed in 30 HCCs using qRT-PCR, imunohistochemistry and Western-Blotting. Whole

transcriptome analyses of different tumor regions as well as tumor-surrounding liver (SL) were performed to identify associated signaling pathways and integrated with our existing HCC database. Results: Expression patterns of established CSC markers were surprisingly heterogeneous. Activation of CSCs was predominantly observed in SL and continuously decreased to the tumor core. Consistently, tran-scriptome profiles between SL and different tumor regions were quite distinct. A generated gene expression-signature showed activation of pathways related to proliferation as well as apop-tosis in the tumor tissue, while the invasive tumor margin (TM) was characterized by inflammatory and EMT-related gene sets as well as activation of pro-survival signaling such as ERK and FOS. Consistently, integration of the different signatures with our database of AZD0530 molecular weight 53 HCC revealed that the TM signature was associated with the survival of

HCC patients. Conclusion: CSCs in HCC are heterogeneous. The CSC phenotype is predominantly determined by the permissive tumor microenvironment. However, pro-oncogenic properties might originate in the TM. The activation of key oncogenic features as well as immune-response signaling indicates that

the cross-talk between tumor and microenvironment might be a promising therapeutic and/ or preventive target. Disclosures: Marcus A. Woerns – Advisory Committees or Review Panels: Bayer, Bayer Peter R. Galle – Advisory Committees or Review medchemexpress Panels: Bayer, BMS, Lilly, Daiichi, Jennerex; Consulting: Medimmune; Grant/Research Support: Roche, Lilly; Speaking and Teaching: Bayer, BMS The following people have nothing to disclose: Michael Fischer, Stefan Heinrich, Jesper B. Andersen, Martin F. Sprinzl, Ines Gockel, Snorri S. Thorgeirsson, Hauke Lang, Jens U. Marquardt BACKGROUND & AIMS: Oral supplementation with branched-chain amino acids (BCAA; leucine, isoleucine, and valine) in patients with liver cirrhosis potentially suppresses incidence of hepatocellular carcinoma and improves event-free survival. However, the detailed mechanisms by which BCAA act on hepatic fibrosis have not been fully elucidated. METHODS: BCAA were administered to atherogenic and high-fat (Ath & HF) diet-induced nonalcoholic steatohepatitis (NASH) model mice and platelet-derived growth factor C transgenic mice (Pdgf-c Tg). Liver histology, tumor incidence, and gene expression profiles were evaluated. RESULTS: Ath & HF diet mice developed hepatic tumors at a high frequency at 68 weeks.

Thus, high dietary cholesterol matched with increased intestinal cholesterol absorption both appear to be key and independent risk factors for the formation of cholesterol gallstones.9 This mechanism might be actively operating in subgroups of subjects who are at lower genetic risk of developing gallstones but are victims of environmental dietary factors. Indeed, the potent and selective inhibitor of Decitabine cost NPC1L1 ezetimibe reduced biliary cholesterol secretion by suppressing intestinal cholesterol absorption and protected gallbladder motor function by desaturating bile, thus preventing the formation of cholesterol gallstones in mice.11, 18 The results are straightforward,

since in find more mice NPC1L1 is expressed only in the intestine. In hamsters and humans, however, NPC1L1 is also detected at a significantly lower expression level in the liver compared with the intestine. Because NPC1L1 is a cholesterol transporter that is expressed on the canalicular membrane of hepatocytes, it could function to limit cholesterol excretion, presumably by reabsorbing cholesterol from bile.19 However, it was found that ezetimibe can

significantly reduce hepatic secretion of biliary cholesterol in cholesterol-fed hamsters.20 Furthermore, in gallbladder biles of Mexican patients with gallstones, ezetimibe reduced biliary cholesterol saturation and retarded cholesterol crystallization.11 These results strongly suggest that the secretion efficiency of biliary cholesterol is most likely determined by the net effect between the efflux and influx of cholesterol molecules across the canalicular membrane of hepatocyte, which could be regulated by ABCG5/G8 and the NPC1L1 pathways.11 It is highly likely that because biliary cholesterol secretion is a unique path for excretion of cholesterol from the body

in humans and hamsters, hepatic ABCG5/G8 may play a stronger role in the regulation of biliary cholesterol secretion than NPC1L1. In addition, in the gut-liver axis, the intestinal NPC1L1 plays a significant MCE role in providing dietary and reabsorbed biliary cholesterol to the body, and the inhibition of its functions by ezetimibe significantly reduces cholesterol absorption. Consequently, the bio-availability of cholesterol from intestinal sources for biliary secretion is decreased significantly.9 Moreover, intestinal absorption of dietary cholesterol and reabsorption of biliary cholesterol could play a major role in a subgroup of patients with cholesterol gallstones. Such aspects need to be prospectively investigated. Because of some gallstone patients with increased hepatic de novo cholesterol synthesis, the results of Krawczyk et al. suggested a potential therapeutic role for statins.

