The objective was to examine whether the genetic variability of 20 candidate genes and red blood cells (RBCs) percentage of total n-3 PUFA, and the interaction term to take into account the effect on plasma lipid and apolipoporotein levels.Data from the Qanuippitaa Nunavik Health Survey (n = 553) were analysed via multivariate regression models with 40 known polymorphisms, RBCs percentage of CETP C-4502T (rs183130) or G-971A (rs4783961) together with higher n-3 PUFA had lower triacylglycerol (TG) concentrations compared to homozygotes for the minor allele. Further, effects of a stronger beneficial association between n-3 PUFA in RBCs and plasma lipid parameters- including lower total cholesterol (TC), lower low-density lipoprotein cholesterol (LDL-C) or higher high-density lipoprotein cholesterol (HDL-C) concentrations- were associated with AGT M235T (rs699) TT genotype, CETP G-971A (rs4783961) AG genotype, T allele carriers of CETP C-4502T (rs183130), and T allele carriers of CETP Ile405Val (rs5882). In contrast, higher n-3 PUFA in RBCs were associated with adverse lipid profiles- including increased LDL-C, increased apolipoprotein B100 or decreased HDL-C concentrations- in G allele carriers of the APOA5 -3 A/G (rs651821), C allele carriers of APOA5 T-1131C (rs662799), G carriers of APOC3 SstI (rs5128) and G carriers of APOA4 Asn147Ser (rs5104).Individuals being heterozygotes for n-3 PUFA of RBCs are associated with lipids related CVD risk factors conferred by genetic variations in the Inuit population.Overall, these results suggest that percentage of total  The lifestyle of the Inuit is rapidly changing towards an increased cardiovascular (CVD) risk factor profile . The CVDn-3 PUFAs represents an important dietary component for CVD risk prevention in the Inuit population [n-3 polyunsaturated fatty acids (n-3 PUFAs), including eicosapentaenoic acid (EPA) (20:5(n-3)) and docosahexaenoic acid (DHA) (22:6(n-3)), has been shown to exert favorable effects on plasma lipid profiles [n-3 PUFA content in red blood cells (RBCs) were indicative of higher n-3 PUFA intake in the Inuit population [n-3 PUFAs [The consumption of fish and marine mammals rich in profiles . Recentln-3 PUFA intake. Thus, gene-nutrient interactions may provide important insights into the inter-individual variability observed in plasma lipid levels and thus on the risk of CVD. Consequently, the main objective of this study was to examine whether the genetic variability of 20 candidate genes in lipid metabolism and percentage of total n-3 PUFA in RBCs - a biomarker of dietary n-3 PUFA intake, are associated with CVD risk factors including TG, TC, LDL-C, HDL-C, ratio TC/HDL-C and apoB100 concentrations.The Inuit populations also carry a unique distinct genetic background . This geThe data were collected in the framework of the Nunavik Inuit Health Survey “Qanuippitaa? How are we?” conducted among the Inuit of Nunavik in 2004. The survey covered all 14 Nunavik communities . In the 2), and internationally recommended cutoffs were used [Height was measured using a rigid square and a measuring tape in a standardized standing position, with the participant's back against the wall looking straight ahead, and arms hanging down freely. Body weight was measured with a scale . Body mass index (BMI) was calculated as weight per meter squared .Blood samples were collected from an antecubital vein into vacutainer tubes containing EDTA. Plasma was separated by centrifugation (2500 x g for 10 min at 4°C) and samples were portioned and frozen for subsequent measurements. Plasma TC and TG concentrations were determined by enzymatic methods using a Hitachi 917 autoanalyser and reagents from Roche Diagnostics (Canada) . The HDLA food-frequency questionnaire (FFQ)  was admin-3 PUFAs in RBCs was calculated from the sum of C18:3 (n-3), C18:4 (n-3), C20:4 (n-3), C20:5 (n-3) (EPA), C22:3 (n-3), C22:5 (n-3) (docosapentaenoic acid (DPA)), and C22:6 (n-3) (DHA).Fatty acid (FA) composition of erythrocyte membranes was determined by gas chromatographic analysis. Membranes of lysed erythrocytes were isolated by centrifugation  and washed twice with 0.9% NaCl solution. Cell membranes were resuspended in 200 μL of the NaCl solution and were spiked with phosphatidylcholine C:15 , used as internal standard. Lipids were extracted using a mixture of chloroform-methanol (2:1 v/v) according to a modified Folch method . FA profDNA was extracted from 100 μl of buffy coat using the QIAamp 96 DNA Blood Kit . Quant-iT PicoGreen® dsDNA Assay Kit (Invitrogen) was used to quantify DNA. DNA was analysed with TaqMan® Pre-Designed SNP Genotyping Assays according to the manufacturer's instructions at the McGill University/Génome Québec Innovation Center .Angiotensin I converting enzyme (ACE) I/D alleles (rs4343), Angiotensinogen (AGT) M235T (rs699), T174M (rs4762), A-20C (rs5050), A-6G (rs5051); Apolipoprotein A-I (APOA1) G-75A (rs670), 84 T/C (rs5070); Apolipoprotein A-IV (APOA4) Asn147Ser (rs5104); Apolipoprotein A-V (APOA5) T-1131C (rs662799), -3 A/G (rs651821), S19W (rs3135506), Gly185Cys (rs2075291); Apolipoprotein B (APOB) XbaI (rs693); Apolipoprotein C-III (APOC3) SstI (rs5128), T-455C (rs2854116); Apolipoprotein E (APOE) Cys112Arg (rs429358), Arg158Cys (rs7412), G-219T (rs405509); ATP-binding cassette, sub-family A (ABC1), member 1 (ABC1A) Arg219Lys (rs2230806); Cholesteryl ester transfer protein (CETP) TaqIB (rs708272), C-629A (rs1800775), C-4502T (rs183130), G-971A (rs4783961), Ile405Val (rs5882); Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) Msp1 (rs4646903), A4889G (rs1048943); Fat mass and obesity associated (FTO) (rs9939609); Glucokinase (hexokinase 4) regulator (GCKR) (rs780094); Insulin induced gene 2 (INSIG2) (rs7566605); Lipoprotein lipase (LPL) HindIII (rs320), Ser447Ter (rs328); Hepatic lipase (HL or LIPC) C-514T (rs1800588), G-250A (rs2070895); Methylenetetrahydrofolate reductase (NAD(P)H) (MTHFR) C677T (rs1801133); Paraoxonase 1 (PON1) L55M (rs854560), Gln192Arg (rs662), C-107T (rs705379); Peroxisome proliferator-activated receptor gamma 2 (PPARG2) Pro12Ala (rs1801282), 681 C/G (rs10865710); Transcription factor 7-like 2 (TCF7L2) C47833T (rs7903146).From a literature review, 40 polymorphisms in 20 genes relating to lipid metabolism were identified Table  namely: Hardy-Weinberg equilibrium (HWE) was tested with the Allele Procedure in SAS, version 9.2 . Distribution of alleles between the Inuit and Caucasian populations has previously been reported . After cn-3 PUFA as a continuous variable, and the polymorphism by RBCs percentage of total n-3 PUFA interaction effect, adjusted for the effects of age, sex, BMI, and total energy intake  on each of the lipid variables. Then, the absolute values of the regression beta- (β) coefficient were derived to estimate the phenotypic difference contributed by each of the genotype by RBC percentage of total n-3 PUFA interaction effect. In addition, to test for the additive effects of multiple polymorphisms, a “risk score” was calculated based on the number of risk genotypes an individual carried from all the significant polymorphism by RBCs percentage of total n-3 PUFA interactions for each lipid parameter. A statistical model was used to evaluate the effect of the “risk score”, RBCs percentage of total n-3 PUFA as a continuous variable, and the risk score by RBCs percentage of total n-3 PUFA interaction effect, adjusted for the effects of age, sex, BMI and energy intake  on each of the lipid variables. Again, the absolute value of the regression β- coefficient was calculated to estimate the phenotypic difference contributed by the number of risk genotypes and percentage of total n-3 PUFA in RBCs interaction effects. When significant differences were found, a comparison pairwise was performed in a global analysis . Statistical analyses were performed with SAS statistical software, version 9.2 .Data are shown as mean ± standard error of mean (S.E.M). Further, variables were checked for normality. Plasma TG and ratio TC/ HDL-C were transformed by natural logarithm to normalize their distribution. A statistical model was used to evaluate the effect of the polymorphism, RBC percentage of total n-3 PUFA in RBCs, energy intake and genotypes were available. Therefore, 553 participants were included. The baseline characteristics, plasma lipids levels, percentage of total n-3 PUFAs in RBCs, and daily energy intake are described in Table n-3 PUFA in the Inuit population were associated to lower TG concentrations (P = 0.0119), higher TC (P = 0.0001) including higher HDL-C (P < 0.0001) and higher LDL-C concentrations (P = 0.0146) without changing the TC/HDL ratio and plasma apoB100 levels when adjusted for age, sex, BMI and energy intake.In summary, 677 households were contacted of which 521 agreed to participate including 1056 individuals who signed a consent form and 917 who agreed to the collection of blood samples. In addition, 769 participants completed the FFQ. For the additional genetic component, 658 individuals from the original individuals gave consent. Consequently, the statistical analyses were done for all individuals where plasma lipid concentrations, percentage of total LPL HindIII (rs320), LPL Ser447Ter (rs328) and TCF7L2 C47833T (rs7903146) which were excluded from statistical analyses. The minor alleles of the following SNPs: AGT T174M (rs4762), AGT A-20C (rs5050), APOA5 Gly185Cys (rs2075291), APOA5 S19W (rs3135506), APOE Cys112Arg (rs429358), and FTO (rs9939609) had low frequencies (MAF < 0.10) in the Inuit population and were also excluded from analyses. In addition, allele frequencies were significantly different (P ≤ 0.05) in 18 SNPs in the Inuit population comparatively to the Caucasian population, as previously described [Genotype frequencies did not deviate from those predicted by the Hardy-Weinberg equilibrium except for the following 3 SNPs: n-3 PUFA in RBCs interactions for plasma lipid concentrations  or G-971A (rs4783961) had significantly lower plasma TG concentrations with higher n-3 PUFA percentage in RBCs compared to homozygotes for the minor allele. In addition, a more favorable plasma lipid profiles when higher percentages of n-3 PUFA were observed: lower plasma TC for T carriers of CETP C4502T (rs183130) or lower plasma TC/HDL ratio for CETP G-971A (rs4783961) for AG genotype. Participants homozygous for the T allele of CETP Ile405Val (rs5882) had lower plasma TC and T carriers of CETP Ile405Val (rs5882) had higher plasma HDL-C concentrations when higher percentage of n-3 PUFA in RBCs were observed. Secondly, for APOA5, G carriers of −3 A/G (rs651821) or C carriers of T-1131C (rs662799) exhibited higher plasma LDL-C concentrations, lower plasma HDL-C concentrations, a higher plasma TC/HDL ratio as well as higher apoB100 levels when they had a higher percentage of total n-3 PUFA in RBCs. Thirdly, carriers of the mutated allele of AGT M235T (rs699), compared to wild-type genotype, had decreased plasma TC and LDL-C concentrations when higher n-3 PUFA percentage were observed. Forth, G allele carriers of APOA4 Asn147Ser (rs5104) had greater apoB100 concentrations with higher percentage of n-3 PUFA. Finally, subjects carrying the G allele of APOC3 SstI (rs5128) had increased TC/ HDL ratio and apoB100 concentrations with higher percentage of n-3 PUFA. Additionally, a higher number of these above “risk alleles” in interaction with percentage of total n-3 PUFA were associated with significantly higher TG (P = 0.0069), TC (P = 0.0010), LDL-C (P = 0.0025) and apoB100 (P = 0.0044) concentrations as well as TC/HDL-C ratio (P = 0.0004) together with lower HDL-C (P = 0.0030) concentrations. Overall, polymorphisms in CETP, AGT, APOA5, APOA4 and APOC3 and the percentage of n-3 PUFA in RBCs are associated with plasma lipid concentrations in the Inuit population.Statistical analyses revealed 8 SNPs by percentage of n-3 PUFA in RBCs influence CVD risk factors in the