All nicotine doses are expressed as free base concentrations. Saline or nicotine was injected subcutaneously (s.c., 0.05 ml/10 g body weight) 5 min before the test. Experimental Design Cognitive, Affective, and Locomotor Behaviors All behavioral procedures are described in detail in the Supplementary material online. Two cohorts of mice were tested, one (15 ��4+/+ and 14 customer reviews ��4?/? females, 10 ��4+/+ and 11 ��4?/? males) in a battery of cognitive tasks and the other (9 ��4+/+ and 9 ��4?/? females, 8 ��4+/+ and 11 ��4?/? males) to replicate the cognitive tests and examine locomotor activity and affective-like behaviors. For behavioral testing, a within-subjects design was used so that mice of each genotype were subjected to testing in the following order: Y maze, Barnes maze training, Barnes maze probe test, cue- and context-induced conditioned fear, Barnes maze retention, Barnes maze reversal, conditioned fear retention.

After the completion of the cognitive test battery, the second mouse cohort was tested in additional procedures assessing locomotor activity and affective-like behavior. Again, a within-subjects design was employed such that mice of each genotype were subjected to testing in the following order: light�Cdark transfer test, marble burying test, locomotor activity test, tail suspension test, and forced swim test. Nicotine-Induced Analgesia Two additional cohorts of na?ve ��4+/+ and ��4?/? mice were tested for nicotine-induced analgesia using the tail immersion and hot plate tests (see Supplementary material online for details).

The first cohort contained 21 ��4+/+ and 21 ��4?/? females, and the second cohort contained 12 ��4+/+ and 15 ��4?/? females, as well as 17 ��4+/+ and 28 ��4?/? males. In each cohort, mice were first tested in the tail immersion test. Nicotine (0, 0.5, 1, 2, and 3 mg/kg, s.c., 5 min before test) was administered according to a within-subjects Latin square design during 5 consecutive days. One week later, the same mice were tested in the hot plate test. For each sex and genotype, mice were split into three subgroups, and nicotine (0, 1, and 3 mg/kg, s.c., 5 min before test) was administered according to a between-subjects design. Each mouse was only tested once in the hot plate test. Statistical Analyses Statistical analyses were performed using the Biomedical Computer Programs for Personal Computers Statistical Package (BMDP, Los Angeles, CA), the GraphPad Prism statistical package (GraphPad, San Diego, CA), and Statview v.

5.0 (SAS Institute Inc., Cary, NC). Two-way repeated-measures analysis of variance (ANOVA) was used to analyze the data collected from the cognitive test battery. The between-subject factors were Genotype and Sex, and the within-subject factors were Time Intervals (cued and contextual conditioning) and Trials/Blocks (the Barnes maze Carfilzomib test).

Within this group, 53% (10/19) of those who tested positive at intake also tested positive at FAP. Within the group where both intake and FAP specimens were available, of the 20 that ever tested drug positive, half showed the same usage at both timepoints (eight positive for THC, one for both THC and opioids, and one for benzodiazepines). Additionally, one woman tested customer reviews positive for cocaine and opioids, and another for THC, at FAP but not intake. Seven women tested positive for THC at intake, but not at FAP. Effects of Drug Use on Smoking Cessation No significant differences were found in smoking abstinence rates between those who ever versus never tested drug positive across the two assessments (CMH ��2 (1) = 0.07, p = .79) or at FAP (CMH ��2 (1) = 2.78, p = .10).

There was no evidence that the effect of drug use on smoking abstinence was heterogenic across treatment groups at any time during pregnancy (Breslow�CDay ��2(1) = 0.33, p = .57) or at FAP (Breslow�CDay ��2(1) = 0.77, p = .38). Although differences in smoking abstinence for those testing drug-positive at FAP is not significant, 72% (13/18) of those who tested positive for drug use at the FAP assessment were also positive for cotinine (i.e., recent cigarette smoking) as compared with 51% (28/55) of those testing drug negative. CONCLUSIONS The present results demonstrate that illicit drug use, especially marijuana, is common in a sample of pregnant women enrolled in smoking-cessation treatment. The approximately 30% prevalence rate for marijuana use in this study corresponds well with the 23% of pregnant smokers reporting use in the last month in a U.