Hepatic leukocytes were recovered as described previously. 9 Cells were analyzed by flow cytometry, were cultured for cytokine determination, or were centrifuged onto glass slides with a Shandon Cytospin 2 (Thermo Fisher Scientific, Waltham, MA) for differential cell counting

(300 cells per sample counted). Cells were restimulated ex vivo, stained, and analyzed as described. 9, 12 The employed antibodies were specific for CD4 (clone RM4-5), CD62L (clone MEL-14), CD11b (clone M1/70), chemokine (C-C motif) receptor 9 (CCR9; clone CD-1.2; eBioscience, San Diego, CA), α4β7 (clone DATK32), lymphocyte antigen 6 complex locus G (Ly6-G; clone 1A8), IL-4 (clone 11B11; BD Pharmingen, San Jose, CA), and F4/80 (clone BM8; Caltag, Carlsbad, CA).

Appropriate Erlotinib purchase isotype-matched clones served as controls and were used to set analysis gates. Hepatic leukocytes were restimulated in vitro with medium or somatic larval antigens at 10 μg per well. After 3 days, supernatants were collected, and IL-4 levels were determined by enzyme-linked immunosorbent assay as described. 9 ALT activity was measured in individual serum samples with a commercially available kit from Pointe Scientific (Canton, MI). Liver tissue was fixed in 10% neutral-buffered formalin and embedded in paraffin. Six-micrometer sections were stained with hematoxylin and eosin for microscopic examination. Photomicrographs were created with a BX51 microscope and a DP12 digital camera system from Olympus (Center Valley, PA). Mesenteric lymph nodes from WT mice were obtained 5 days after oral infection. CD4+ T cells were purified by negative selection with the CD4+ T cell isolation 3MA kit from Miltenyi Biotec (Auburn, CA). The percentage of CD4+ cells was determined to be ≥95% by flow cytometry. Cells (2 × 106) in 0.5 mL of phosphate-buffered saline (PBS) or PBS alone were injected intraperitoneally into IL-10 KO recipients 1 day prior to their infection.

In some groups of recipients, a control IgG or α-IL-10R (clone 1B1.3a) antibody (300 μg intraperitoneally every other day beginning 1 day before infection) was administered. Other groups included mice that were given cells and PBS or PBS only. To aid in the interpretation of the effects of the α-IL-10R treatment, we also included a group MCE公司 of WT recipients that were given the same dose and regimen as the IL-10 KO mice. Twelve days later, mice were evaluated for ALT activity, liver histology, hepatic leukocyte content (total, CD4+α4β7+ cells, and Ly6-G+F4/80− cells), and cytokine production. Each experiment was performed three to five times, and each group contained three to five mice. Means and standard deviations were calculated from values obtained from individual mice in a treatment group. Means were compared by the Student t test or analysis of variance followed by an appropriate posttest with GraphPad Prism software (San Diego, CA). Significance was assessed at P < 0.05.

Conversely, VD supplementation has been proposed as an adjunct to current standard cares for treatment of hepatitis C.[60] A clinical study found that 25(OH)D serum levels Ensartinib were significantly lower in chronic

hepatitis C (25 μg/L) than in the controls (43 μg/L).[61] Expression levels of CYP27A1 correlated with 25(OH)D levels, but they were inversely related to necroinflammation. Moreover, low VD is linked to severe fibrosis and impaired sustained virologic response (SVR) in IFN-based therapy. One clinical trial showed that adding VD to the standard IFN plus ribavirin treatment significantly increased SVR in patients with chronic hepatitis C virus (HCV) genotype 1.[62] The SVR was defined as undetectable HCV-RNA at 24 weeks post-treatment.