Inuit population. Specifically, results demonstrate that 8 SNPs in 5 candidate genes: CETP ((rs5882) (rs183130) and (rs4783961)), AGT (rs699), APOA5 ((rs651821) and (rs662799)), APOA4 Asn147Ser (rs5104), and APOC3 (rs5128), are associated with plasma lipid concentrations in interaction with the percentages of n-3 PUFA in RBCs in the Inuit population.Results suggest that genetic factors and the percentages of n-3 PUFA in the Inuit population were associated with lower TG levels, higher TC including HDL-C and LDL-C concentrations without affecting the TC/HDL ratio and plasma apoB100 levels. In the same way, a meta-analysis that combined 21 trials with consumption of fish oil, reported a decrease in plasma TG concentrations and an increase in HDL-C and LDL-C concentrations without changes in TC concentrations [n-3 PUFA in RBCs on CVD risk factors in the Inuit population are similar to those previously reported; however, gene-nutrient interactions contribute the inter-individual variability observed in the plasma lipid levels in this population.Higher percentage of trations . Thus, tCETP C4502T (rs183130) or G-971A (rs4783961) had lower plasma TG concentrations with higher percentages of n-3 PUFA compared to the homozygotes for the minor allele. In addition, these heterozygotes for CETP C4502T or G-971A had lower plasma TC levels or TC/HDL ratio with higher percentages of n-3 PUFA compared to homozygotes for the rare allele, respectively. CETP plays a central role in HDL-C metabolism by shuttling cholesteryl esters from HDL particles to apoB- containing particles, partly in exchange for TG [CETP gene are associated with altered plasma HDL-C concentrations, lipoprotein particle sizes, CETP plasma concentrations and activity, and perhaps the risk of coronary artery disease [CETP C4502T SNP on HDL-C concentrations. We have also demonstrated no interaction with total fat or saturated fat intake with this SNP on plasma lipid levels [n-3 PUFA in the diets of wild-type or heterozygotes for CETP would favor an improved cardiovascular risk profile compare to carriers of the minor allele.Heterozygotes for e for TG . It is w disease . Earlier disease  demonstrCETP Ile405Val (rs5882) decreased plasma TC and increased plasma HDL-C concentrations with higher percentages of n-3 PUFA. This functional SNP, corresponding to the substitution of a valine for an isoleucine caused by a C → T mutation, has been associated with lower CVD risk via lowering of CETP protein concentration and activity; consequently, increasing HDL-C levels and lipoprotein particle sizes [CETP Ile405Val had greater reduction in plasma HDL-C and apoAI concentrations than subjects carrying a T allele when a high-PUFA: saturated diet was replaced with a low- PUFA: saturated diet [CETP Ile405Val polymorphism contributes to the variability of plasma lipids concentrations.Further, carriers of the T allele for le sizes . Previoule sizes  demonstrted diet . Togethen-3 PUFA in carriers of the T allele of AGT M235T (rs699) were associated with lower plasma TC and LDL-C concentrations than carriers of the wild-type allele. The M235T polymorphism in the AGT gene has been associated to an increased CVD risk in the presence of hypercholesterolemia [AGT with high total and saturated fat intake exhibited higher plasma TC and LDL-C concentrations [AGT M235T should consume a diet high in n-3 PUFA and low in saturated fat, to reduce their risk of CVD.Similarly, higher percentage of trations . Taken tAPOA5 -3 A/G (rs651821) or C allele carriers of APOA5 T-1131C (rs662799) had a more deteriorated lipid profile including higher plasma LDL-C and apoB100 concentrations, a higher TC/HDL ratio together with lower plasma HDL-C concentrations with higher percentage of n-3 PUFA in RBCs. APOA5 regulates TG metabolism; therefore, different SNPs in APOA5 may be associated with TG concentrations and CVD risk factors [APOA5 T-1131C polymorphism with energy intake and total fat was observed for plasma TG and TC concentrations in a population of Puerto Rican older adults [APOA5 T-1131C, whereas in subjects carrying the APOA5 T-1131C polymorphism, higher fat intakes were not associated with higher BMI and TG-rich lipoproteins [n-6 PUFAs, are related to a more atherogenic lipid profile in individuals of the Framingham Heart Study with the APOA5 T-1131C polymorphism. Overall, results suggest that SNPs in APOA5 are determinants of variation in lipid response to n-3 PUFA intake; however, the outcome of these gene-nutrient interactions needs to be investigated further.In contrast, G allele carriers of  factors . Variatir adults . Secondlproteins . Finally131C rs6699 had a APOA4 Asn147Ser (rs5104) had higher apoB100 levels with higher percentages of n-3 PUFA in RBCs. It has been suggested that APOA4 plays an important role in the metabolism of TG-rich lipoproteins [APOA4 variants may alter protein and thus have been linked to baseline TG concentrations [APOA4 Asn147Ser had lower plasma apoA1 concentrations in a postprandial state after a high saturated meal [APOA4 Asn147Ser may be more sensitive to changes in dietary fat.Further, carriers of the G allele of proteins . APOA4 vtrations  and TG itrations . A studyted meal . TogetheAPOC3 G minor allele of SstI (rs5128) had increased TC/HDL ratio and plasma apoB100 concentrations with higher percentages of n-3 PUFA. APOC3 increases plasma TG concentrations by inhibiting the lipoprotein lipase activity and by altering the Apo-E mediated uptake of TGs. The G- allele has been associated with elevated plasma TC, TG, and apoC-III concentrations [APOC3 G allele of SstI may benefit more from a high monounsaturated diet compared to an n-3 PUFA diet.Similarly, carriers of the trations . Lopez-Mtrations  demonstrn-3 PUFA for all CVD risk factors. Thus, this demonstrates that it is possible to develop models with advantageous and disadvantageous alleles together with dietary intake data and predict the risk of a deteriorated lipid