S. national survey of drug use (SAMHSA, 2010) and is considerably higher than the 2%�C4% of pregnant women in general who report marijuana use (e.g., Havens, Simmons, Shannon, & Hansen, 2009). Additionally, the majority of women who tested positive for illicit drug use at the beginning of their pregnancy in this study also tested positive near the end of their pregnancy, suggesting ongoing use. It should be noted that our measurements of drug use likely underestimate illicit use in pregnant smokers, as those who were receiving opioid-maintenance therapy (buprenorphine or methadone) were excluded from the current trials as opioids are known to alter smoking rates (e.g., Mello, Mendelson, Sellers, & Kuehnle, 1980).

As opioid-maintained pregnant smokers have relatively high rates of illicit drug use (Choo, Huestis, Schroeder, Shin, & Cilengitide Jones, 2004; Haug, Stitzer, & Svikis, 2001; Jones et al., 2009), any trial that excludes them will likely underestimate illicit drug use in pregnant smokers. One concern about drug use during pregnancy is possible detrimental effects on smoking cessation. While this study was not powered to investigate effects on treatment success, many abused drugs are known to increase smoking rates (e.g.

Using a variety Z-VAD-FMK of smoking status definitions (typically nondaily or 1�C5 CPD), existing longitudinal studies suggest that the lightest smokers are more likely than heavier smokers to either increase or decrease consumption (Hassmiller, Warner, Mendez, Levy, & Romano, 2003; Hennrikus, Jeffery, & Lando, 1996; Hyland, Rezaishiraz, Bauer, Giovino, & Cummings, 2005; Lindstrom & Isacsson, 2002; McDermott, Dobson, & Owen, 2007; Stanton, Papandonatos, Lloyd-Richardson, & Niaura, 2007; Wetter et al., 2004; Zhu, Sun, Hawkins, Pierce, & Cummins, 2003). However, these studies are of limited utility for identifying factors associated with transitions in smoking status in the U.S. context. They focus on non-U.S. populations (Lindstrom & Isacsson, 2002; McDermott et al., 2007) or narrowly defined U.S.

populations (e.g., adolescents, college students, or employees; Hennrikus et al., 1996; Stanton et al., 2007; Wetter et al., 2004), lack a prospective design (e.g., using recall of prior year’s smoking; Hassmiller et al., 2003), or do not focus on determinants of increased or decreased cigarette consumption (Hyland et al., 2005; Zhu et al., 2003). We addressed these research gaps by analyzing survey data from a recent large population-based cohort of adult smokers in Massachusetts who were interviewed three times over a 4-year follow-up period. We disaggregated smokers into four groups by consumption (CPD) and frequency (daily vs. nondaily smoking), examined changes in smoking status over time in each of these groups, and identified factors associated with progression to heavier smoking and to quitting or reduction for different groups of light smokers (defined in this study as smoking ��10 CPD).

Methods Sample A probability-sample random-digit�Cdialed survey was administered to residents of Massachusetts in 2001�C2002 to study smoking practices, attitudes, and support for tobacco control policies in the state. The survey oversampled smokers, young adults, and recent quitters to ensure adequate power to study those subpopulations. Professional interviewers from the University of Massachusetts�CBoston Center for Survey Research attempted to interview one systematically selected adult aged at least 18 years in each eligible household identified through an initial screening interview with an adult household resident.

They successfully screened 66% of sampled households and interviewed 70% of selected adults for a final sample size at baseline of 6,739. All 3,083 respondents who indicated at baseline that they had smoked at least 100 cigarettes in their lives and currently smoked every day or some days were contacted again 2 and 4 years later. Of the Drug_discovery baseline smokers, 1,726 (56.0%) completed follow-up interviews in 2003�C2004 and 1,319 (42.8%) completed interviews in 2005�C2006.

Okuyemi, Faseru, Sanderson Cox, Bronars, and Ahluwalia (2007) compared smoking abstinence rates for nonmenthol and menthol cigarette smokers among 755 Blacks in a double-blind, placebo-controlled randomized trial examining placebo check details and nicotine gum. All participants were light smokers with a rate of ��10 cigarettes/day, and 81.7% smoked menthol cigarettes. At 26 weeks postrandomization, nonmenthol smokers were more likely to quit than menthol cigarette smokers, 18.8% and 11.2%, respectively (p = .015). Data from the 2005 U.S. National Health Interview Survey were used to examine the relationship between race/ethnicity, menthol smoking, and cessation in a nationally representative sample of 7,815 adults who were current and former cigarette smokers and had made a quit attempt (Gundersen, Delnevo, & Wackowski, 2009).