The increased SVR attained by VD treatment was found to be even better for patients infected with HCV genotypes 2 and 3.[63] Another clinical buy CH5424802 study showed that the levels of VD and of its active form were significantly lower in advanced liver disease (hepatic cirrhosis and/or carcinoma) patients.[64] Conversely, low VD is associated with inflammation, as shown by elevated IL-17 and IL-23 levels in advanced patients. Regarding the underlying molecular mechanisms, an in vitro study showed that vitamin D3 remarkably inhibits HCV production in Huh7.5 hepatoma cells.[65] These cells express VD hydroxlases and can eventually generate calcitriol. Notably, treatment with calcitriol resulted in HCV inhibition through induction of IFN-beta. Intriguingly, HCV infection increased calcitriol production by inhibiting CYP24A1, implying that viral-induced immune tolerance may favor viral chronicity. A recent study surprisingly found that 25-hydroxyvitamin D3, but not 1,25-dihydroxyvitamin D3, is capable of reducing HCV by inhibiting infectious virus assembly.[66] VDBP gene polymorphisms may also determine the VD levels. By examining HCV patients treated with a combination therapy of pegylated interferon alpha (PEG–IFN) plus ribavirin, one study found that good responses to the treatment were related to both the VD levels greater than 20 ng/mL and

the wild-type VDBP polymorphisms.[67] VDR polymorphisms have also been associated with liver diseases, such as primary biliary cirrhosis.[68, 69] A recent clinical study medchemexpress measured the effects of 25-OH VD plasma levels and VDR polymorphisms on fibrosis progression in HCV patients. Results showed that the bAt(CCA)-haplotype was significantly associated with fibrosis progression.[70] VD deficiency or insufficiency is well recognized for the association with variety of chronic degenerative diseases, including chronic hepatitis, viral persistence, ALD, NASH, and poor responsiveness for antiviral treatment. Among its multiple functions, immune modulation/regulation by VD is essential for tissue homeostasis and health physiologic response in addition to its job in calcium adsorption.

Conversely, VD supplementation has been proposed as an adjunct to current standard cares for treatment of hepatitis C.[60] A clinical study found that 25(OH)D serum levels Selleckchem p38 MAPK inhibitor were significantly lower in chronic

hepatitis C (25 μg/L) than in the controls (43 μg/L).[61] Expression levels of CYP27A1 correlated with 25(OH)D levels, but they were inversely related to necroinflammation. Moreover, low VD is linked to severe fibrosis and impaired sustained virologic response (SVR) in IFN-based therapy. One clinical trial showed that adding VD to the standard IFN plus ribavirin treatment significantly increased SVR in patients with chronic hepatitis C virus (HCV) genotype 1.[62] The SVR was defined as undetectable HCV-RNA at 24 weeks post-treatment.

The increased SVR attained by VD treatment was found to be even better for patients infected with HCV genotypes 2 and 3.[63] Another clinical Z IETD FMK study showed that the levels of VD and of its active form were significantly lower in advanced liver disease (hepatic cirrhosis and/or carcinoma) patients.[64] Conversely, low VD is associated with inflammation, as shown by elevated IL-17 and IL-23 levels in advanced patients. Regarding the underlying molecular mechanisms, an in vitro study showed that vitamin D3 remarkably inhibits HCV production in Huh7.5 hepatoma cells.[65] These cells express VD hydroxlases and can eventually generate calcitriol. Notably, treatment with calcitriol resulted in HCV inhibition through induction of IFN-beta. Intriguingly, HCV infection increased calcitriol production by inhibiting CYP24A1, implying that viral-induced immune tolerance may favor viral chronicity. A recent study surprisingly found that 25-hydroxyvitamin D3, but not 1,25-dihydroxyvitamin D3, is capable of reducing HCV by inhibiting infectious virus assembly.[66] VDBP gene polymorphisms may also determine the VD levels. By examining HCV patients treated with a combination therapy of pegylated interferon alpha (PEG–IFN) plus ribavirin, one study found that good responses to the treatment were related to both the VD levels greater than 20 ng/mL and

the wild-type VDBP polymorphisms.[67] VDR polymorphisms have also been associated with liver diseases, such as primary biliary cirrhosis.[68, 69] A recent clinical study MCE公司 measured the effects of 25-OH VD plasma levels and VDR polymorphisms on fibrosis progression in HCV patients. Results showed that the bAt(CCA)-haplotype was significantly associated with fibrosis progression.[70] VD deficiency or insufficiency is well recognized for the association with variety of chronic degenerative diseases, including chronic hepatitis, viral persistence, ALD, NASH, and poor responsiveness for antiviral treatment. Among its multiple functions, immune modulation/regulation by VD is essential for tissue homeostasis and health physiologic response in addition to its job in calcium adsorption.