profile in a specific population. However, the major limitation for the study is the limited number of SNPs chosen based on previous research findings. If such models would be developed, large studies using numerous genetic variations should be included to be able to assess the greatest variability.Since the effect size of each SNP was small even if the interaction was statistically significant, we demonstrated an additive effect of these SNPs in interaction with percentage of n-3 PUFA in RBCs, as an indicator of n-3 PUFA intake and genetic variations in CETP, AGT, APOA5, APOA4 and APOC3, in the Inuit population are associated with CVD risk factors. In general, n-3 PUFA consumption has favorable effects on lipid related CVD risk factors; however, some genetic subgroups may benefit to greater extent from a higher intake of n-3 PUFA for lipid related CVD risk factors.Overall, these results suggest that the percentages of FAs: Fatty acids; CVD: Cardiovascular disease; RBCs: Red blood cells; PUFA: Polyunsaturated fatty acids; TG: Triacylglycerol; TC: Total cholesterol; LDL-C: Low-density lipoprotein cholesterol; HDL-C: High-density lipoprotein cholesterol; IHD: Ischemic heart disease; apo: Apolipoprotein; EPA: Eicosapentaenoic acid; DHA: Docosahexaenoic acid; DPA: Docosapentaenoic acid; ACE: Angiotensin I converting enzyme; AGT: Angiotensinogen; APOA1: Apolipoprotein A-I; APOA4: Apolipoprotein A-IV; APOA5: Apolipoprotein A-V; APOB: Apolipoprotein B; APOC3: Apolipoprotein C-III; APOE: Apolipoprotein E; ABC1: ATP-binding cassette, sub-family A; ABC1A: Member 1; CETP: Cholesteryl ester transfer protein; CYP1A1: Cytochrome P450, family 1, subfamily A, polypeptide 1; FTO: Fat mass and obesity associated; GCKR: Glucokinase (hexokinase 4) regulator; INSIG2: Insulin induced gene 2; LPL: Lipoprotein lipase; HL or LIPC: Hepatic lipase; NAD(P)H MTHFR: Methylenetetrahydrofolate reductase; PON1: Paraoxonase 1; PPARG2: Peroxisome proliferator-activated receptor gamma 2; TCF7L2: Transcription factor 7-like 2; HWE: Hardy-Weinberg equilibrium.The authors declare that they have no competing interests.IR performed statistical analysis, interpreted data and wrote the paper; CO performed statistical analysis; VB, ADL, BA, FP, YG, and MLCD were responsible for data collection and analysis including genotyping, nutrient intakes and lipid parameters in the original study; ED, RAH and MCV were responsible for the study design; all authors contributed to the critical revision of the article; IR and MCV have primary responsibility for final content. All authors read and approved the final manuscript.


Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80–89) or site #4 (aa 280–289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications.Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast  Hantavirus is a genus of the family Bunyaviridae, containing more than 20 different hantavirus species [C-terminal to a conserved WAASA sequence by the cellular signal peptidase complex gives rise to glycoproteins Gn and Gc  [ species . Hantavi species . The han species . GPC is  species . Cotransctively) . The hanctively) .Bunyaviridae [The viral RNA segments are associated with the nucleocapsid protein to form ribonucleoprotein (RNP) complexes . The hanaviridae . E. coli, yeast, or insect cells [E. coli, yeast, insect and mammalian cell expression systems and their use for serologic detection of hantavirus infections and as potential vaccines [Hantavirus infection in humans can cause two diseases with some similarities in their symptoms, hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) ,16,17. Tct cells ,26,27,28vaccines ,30,31,32vaccines ,34,35,36E. coli-derived fragments (aa 647–886 and aa 829–1138) of Andes virus (ANDV) Gc protein and used for detection of ANDV antigens in urine samples [Monoclonal antibodies (MAbs) raised against hantavirus antigens are widely used as diagnostic tools for hantavirus infection, either for the detection of virus-specific antibodies by different capture ELISA formats ,26,37,38 samples .Active immunisation with recombinant glycoproteins or glycoprotein-encoding DNA vaccine constructs have demonstrated their protective potential in animal models ,48. In aAs hantavirus Gc glycoprotein represents a target for neutralizing antibodies and an important antigen for serologic tests ,46, therIn the current study, we attempted to generate Gc-specific MAbs by using an entire hantavirus Gc protein and alternatively by using recombinant virus-like particles (VLPs) harbouring a selected hantavirus Gc protein segment. For the insertion into VLPs, we selected a 99 aa-long segment of PUUV-Gc protein spanning aa 880–978 of PUUV GPC that contained target epitopes for neutralizing antibodies ,46. We hN-terminally hexahistidin-tagged full-length PUUV Kazan Gc (PUUV-Gc) protein  demonstrated that recombinant PUUV-Gc protein is found predominantly in the insoluble fraction (data not shown). The identity of PUUV-Gc protein was confirmed by its immunoreactivity with anti-tetra histidine MAb and rabbit polyclonal anti-PUUV antibodies (data not shown).  