The proportion of the total sample who were menthol cigarette smokers was 26.5%. Findings indicated that the association between menthol smoking and cessation differed between Whites and Blacks and Whites and Hispanics. Non-White menthol smokers were significantly less likely to have quit smoking (adjusted odds ratio [AOR] = 0.55, p < 0.01) compared with their nonmenthol smoking counterparts. In contrast, among Whites, menthol smokers were more likely to be former smokers than nonmenthol smokers (AOR = 1.17, p < 0.05). In the Coronary Artery Risk Development in Young Adults (CARDIA) Study, smoking cessation behavior and risk factors for coronary artery disease were measured from 1985 to 2000 among 1,544 smokers aged 18�C30 years at enrollment.

Among Blacks, 89% preferred menthol cigarettes compared with 29% of European Americans (Pletcher et al., 2006). While there were no differences in cessation rates by cigarette type, there was a significant increase in relapse risk among menthol versus nonmenthol cigarette smokers (OR = 1.89, p = .009). In a secondary analysis of a bupropion clinical trial with 600 Black smokers, nonmenthol smokers less than 50 years of age were more likely to have quit smoking (OR = 2.0, CI = 1.03�C3.95) at 6 weeks postenrollment than menthol cigarette smokers (Okuyemi et al., 2003). The sample was composed of 78.5% menthol cigarette smokers and 22% nonmenthol smokers. In summary, five of these eight studies assessing smoking cessation and menthol cigarette use provide evidence of lower quit rates or higher relapse rates among menthol cigarette smokers.

Studies with negative findings were with unique samples of female prisoners, male veterans, and individuals with chronic Carfilzomib lung impairment. Discussion The sensory effects of menthol cigarettes in increasing the reinforcing effects of nicotine in cigarettes was evidenced in studies that investigated the qualitative sensory effects of menthol cigarettes, subjective measures of menthol cigarette use on nicotine dependence, and the impact of menthol cigarette use on smoking cessation.

, 2010; Picciotto et al., 2002; Shytle et al., 2002). Many studies in our review excluded smokers taking antidepressants. selleck inhibitor We previously found that few studies examine antidepressant drug response by smoking status (Weinberger, McKee, Picciotto, & Mazure, 2011) and, conversely, few studies examine the impact of antidepressants on smoking cessation outcomes although preliminary data suggest that smokers taking antidepressants have more trouble quitting (Gravely-Witte, Stewart, Suskin, & Grace, 2009; Japuntich et al., 2007; Weinberger, McKee, & George, 2012). It remains unclear how smokers taking antidepressants respond to pharmacological and behavioral smoking cessation treatments that have been found to be effective in general populations (Fiore et al., 2008).

Second, few studies have examined the interactive impact of gender and depression on smoking cessation outcomes consistent with reviews showing that smoking treatment research rarely examine outcomes by gender (Dickerson et al., 2009; Piper et al., 2001). Women appear to have more trouble quitting smoking than men (Perkins, 2001; Perkins & Scott, 2008; Schnoll, Patterson, & Lerman, 2007; Wetter et al., 1999) and the majority of studies in this review that considered gender reported that depression had a greater negative impact on smoking cessation outcomes for women as compared with men. Gender differences were found for several pharmacological agents (e.g., naltrexone, clonidine, and nortriptyline; Covey et al., 1999; Glassman et al., 1993; Hall et al.

, 1998) and additional research examining gender difference is needed for both pharmacological and behavioral treatments. Our analyses of epidemiological data (Weinberger et al., 2012a, 2012b) found that overall smoking cessation rates over a 3-year period in the general U.S. adult population were similarly impacted by depression for men and women, however, depression would still be expected to have a greater impact on female smokers due to the fact that women experience depression at higher rates than men (Grant et al., 2004). At this time, treatments and treatment-related variables that can be used to best help women with depression to successfully quit smoking are still not identified. Third, no study included in this review examined racial differences in the relationship between depression and smoking cessation.

Most studies included a small number of participants from minority groups, or included samples that were entirely from a minority population, both of which preclude GSK-3 the statistical analysis of outcomes by race. One study published in 2011 (Castro et al., 2011) found that higher baseline depressive symptoms were associated with lower quit rates at 1, 2, and 4 weeks for Caucasian and African-American smokers, but not Hispanic smokers, in a sample of 389 adults (n = 133 Caucasian, n = 130 African Americans, n = 126 Hispanic) receiving counseling, self-help materials, and transdermal nicotine patch.