Conversely, VD supplementation has been proposed as an adjunct to current standard cares for treatment of hepatitis C.[60] A clinical study found that 25(OH)D serum levels BAY 57-1293 cost were significantly lower in chronic

hepatitis C (25 μg/L) than in the controls (43 μg/L).[61] Expression levels of CYP27A1 correlated with 25(OH)D levels, but they were inversely related to necroinflammation. Moreover, low VD is linked to severe fibrosis and impaired sustained virologic response (SVR) in IFN-based therapy. One clinical trial showed that adding VD to the standard IFN plus ribavirin treatment significantly increased SVR in patients with chronic hepatitis C virus (HCV) genotype 1.[62] The SVR was defined as undetectable HCV-RNA at 24 weeks post-treatment.

The increased SVR attained by VD treatment was found to be even better for patients infected with HCV genotypes 2 and 3.[63] Another clinical Erastin study showed that the levels of VD and of its active form were significantly lower in advanced liver disease (hepatic cirrhosis and/or carcinoma) patients.[64] Conversely, low VD is associated with inflammation, as shown by elevated IL-17 and IL-23 levels in advanced patients. Regarding the underlying molecular mechanisms, an in vitro study showed that vitamin D3 remarkably inhibits HCV production in Huh7.5 hepatoma cells.[65] These cells express VD hydroxlases and can eventually generate calcitriol. Notably, treatment with calcitriol resulted in HCV inhibition through induction of IFN-beta. Intriguingly, HCV infection increased calcitriol production by inhibiting CYP24A1, implying that viral-induced immune tolerance may favor viral chronicity. A recent study surprisingly found that 25-hydroxyvitamin D3, but not 1,25-dihydroxyvitamin D3, is capable of reducing HCV by inhibiting infectious virus assembly.[66] VDBP gene polymorphisms may also determine the VD levels. By examining HCV patients treated with a combination therapy of pegylated interferon alpha (PEG–IFN) plus ribavirin, one study found that good responses to the treatment were related to both the VD levels greater than 20 ng/mL and

the wild-type VDBP polymorphisms.[67] VDR polymorphisms have also been associated with liver diseases, such as primary biliary cirrhosis.[68, 69] A recent clinical study MCE公司 measured the effects of 25-OH VD plasma levels and VDR polymorphisms on fibrosis progression in HCV patients. Results showed that the bAt(CCA)-haplotype was significantly associated with fibrosis progression.[70] VD deficiency or insufficiency is well recognized for the association with variety of chronic degenerative diseases, including chronic hepatitis, viral persistence, ALD, NASH, and poor responsiveness for antiviral treatment. Among its multiple functions, immune modulation/regulation by VD is essential for tissue homeostasis and health physiologic response in addition to its job in calcium adsorption.

2%)]. Because there is no specific objective marker of JQ1 ic50 drug-induced liver disease, an accurate diagnosis of hepatotoxicity has constantly been challenging. DILI must always be considered, however, when there is a temporal association between observed liver injury and the receipt

of a drug. The warning signal has been either an acute onset of clinical symptoms (e.g., rash, fever, abdominal pain, or jaundice) or, more commonly, biochemical dysfunction, which includes raised levels of ALT, AST, AP, gamma-glutamyl transpeptidase, and/or serum bilirubin.1, 19, 20 Although these abnormalities strongly suggest liver disease, they are in fact nonspecific indicators and, moreover, do not provide an etiological diagnosis. The use of expert opinion to identify DILI has long been regarded as the gold standard for diagnosing hepatotoxicity, especially when reports come

from well-recognized authorities rather than from an inexperienced occasional observer.2-5, 21, 22 However, because this approach is subjective and lacks defined criteria, a group of international experts convened a meeting under the auspices of the Council for International Organizations of Medical Scientists with the goal of introducing structure and uniformity to the causality process selleck kinase inhibitor through the development of highly defined diagnostic criteria for drug-induced liver disease. The meeting was supported by Roussel-Uclaf Pharmaceuticals,