protein  was exprThe His-tagged PUUV-Gc protein was purified from the insoluble fraction of yeast cell lysate using nickel chelate affinity chromatography A, lane 1We assumed that the high molecular mass smear observed by Western blot analysis of yeast-expressed Gc protein might be explained by its ubiquitinylation. A Western blot analysis with polyclonal anti-ubiquitin antibody revealed a characteristic pattern of a polyubiquitinylated protein B, lane 1Thus, our attempts to generate Gc-specific MAbs using recombinant full-length Gc protein were unsuccessful.S. cerevisae were transformed with recombinant plasmids pFX7-VP1/L/Kaz-Gc99#1 and pFX7-VP1/L/Kaz-Gc99#4. Galactose induction of yeast cells resulted in the production of chimeric proteins VP1-PUUV-Gc99#1 and VP1-PUUV-Gc99#4 at amounts detectable by SDS-PAGE (To generate chimeric proteins consisting of hamster polyomavirus (HaPyV) major capsid protein VP1 and PUUV Gc protein segment, yeast 99#1 and VP1-PUUV-Gc99#4 fusion proteins confirmed the formation of VLPs similar in size and shape to unmodified VP1 VLPs. The diameter of these chimeric VLPs was 45–50 nm, which is typical for polyomavirus capsids . The same three serum specimens showed positivity for recombinant full-length PUUV-Gc protein. The reactivity of chimeric VLPs with PUUV-positive human serum specimens confirms surface exposure and accessibility of the inserted Gc sequence. Antigenicity of chimeric VLPs harbouring PUUV-Gc protein segment was investigated by an indirect ELISA using human serum specimens previously found to be positive for PUUV N protein-specific antibodies . The mic99#1 and VP1-PUUV-Gc99#4. To achieve a reduced VP1-carrier-specific immune response, subsequent immunizations were performed by alternative use of the two different constructs harbouring the PUUV-Gc protein segment either at position #1 or #4. This approach has been proven successful in our previous study for the generation of the MAbs against a hantavirus N protein segment presented on VLPs [4 (data not shown). For immunogenicity testing and generation of Gc-specific MAbs, BALB/c mice were immunized with chimeric proteinsVP1-PUUV-Gc on VLPs . After e99#1 and VP1-PUUV-Gc99#4) and full-length Gc protein. Hybrid clones specific both for chimeric VLPs and PUUV-Gc proteins were subjected to isotyping and recloning. After these procedures, one stable hybridoma clone producing Gc-specific MAb of IgG1 subtype was obtained (clone #10B8). The specificity of #10B8 antibody was proven both by ELISA and Western blot analysis using different yeast-expressed hantavirus antigens. Antibody #10B8 was reactive with recombinant full-length PUUV-Gc protein (data not shown) and chimeric VLPs harbouring PUUV-Gc99 segment , Prospect Hill virus (PHV) and Sin Nombre virus (SNV)  C.To evaluate the diagnostic potential of the MAb #10B8, its reactivity with hantavirus-infected cells was tested by an immunofluorescence assay (IFA) using commercially available slides. The MAb #10B8 was reactive with PUUV-infected Vero E6 cells and demonstrated a broad cross-reactivity with other hantaviruses, including HTNV, SEOV, DOBV, SNV and Saaremaa virus (SAAV) although there was a slightly different immunoreactivity pattern dependently on hantavirus strain . The strTo prove whether the MAb #10B8 has hantavirus-neutralizing activity, it was tested by the chemiluminescence focus reduction neutralization test on PUUV- and DOBV-infected cells. Even at the highest concentrations used, the MAb #10B8 did not cause reduction in the number of foci (data not shown). Therefore, it was concluded that the MAb #10B8 is non-neutralizing.Antibodies against viral glycoproteins have important diagnostic and therapeutic applications. However, in most cases the MAbs specific to viral glycoproteins are raised against an intact virus or virus-infected cells ,42,43. T99 protein sequence. Previous studies have shown that HaPyV VP1-based VLPs tolerate inserts of different size and origin and mediate the induction of high-titered insert-specific antibodies [99 segment were efficiently expressed in yeast S. cerevisiae and self-assembled to VLPs. The surface exposure and accessibility of the inserted Gc99 protein segment was confirmed by the reactivity of native chimeric VLPs with human serum specimens collected from patients seropositive for hantavirus N protein [99 segment was reactive with hantavirus-infected cells. Although the selected 99 aa-long segment of PUUV-Gc protein (spanning aa 880–978 of PUUV GPC) contained the epitopes for neutralizing antibodies [in vitro. This is in line with the epitope mapping of MAb #10B8 at aa 880–887 of PUUV Kazan GPC. This sequence is located outside the previously identified neutralizing epitopes spanning aa 916–924 and aa 954–963 of HTNV GPC [To overcome problems related to low yield and stability of recombinant glycoprotein, we have used an alternative approach for presenting epitopes of hantavirus Gc protein to B cells and inducing an efficient B cell response. We have inserted 99 aa-long segments of PUUV-Gc protein into HaPyV VP1 protein to obtain chimeric VLPs with a surface-exposed Gctibodies ,66,67,68tibodies ,64. For tibodies . Chimeri protein . Chimeri protein ,19,20. Ttibodies  the geneHTNV GPC  as well HTNV GPC . Five huHTNV GPC .In conclusion, our study has demonstrated the potential of hamster polyomavirus-derived VP1 protein as a highly efficient carrier for difficult-to-express antigens, such as viral glycoproteins. To our knowledge, this is the first example of a successful expression of chimeric VLPs with inserted large segment of a viral glycoprotein. Chimeric VLPs harbouring surface-exposed glycoprotein segments might be exploited as antigens for detection of glycoprotein-specific antibodies. Moreover, we have demonstrated that chimeric VLPs represent promising immunogens for generation of glycoprotein-specific antibodies.The immunodetection was performed using an anti-His MAb , polyclonal rabbit anti-ubiquitin antibody , a rabbit antiserum raised against Gc protein segment aa 659–794 of PUUV strain CG18–20  and a coPfu DNA polymerase  and using the following primers, containing SpeI restriction sites and six histidine codons (underlined):The entire PUUV, strain Kazan, Gc protein-encoding open reading frame (GenBank Acc. No. Z84205.1) was amplified by PCR using pCR-BluntII-TOPO-PUUV-Kaz-Gc plasmid as a template. The PCR amplification was performed according to standard protocols using ACTAGTACAATGCACCATCACCATCACCATGAGACACAGAATTTGAATGCA-3'5'-TGCACTAGTCTAAGGTTTATGCTCTTTTCTG-3'5'-GCN-terminal hexahistidin (His) tag and the Gc-protein was excised using Spe I and subcloned into the Xba I site of the yeast expression vector pFX7 [Saccharomyces cerevisiae strain 8188 4D . As a negative control, the plasmid pFX7 without any insert was used.The obtained PCR amplification product was inserted into the vector pCR-Blunt II-TOPO . Recombinant plasmids were screened in Top-10 cells (Invitrogen). The hantavirus Gc sequence insert within the pCR-Blunt II-TOPO plasmid was verified by DNA sequencing. The obtained sequence coding for an tor pFX7  under th2PO4, 0.01 M Tris, pH 4.5). Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were accomplished to prove the identity and purity of the eluted recombinant Gc protein.Transformed yeast cells were cultivated and disrupted as previously described . PurificN proteins of PUUV, strains Kazan and Sotkamo, of DOBV, genotype Dobrava strain Slovenia, and Kurkino strain SK/Aa, and of HTNV, strain Fojnica, were purified as previously described .BamH I restriction sites (underlined):A DNA fragment encoding a segment of PUUV, strain Kazan Gc protein spanning aa residues 880–978 was amplified by PCR using pCR-Blunt II-TOPO-PUUV-Kaz-Gc as template and using the following primers, containing GGATCCTGGAGATATAATGAGCACAC-3'Kaz-Gc-BD 5'-GCGGATCTGTTTCACTTAAATCTTG-3'Kaz-Gc-RB 5'-GCBgl II cloning site within the gene encoding hamster polyomavirus (HaPyV) major capsid protein VP1 either at position #1 (codons 80–89) or position #4 (codons 280–289) [99#1 and pFX7-VP1/L/Kaz-Gc99#4 where the PUUV-Kazan-Gc99 protein-encoding sequence is inserted either at position #1 or #4, and flanked by GSSG-encoding linkers (L) [17#1 and pFX7-∆VP1/L/Kaz-Gc80#4 used for MAb #10B8 eptope mapping were created after removing part VP1 and PUUV-Gc99-encoding sequence by cleavage with restriction enzymes Smi I and Acc 65I, blunting and religation of plasmid pFX7-VP1/L/Kaz-Gc99#1 and by cleavage with Smi I and Bsp 119, blunting and religation of plasmid pFX7-VP1/L/Kaz-Gc99#4, respectively (The PCR product was inserted into the 280–289) . This clkers (L) ; see Figectively .S. cerevisiae strain AH22–214  and their purification was performed as described previously for VP1 and other chimeric proteins [99 proteins were placed on 200-mesh carbon coated Cu grids, negatively stained with 2% aqueous uranyl acetate solution and examined by JEM-100S electron microscope . For comparison, authentic HaPyV-VP1-derived VLPs were examined. The expression of the VP1 and VP1-PUUV-Gc fusion proteins in yeast proteins ,72. The ready-to-use substrate .Purified proteins (100 ng/lane) or crude cell lysates  were fractionated by SDS-PAGE in 10% mini-gels. Proteins were detected in the SDS PAGE by Coomassie blue staining . For Western blot, separated proteins were transferred to polyvinyldifluoride (PVDF) membranes  under semidry conditions. Membranes were blocked by incubation in 5% dry milk/phosphate-buffered saline (PBS) for 1 h at RT, washed in PBS containing 0.1% Tween 20 (PBS-T) and incubated for 1 h at RT with the respective primary antibodies. His-tagged Gc protein was detected using anti-tetra-His MAb  and rabbit anti-PUUV Gc protein antibodies. VP1 and chimeric VP1-derived proteins were detected using the MAb #6D11 raised against HaPyV VP1 . The potAll procedures involving laboratory mice were performed at the breeding colony of the Center for Innovative Medicine  under controlled laboratory conditions in strict accordance with Lithuanian and European legislation. 99#1 protein, the 2nd immunization was done with VP1-PUUV-Gc99#4 protein. For a primary immunization, the antigen was emulsified in Complete Freund’s adjuvant (Sigma-Aldrich Co.), the subsequent immunizations were performed with antigens dissolved in PBS without adjuvant. After the 3rd immunization, serum specimens were collected to determine the titers of specific antibodies. Finally, the mouse with the highest anti-Gc titer was boosted with the same dose of VP1-PUUV-Gc99#4 protein in PBS and sacrificed for hybridoma development. To generate MAbs against the recombinant PUUV-Gc protein, three BALB/c mice were injected subcutaneously 3 times with 50 µg of recombinant PUUV-Gc protein at 21-day intervals. To generate the MAbs against the PUUV-Gc segment inserted into VLPs, the 1st and the 3rd immunizations were done with VP1-PUUV-Gc99#1 or VP1-PUUV-Gc99#4 protein. Positive clones were stabilized by limiting dilution cloning on a macrophage feeder layer. Hybridoma cells were maintained in complete Dulbecco’s modified Eagle’s medium (DMEM) containing 15% fetal calf serum  and antibiotics. Heavy chain types of MAbs were determined by ELISA using Monoclonal Antibody Isotyping Kit . Hybridomas were generated essentially as described previously . Four daready-to-use substrate (Sigma-Aldrich Co.). The optical density (OD) was measured at 450 nm in a microtiter plate reader . The specificity of mouse antisera, MAbs and human sera was tested by an indirect ELISA. Microtiter plates  were coated with the respective antigens by adding 100 μL of the antigen solution (2 μg/mL) in coating buffer  and incubation overnight at 4 °C. The plates were blocked for 30 min at RT with 1% bovine serum albumin (BSA) in PBS and then incubated with either serially diluted serum sample or undiluted hybridoma supernatant for 1 h at RT. After washing, the plates were incubated with HRP-labeled anti-mouse IgG  or anti-human IgG (Bio-Rad) for 1 h at RT. The immune reaction was detected with TMB GDPGDIMSTPTGMKCPDL-OH; (#2) biotin-SGSGGDPGDIMSTPT-OH; (#3) biotin-SGSGGDPGDIMS-OH. Peptide harbouring human hepatitis B virus surface protein sequence (underlined) aa 116–127 (biotin-SGSGSTGPCRTCTTPA-OH) was used as a negative control. To test the reactivity of MAb with biotinylated synthetic peptides, the microtiter plates  were coated with 100 μL of avidin  at 5 μg/mL in deionized water and air-dried overnight at 37 °C. After washing, 100 μL of biotinylated peptides (10 μg/mL) were added to the avidin-coated wells and incubated for 1 h at 37 °C. The plates were then incubated with 100 μL of undiluted hybridoma supernatant and developed as above.Epitope mapping was performed by an indirect ELISA using biotinylated synthetic