The amplified ISX cDNA product then was cloned into the pCDNA 3.1 V5/His TOPO vector by following the manufacturer��s instructions (Invitrogen) and transformed into selleck bio TOP10 cells according to the manufacturer��s instructions (Invitrogen). Positive clones were selected and subjected to plasmid DNA minipreparation according to manufacturer��s instructions (Qiagen, Valencia, CA, USA). Finally, appropriate construction of wild-type (WT) ISX ORF (pISX-WT) in the pCDNA 3.1 V5/His TOPO vector was verified by sequence analysis on both strands (Genomics Core Sequencing Facility, Case Western Reserve University, Cleveland, OH, USA). Indirect immunofluorosence and confocal microscopy For immunostaining experiments, CaCo-2 cells were seeded at a density of 2 �� 105 cells on coverslips in 6-well plates and allowed to adhere for 24 h.

Cells were treated with RA (1 ��M) or control vehicle (ethanol) and incubated for 12 h. Then cells were fixed in a freshly prepared mixture of 4% formalin in phosphate buffered saline (PBS; pH 7.4) for 20 min at room temperature. After multiple washes with PBS, cells were incubated with blocking buffer (2% BSA and 0.2% Triton-X 100 in PBS) for 15 min at room temperature. The primary polyclonal rabbit antiserum anti-ISX prepared at a 1:500 dilution in blocking buffer was then added to these cells and incubated for 1 h at room temperature. The primary antibody was removed, and cells were washed gently 3 times with PBS for a total of 30 min. The secondary antibody anti-rabbit conjugated Alexa 488 was diluted 1:500 in blocking buffer, added to the cells, and incubated for 1 h at room temperature in darkness.

After further washing in PBS, coverslips with cells were mounted facedown onto glass slides (Labtek, Scotts Valley, CA, USA) with a small drop of ProLong Gold antifade mounting medium containing DAPI (Molecular Probes). The next day, cells were examined at room temperature under a Zeiss LSM 510 UVMETA confocal microscope with an HCX Plan ��40 numerical aperture 1.4 oil-immersion objective lens (Zeiss, Jena, Germany). Images were acquired with Zeiss Anacetrapib confocal software version 2.0. All experiments were performed in triplicate, and ~100 cells/experiment were counted. ChIP assay and PCR CaCo-2 cells were cultured in 150-cm2 dishes and, on 90% confluence, were treated with 1% formaldehyde at 37��C with gentle swirling for 10 min to enable crosslinking of nuclear proteins with genomic DNA. Then the ChIP assay, to evaluate binding of RARs to the ISX promoter, was performed essentially as described by the manufacturer (Millipore). About 4 ��g of RAR antibody (M-454; Santa Cruz Biotechnologies) was used for immunoprecipitation, and IgG was used as the negative control.

Livers from animals administered with AAV were collected at P90 and levels of transgene expression were assessed by both analysis of eGFP-positive cells on cryo-sections (Fig. 1A and Fig. Afatinib clinical S1A) and anti-eGFP Western blot (Fig. 1B, Fig. S1B and Fig. S2A). We observed a higher number of eGFP-expressing cells (Fig. 1A, right panels and Fig. S1A) and a stronger (but not significantly different) intensity of eGFP bands (Fig. 1B, P90 white and grey bars, Fig. S1B and Fig. S2A) in livers from animals injected at P30 compared to those injected at P4. Concordantly, the number of AAV genome copies/molecule of diploid genome (gc/mdg) was significantly higher in livers from animals injected at P30 than in those injected at P4 (Fig. 1C, P90 white and grey bars).

Figure 1 AAV2/8 administration to newborn rats is associated with vector genome dilution. To confirm that the reduced amount of vector genomes and the lower transgene expression levels observed in livers of rats injected at P4, as opposed to those injected at P30, were due to the proliferation-related dilution of episomal AAV vector genomes, we injected rats at P4 with AAV2/8-TBG-eGFP and collected livers at P15, P30 and P90. We observed the highest number of eGFP-positive cells (Fig. 1A), highest eGFP expression levels (Fig. 1B and Fig. S2B) and vector gc/mdg (Fig. 1C) in livers collected at P15. These were reduced in animals sacrificed at later time points with the strongest reduction observed between P15 and P30.