and hence the instrument is called RUCAM. The strategy awards points for seven different domains.10 Other attempts have been made to develop causality instruments with the hope of simplifying the adjudication process,11, 23 but the value of 上海皓元 some has been questioned24; this has left RUCAM as the preferred causality instrument. Although used by some experts in the field and often referred to in discussing DILI causality, RUCAM has not been adopted in general clinical practice or, in fact, by most practicing hepatologists and gastroenterologists. The chief reason is that it is a time-consuming process with insufficient and sometimes confusing information on how to score some of the elements of its domains. Nevertheless, during the planning for DILIN, the decision was made to use and compare two approaches for establishing causality: a refined and highly structured expert opinion method and the RUCAM instrument. A direct comparison of the DILIN structured expert opinion and RUCAM revealed that the DILIN process was more likely than RUCAM to generate a score supportive of drug-induced liver disease. Using the DILIN system, reviewers scored 73% of cases (405/557) as definite or highly likely, whereas only 24% of the cases (132/557) were scored as highly probable in the corresponding RUCAM category (Table 6).

2%)]. Because there is no specific objective marker of Neratinib manufacturer drug-induced liver disease, an accurate diagnosis of hepatotoxicity has constantly been challenging. DILI must always be considered, however, when there is a temporal association between observed liver injury and the receipt

of a drug. The warning signal has been either an acute onset of clinical symptoms (e.g., rash, fever, abdominal pain, or jaundice) or, more commonly, biochemical dysfunction, which includes raised levels of ALT, AST, AP, gamma-glutamyl transpeptidase, and/or serum bilirubin.1, 19, 20 Although these abnormalities strongly suggest liver disease, they are in fact nonspecific indicators and, moreover, do not provide an etiological diagnosis. The use of expert opinion to identify DILI has long been regarded as the gold standard for diagnosing hepatotoxicity, especially when reports come

from well-recognized authorities rather than from an inexperienced occasional observer.2-5, 21, 22 However, because this approach is subjective and lacks defined criteria, a group of international experts convened a meeting under the auspices of the Council for International Organizations of Medical Scientists with the goal of introducing structure and uniformity to the causality process buy LY294002 through the development of highly defined diagnostic criteria for drug-induced liver disease. The meeting was supported by Roussel-Uclaf Pharmaceuticals,

and hence the instrument is called RUCAM. The strategy awards points for seven different domains.10 Other attempts have been made to develop causality instruments with the hope of simplifying the adjudication process,11, 23 but the value of 上海皓元医药股份有限公司 some has been questioned24; this has left RUCAM as the preferred causality instrument. Although used by some experts in the field and often referred to in discussing DILI causality, RUCAM has not been adopted in general clinical practice or, in fact, by most practicing hepatologists and gastroenterologists. The chief reason is that it is a time-consuming process with insufficient and sometimes confusing information on how to score some of the elements of its domains. Nevertheless, during the planning for DILIN, the decision was made to use and compare two approaches for establishing causality: a refined and highly structured expert opinion method and the RUCAM instrument. A direct comparison of the DILIN structured expert opinion and RUCAM revealed that the DILIN process was more likely than RUCAM to generate a score supportive of drug-induced liver disease. Using the DILIN system, reviewers scored 73% of cases (405/557) as definite or highly likely, whereas only 24% of the cases (132/557) were scored as highly probable in the corresponding RUCAM category (Table 6).

Mean displacement (MD) values in white NVP-BEZ235 molecular weight matter (WM), gray matter (GM), and lateral ventricle (cerebrospinal fluid [CSF]) of normal subjects,

plaques, and normal appearing WM (NAWM) of MS subjects and glioma lesions were calculated. Mann-Whitney U test was used for comparison. In normal subjects, MD values were 6.6 ± 0.2, 8.44 ± 0.41, and 17.08 ± 0.80 μm for WM, GM, and CSF, respectively, while those for NAWM and WM plaques in MS, and glioma lesions were significantly higher at 7.0 ± 0.17, 9.3 ± 2.3, and 9.6 ± 0.40 μm, respectively, compared to WM in normal subjects. We propose that the relative values of MD obtained by QSI in control and diseased tissues can be useful for diagnosing various WM abnormalities. “
“To evaluate the safety of thrombolysis with rt-PA in acute ischemic strokes during GSK1120212 mouse a 12-hour time window using an ultrafast MR protocol. Forty-six patients

who met the clinical criteria (acute ischemic stroke within 12 hours after symptom onset; National Institutes of Health stroke scale score (NIHSS) of 4 to 22 and no intracranial hemorrhage on CT) and MRI selection criteria (acute ischemic stroke except lacunar and large DWI lesion) were treated with intravenous rt-PA. MRI was performed before rt-PA, and at 24 hours, 7 days, and 14 days after stroke. Clinical status was assessed using the NIHSS and Modified Rankin scale (mRS). From 46 MRI-selected rt-PA patients, 43.5% (n= 20) were treated ≤3 hours (group A) and 56.5% (n= 26) after 3 to 12 hours (group B). No patients experienced symptomatic MCE公司 intracranial hemorrhage and the mortality rate was zero. No significant differences in age, gender, MRI lesion volumes, NIHSS score, and mRS were found between the 2 groups. Forty-five percent of the patients in group A and 46% in group B experienced a favorable outcome (P= .938). Our results demonstrated the safety of thrombolysis with rt-PA in selected stroke patients within a 12-hour time window using an ultrafast MR protocol. Neuroimaging 2011;21:370-374. “
“Real-time functional MRI feedback (RTfMRIf) is a developing technique, with

unanswered methodological questions. Given a delay of seconds between neural activity and the measurable hemodynamic response, one issue is the optimal method for presentation of neurofeedback to subjects. The primary objective of this preliminary study was to compare the methods of continuous and intermittent presentation of neural feedback on targeted brain activity. Thirteen participants performed a motor imagery task and were instructed to increase activation in an individually defined region of left premotor cortex using RTfMRIf. The fMRI signal change was compared between real and false feedback for scans with either continuous or intermittent feedback presentation. More individuals were able to increase their fMRI signal with intermittent feedback, while some individuals had decreased signal with continuous feedback.

,22 and siRNA against GFP was designed from RiboBio Co., Ltd. (Guangzhou, China). The inhibitor of Myc/Max dimerization 10058-F4 (sc-213577) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For transfection, plasmids (1-5 μg), siRNAs (100 nM final concentration), miRNA mimics (50 nM final concentration), or miRNA inhibitors (100 nM final concentration) were transfected into appropriate cells using Lipofectamine

2000 (Invitrogen), 48 hours after transfection cell pellets were collected Selleckchem STI571 and subjected to RNA isolation and immunoblot analysis. RNA was extracted using TRIzol (Invitrogen) from the indicated cell lines according to the manufacturer’s protocol. DNA contamination was removed with RNAse-free DNase I. All reagents

for stem-loop RT were obtained from Promega, Inc. (Madison, WI) and RiboBio. PCR products were analyzed on 3% agarose gels. Small RNA U6 was used as an internal control. Real-time PCR was performed with SYBRGreen (Bio-Rad). Primers and other reagents of mature miRNA assays were purchased from RiboBio. Primers of real-time PCR for other genes are listed in Supporting Table 1. Chromatin immunoprecipitation (IP) assays were performed according to Yi et al.23 Briefly, intracellular protein-DNA complexes were cross-linked in situ by the addition of 1% of formaldehyde. Total lysates were then sonicated and subjected to chromatin-conjugated IP using specific antibodies. After reversal of cross-links, Pembrolizumab clinical trial precipitated DNA was purified and analyzed by real-time PCR with specific primers (Supporting Table 1.). Cell cycle analyses were performed on propidium iodide–stained 上海皓元医药股份有限公司 nuclei using a MoFlo XDP-Flow Cytometer (Beckman Coulter, Inc). Data were analyzed by single-histogram statistics.20, 24 For colony assays, 1 × 103 cells of the indicated type were plated in triplicate in soft agar (0.35% low melting point agarose on top of 0.7% bottom agarose) in six-well plates and fed intermittently with DMEM. Colonies were

enumerate after 2 weeks by staining with methylene blue after methanol fixation.25 Four-week-old male BALB/c nude mice were purchased from Shanghai SLAC Laboratory Animal Co. and maintained in microisolator cages. Tumorigenicity assays and tumor volume measurements were performed as previously described.20 Briefly a total of 1 × 107 indicated cells were suspended in 100 μL serum-free DMEM and injected subcutaneously in the flanks of animals. Tumor growth was monitored every three days for a total period of 30 to 40 days. Tumor volumes were calculated by the equation V (mm3) = a × b × c/2, where a is the length, b is the width, and c is the height. Informed consent was obtained at the Union Hospital in Wuhan and at the Eastern Hepatobiliary Surgery Hospital in Shanghai, China. The diagnosis of HCC was confirmed in each case by histological reviews. None of the patients received chemotherapy prior to hepatectomy. Co-IP was performed as described.