In addition, the eGFP-positive cells in livers collected at P90 appeared in clusters when rats were injected at P4 but not at P30 (data not shown), possibly representing clones from a single cell containing integrated vector genomes, as previously described in mice injected at birth with AAV2/8 [30]. Thus, as previously reported [9], [30], [31], hepatocyte proliferation results in dilution of AAV vector genomes in transduced cells and reduced efficacy of long-term liver transduction in newborn rats. To test if AAV vector genome dilution occurs in liver of adult animals, we injected rats with AAV2/8-TBG-eGFP at P30 and collected their livers at P41 and at P90. Western blot analysis showed in some but not all the livers collected at P41 slightly higher levels of eGFP expression than at P90 (Fig. S2C). However the quantification Batimastat of the corresponding eGFP band intensity did not evidence statistically significant differences (Fig. 1B, white and dark grey bars). Thus, although we can’t exclude that in some animals vector dilution occurs in liver of rats injected at P30, overall this was not significant. Interestingly, the amount of liver vector genomes at P41 is significantly higher than at P90 (Fig. 1C, white and dark grey bars).

9% (Table 2). Compared with participants who did not have peripheral artery disease, participants with peripheral artery disease kinase inhibitor Tipifarnib were more likely to be women, older, less educated, Black, and either former or current smokers. They were also more likely to have a lower estimated glomerular filtration rate (eGFR), diabetes mellitus or hypertension, and higher serum cotinine and blood cadmium concentrations (Table 2). Table 2. Participant Characteristics by the Presence or Absence of PAD Peripheral Artery Disease After adjustment for demographics and risk factors for cardiovascular disease, the odds ratios for peripheral artery disease were 1.98 (95% CI: 1.41, 2.79), 5.23 (95% CI: 3.40, 8.04), and 3.36 (95% CI: 1.86, 6.10) for former, nonmenthol, and menthol smokers, respectively, compared to never-smokers (Table 3, Model 2).

Further adjustment for pack-years of smoking and serum cotinine attenuated the odds ratios for peripheral artery disease to 1.44, 3.65, and 2.51 for former, nonmenthol, and menthol smokers, respectively, compared to never-smokers (Table 3, Model 3). We observed no significant difference in the association between smoking and peripheral artery disease for current smokers of nonmenthol and menthol cigarettes (p value for heterogeneity = .59). Adjustment for blood cadmium concentrations attenuated the odds ratios for peripheral artery disease by 6%, 25%, and 28% for former, nonmenthol, and menthol smokers, respectively, compared to never-smokers (Table 3, Model 4) Table 3.

Odds Ratios (95% CI) of Periphery Artery Disease (PAD) by Smoking Status After adjustment for demographics, cardiovascular risk factors, smoking status, pack-years of smoking, and serum cotinine, the odds ratios for peripheral artery disease were 2.35 (95% CI: 1.54, 3.59) and 0.74 (95% CI: 0.51, 1.07), respectively, for Black participants and for participants of other races compared to those for White participants (Table 4, Model 3). After further adjustment for current menthol cigarette smoking, the odds ratio for peripheral artery disease for Blacks increased to 2.47 (95% CI: 1.62, 3.77), with no change for other races compared to White participants (Table 4, Model 4). Table 4. Odds Ratios (95% CI) of Periphery Artery Disease (PAD) by Race/Ethnicity DISCUSSION In a representative sample of U.S.

adults who participated in NHANES 1999�C2004, we found increased odds of peripheral artery disease for former and current smokers compared to never-smokers, with no difference in risk between current menthol and nonmenthol cigarette smokers. Adjustment for blood cadmium concentrations attenuated the association between current smoking and peripheral artery disease similarly for Cilengitide smokers of menthol and nonmenthol cigarettes, suggesting that it contributes similarly for both types of cigarette smokers. Smoking is known to be a major risk factor for peripheral artery disease (U.S. Department of Health and Human Services, 2004).

Hepatocytes are known to be in a quiescent status, and the toxic triphosphate processes by the suicide gene HSV-TK after selleck screening library GCV administration theoretically need cell replication to induce apoptosis. The fact that administration of GCV results in depletion of EPO/TK�Cexpressing hepatocytes is probably due to the incorporation of the toxic triphosphates into mitochondrial DNA.30 Braun and colleagues also reported that HSV-TK�Cexpressing hepatocytes were ablated in adult animals in HSV-TK transgenic mice upon GCV treatment.31 A low systemic dissemination of the vector in extrahepatic tissues was pointed out by Alu-long terminal repeat PCR. Alu-long terminal repeat PCR was specifically used to distinguish the integrated provirus from the plasmidic contamination (data not shown).

Dissemination is probably due to noninternalized lentiviral vectors that adhere to hepatocyte membranes resulting in in vivo lentiviral vector spreading.32 Because brain biopsies were taken at the time of killing, i.e., after GCV-mediated ablation of transduced hepatocytes, the brain-detected vector DNA may be due to monocytes/macrophages trafficked into brain after having phagocyted apoptotic transduced hepatocytes and/or being lentivirally transduced.33,34,35 We observed that the vector integration in nonhepatic tissues does not lead to protein expression. This demonstrates the liver specificity of mTTR promoter in nonhuman primates and suggests inactivity of mTTR enhancers in these tissues. This inactivity may be important considering that the in vivo genotoxicity side effects reported for oncoretroviral vectors were mostly related to retroviral enhancer activity.

36 No gross macroscopic or histological anomalies were detected in any organ at the killing of macaques. Nevertheless, because it is important to avoid vector dissemination, we performed preliminary experiments to completely remove noninternalized infectious virus after transduction. Our preliminary results show that the sequential incubation of transduced hepatocytes with a protease Batimastat and human serum efficiently eliminated those infectious virus particles without impairing viability of transduced hepatocytes (J. Birraux, B.E. Wildhaber, C. Jond, D.C. Belli, and O. Menzel, personal communication). Protease treatment would release cell surface�Cbound viruses and human serum was shown to inactivate vesicular stomatitis virus glycoprotein�Cpseudotyped lentiviral vectors.37 In conclusion, this study demonstrates the feasibility of SLIT in the Macaca fascicularis, an animal model very close to human infants, with long-term (more than a year) survival and functionality of the autotransplanted lentivirally transduced hepatocytes.

To examine the role of these pathways in the regulation of DR4 or DR5 function, genes induced or repressed by recombinant human TRAIL (rhTRAIL) and DR5-selective rhTRAIL variants were determined in a colon cancer cell model using cDNA microarray technology. In this report we show that the immediate www.selleckchem.com/products/z-vad-fmk.html early gene, Egr-1, is constitutively expressed in colon cancer cells and further induced in response to rhTRAIL by both DR4 and DR5. Furthermore, we show that the short isoform of c-FLIP controls the activity of the DR5 receptor, but not of DR4. The constitutively expressed Egr-1 inhibits TRAIL-mediated apoptosis, probably by driving constitutive c-FLIP expression. Materials and methods Cell culture and treatments Colo205 cells were obtained from American Tissue Culture Collection (ATCC).

HCT15 and HCA7 cells were a kind gift from Professor L Egan (National University of Ireland, Galway). Colo205 and HCT15 cells were maintained in RPMI-1640 medium and HCA7 in DMEM medium, both media supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, 50Uml�C1 penicillin and 50mgml�C1 streptomycin at 37��C, 5% CO2 in a humidified incubator. Cells were seeded at 2 �� 105cellsml�C1 at 1 day before treatment. To induce apoptosis, cells were treated with rhTRAIL (non-tagged, fragment of amino acids 114�C281, Triskel Therapeutics, Groningen, The Netherlands) DR5-selective mutants D269H, D269H/E195R, agonistic DR4 or DR5 antibodies (Novartis Pharmaceuticals, Basel, Switzerland), recombinant human TNF (PromoCell, Heidelberg, Germany) or agonistic anti-Fas antibody (clone CH-11, MBL International, Woburn, MA, USA) at the concentration and times specified in the figure legends.

All reagents were from Sigma-Aldrich (St Louis, MD, USA) unless otherwise stated. Cell viability assay Cell viability was monitored using 3-(4, 5-dimethylthiazolyl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. After treatment, MTT (0.5mgml�C1) was added to cells and incubated for 3h at 37��C. The reaction was stopped by addition of an MTT stop solution of 20% SDS in 50% dimethyl formamide. The purple formazan precipitate generated was allowed to dissolve for 1h on an orbital shaker. The colour intensity GSK-3 was measured at 550nm on a Wallac Victor 1420 Multilabel counter (PerkinElmer Life Sciences, Waltham, MA, USA). Cell viability was expressed relative to the absorbance of untreated cells, which was taken as 100% viable. Cell death assay Cell death was monitored by labelling of phosphatidyl serine externalised on the surface of apoptotic cells with Annexin-V-FITC (IQ Corporation, Groningen, The Netherlands) or by haematoxylin and eosin staining of cytospins.