peptides  harbouring PUUV Kazan GPC sequences (underlined) aa 880–897 (#1), aa 880–890 (#2) or aa 880–887 (#3): (#1) biotin-SGSGIndirect immunofluorescence assay (IFA) was performed using commercial IFA slides with PUUV (strain Sotkamo)-, DOBV (strain Slovenia)-, Saaremaa virus-, SEOV-, HTNV- and SNV-infected Vero E6 cells  as recommended by the manufacturer. Briefly, cell culture supernatant of hybridoma #10B8 was added to the slides in a dilution of 1:50 in PBS or undiluted. The negative control MAb #2H1directed against parainfluenza type 3 N protein  was usedHantavirus-neutralizing activity of the MAb #10B8 was investigated using the chemiluminescence focus reduction neutralization test (c-FRNT) essentially as described previously . HybridoN-terminally located Gc epitope (aa 880–887 of GPC) conserved among different hantavirus strains. The current study demonstrates the potential of hamster polyomavirus-derived VP1 protein as a carrier for segments of difficult-to-express antigens, such as viral glycoproteins. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications.Hamster polyomavirus VP1 protein-derived chimeric VLPs harboring a 99 aa-long segment of PUUV Gc glycoprotein were efficiently produced in yeast and used to generate a Gc-specific MAb that reacts with hantavirus-infected cells. The generated MAb #10B8 recognizes


Microarray experiments also revealed the enrichment of the gene set involved in p38 MAPK signaling in EpCAM+ cells treated with DSF but not 5-FU. In addition, DSF appeared to downregulate Glypican 3 (GPC3) in a manner independent of ROS-p38 MAPK pathway. GPC3 was co-expressed with EpCAM in HCC cell lines and primary HCC cells and GPC3-knockdown reduced the number of EpCAM+ cells by compromising their self-renewal capability and inducing the apoptosis. These results indicate that DSF impaired the tumorigenicity of tumor-initiating HCC cells through activation of ROS-p38 pathway and in part through the downregulation of GPC3. DSF might be a promising therapeutic agent for the eradication of tumor-initiating HCC cells.Tumor-initiating cells (TICs) play a central role in tumor development, metastasis, and recurrence. In the present study, we investigated the effect of disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, toward tumor-initiating hepatocellular carcinoma (HCC) cells. DSF treatment suppressed the anchorage-independent sphere formation of both HCC cells. Flow cytometric analyses showed that DSF but not 5-fluorouracil (5-FU) drastically reduces the number of tumor-initiating HCC cells. The sphere formation assays of epithelial cell adhesion molecule (EpCAM) Accumulating evidence has revealed that a minor population of tumor cells, called cancer stem cells or tumor-initiating cells (TICs), organizes a cellular hierarchy in a similar fashion to normal stem cells and shows pronounced tumorigenic activity in xenograft transplantations Disulfiram (DSF) is an irreversible inhibitor of aldehyde dehydrogenase and has been clinically used in the treatment of alcohol dependence for roughly 70 years in vitro and in vivo. We found that DSF impaired their tumor-initiating ability and induced apoptosis by activating the reactive oxygen species (ROS)-p38 pathway. Furthermore, the downregulation of Glypican3 (GPC3) expression, which is caused independently of the ROS-p38 pathway, appeared to also be responsible for the anti-TIC effect of DSF.In the present study, we examined the effects of DSF on tumor-initiating HCC cells 6 Huh1 and Huh7 cells into NOD/SCID mice, DSF was administered intraperitoneally every other day. Tumor initiation and growth were apparently suppressed by the DSF treatment in a dose-dependent manner (As shown in a variety of cancer cells t manner . TogetheALDH-knockdown reduced proliferation and motility of lung cancer cells ALDH1-knockdown affected neither cell growth nor tumorigenicity in HCC cells ALDH2 in Huh1 and Huh7 cells with lentivirus-mediated short hairpin RNA (shRNA) against ALDH2 using enhanced red fluorescent protein (ERP) as a marker for infection (ALDH2-knockdown cells and control cells expressing shRNA against luciferase (sh-Luc)  and mitochondrial ALDH2 nfection . No sign(sh-Luc) . Additioof ALDH2 . Taken thigh fraction markedly decreased from 44.4% to 9.8% in Huh1 cells and from 36.7% to 12.5% in Huh7 cells. Concordant with this, real-time RT-PCR analysis showed decreased expression of E-cadherin (CDH1) and alfa-fetoprotein (AFP), hepatic stem/progenitor cell markers, in DSF-treated cells , and CD133 using flow cytometry. The DSF treatment appeared to decrease the number of HCC cells expressing these markers . Among ted cells . In cleaed cells . These r− HCC cells than in EpCAM+ cells .We identified DSF-responsive genes (698 upregulated genes and 605 downregulated genes) and 5-FU-responsive genes  . Of inteand 5-FU  and S2. and 5-FU . Among tanalyses . HoweverGPC3 expression by DSF, we examined GPC3 expression in EpCAM+ HCC cells co-treated with NAC or SB203580. Neither NAC nor SB203580 restored the expression of GPC3  as a marker for viral infection. Western blot analysis of these cells showed that both shRNAs against GPC3 (sh-GPC3-1 and sh-GPC3-2) markedly repressed GPC3 expression, although sh-GPC3-1 was more effective than sh-GPC3-2 .Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice  were bred and maintained in accordance with our institutional guidelines for the use of laboratory animals.2 (0.1 or l µM) or 5-FU . Cells were treated with MG132 , N-Acetyl-L-cysteine (NAC) , and SB203860 .The HCC cell lines were obtained from the Health Science Research Resources Bank . DSF was kindly provided by Mitsubishi Tanabe Pharma Corporation. Cells were treated with DSF/CuClFor the sphere formation assay of Huh1, Huh6 and Huh7 cells, 1,000 cells were plated onto ultra-low attachment 6-well plates . For the assay of PLC/PRF/5 cells, 500 cells were plated onto NanoCulture 24-well plates . The number of spheres (>100 µm in diameter) was counted on day 14 of culture. For the secondary sphere formation, a single cell suspension derived from primary colonies was obtained using a Neurocult chemical dissociation kit . Paraffin-embedded sections of the spheres were subjected to hematoxylin & eosin (H&E) staining and immunohistochemical staining with anti-EpCAM  and anti-AFP  antibodies.Single-cell suspensions were stained with allophycocyanin (APC)-conjugated anti-EpCAM antibody and anti-CD13 antibody  or APC-conjugated anti-CD133/1 antibody . After the incubation, 1 µg/ml of propidium iodide was added to eliminate dead cells. Flow cytometirc cell sorting and analyses were performed using FACSAria or FACSCanto . Intracellular ROS levels were determined by flow cytometry using H2DCFDA (Sigma) and MitoSOX  staining.6 Huh1 and Huh7 cells were suspended in DMEM and Matrigel (BD) (1∶1). The cells were implanted into the subcutaneous space of the backs of NOD/SCID mice. DSF (10 or 50 mg/Kg) was administered intraperitoneally every other day.A total of 2×10ALDH2-knockdown cells and ALDH1-and ALDH2-double knockdown cells were subjected to Western blotting using anti-ALDH1 (BD Biosciences) and anti-ALDH2  antibodies. GPC3-knockdown cells selected by cell sorting for enhanced green fluorescent protein (EGFP) expression were also subjected to Western blot analysis using anti-GPC3 antibody (Santa Cruz Biotechnology).DSF-treated HCC cells were subjected to Western blot analysis using anti-p38 , anti-phospho-p38 , and anti-tubulin  antibodies. ALDH2  was constructed for the double knockdown of ALDH1 and ALDH2. Lentiviral vectors (CS-H1-shRNA-EF-1a-EGFP) expressing shRNAs against murine GPC3  were also constructed. Recombinant lentiviruses were produced as described elsewhere A lentiviral vector carrying ERP (CS-H1-shRNA-RfA-ERP) expressing shRNAs against Human GPC3 cDNA was cloned into a site upstream of IRES-neomycin in the pLP-IRESneo vector . Stable transfection into Huh1 cells with G418 selection was performed.Quantitative RT-PCR was performed with an ABI PRISM 7300 Sequence Detection System (Applied Biosystems) using the Universal Probe Library System (Roche Diagnostics) according to the manufacturer's directions. The sequences of primers are listed in After fixation with 2% paraformaldehyde and blocking in 10% goat serum, the cells were stained with anti-EpCAM  and anti-phospho-p38 MAPK  antibodies. Subsequently, the cells were incubated with Alexa-488–conjugated goat anti-mouse immunoglobulin G (IgG) (Molecular Probes) and Alexa-555–conjugated goat anti-rabbit IgG (Molecular Probes). The cells were coverslipped using a mounting medium containing 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) . For detection of apoptosis, the cells were also stained with an anti-active caspase-3 (CASP3) antibody , followed by incubation with Alexa-555 conjugated goat anti-rabbit IgG (Molecular Probes).+ cells, we selected probe sets that exhibited gene expression changes with statistical significance as follows: (i) genes exhibiting a change greater than 1.5-fold (p<0.05), (ii) genes exhibiting a change from 1.0 to 1.5-fold (p<0.01), and (iii) switch-on type (upregulated from the “absent” to “present” level) and switch-off type genes (downregulated from the “present” to “absent” level) exhibiting a change greater than 4.0-fold (p<0.01). Moreover, functional analyses were performed using Ingenuity Pathway Analysis (IPA) version 12402621 (Ingenuity Systems). To identify gene signatures after DSF or 5-FU treatment, gene set enrichment analysis (GSEA) was also conducted http://www.ncbi.nlm.nih.gov/geo/.Cy3-labeled complementary RNA was hybridized to a SurePrint G3 Human GE 8×60 K microarray . Array images were scanned using a DNA Microarray Scanner (Agilent) and analyzed using Feature Extraction version 10.27.1.1. (Agilent). Normalization was performed using GeneSpring GX11.5.1 (Agilent). The expression value  for each probe set was calculated using GeneSpring GX 12.0 (Agilent). Data were obtained for triplicate samples from three independent experiments. The data were subjected to normalization using GeneSpring normalization algorithms (Agilent). Only gene expression levels with statistical significance (p<0.05) were recorded as being “detected” above background levels, and genes with expression levels below this statistical threshold were considered “absent.” To identify differentially expressed genes in EpCAMData are presented as the mean ± SEM. Statistical differences between 2 groups were analyzed using the Mann-Whitney U test. P values less than 0.05 were considered significant.Figure S1In vitro assays of HCC cells treated with DSF. (A) Dose-dependent and time-dependent inhibition of proliferation in HCC cells treated with DSF. *Statistically significant (p<0.05). (B) Detection of apoptotic cell death by immunostaining for active CASP3. Nuclear DAPI staining is shown in the insets. Scale bar = 100 μm. (C) Quantification of the percentage of apoptotic cells. *Statistically significant (p<0.05).(TIF)Click here for additional data file.Figure S2ALDH2-knockdown and double knockdown of ALDH1 and ALDH2. (A) Cells transduced with the indicated lentiviruses were subjected to Western blotting using anti-ALDH2 and anti-tubulin (loading control) antibodies. (B) Cell proliferation in ALDH2-knockdown HCC cells was monitored by counting cell numbers. (C) Number of primary spheres generated from 1,000 cells at day 14 of culture. (D) Cells co-transduced with the indicated lentiviruses were subjected to Western blotting using anti-ALDH1 antibody, anti-ALDH2 and anti-tubulin (loading control) antibodies. (E) Bright-field (upper panels) images of non-adherent spheres at day 14 of culture. Scale bar = 100 μm. EGFP and RFP expression in double-knockdown spheres are shown in the insets. (F) Number of primary spheres generated from 1,000 cells at day 14 of culture.In vitro assay for (TIF)Click here for additional data file.Figure S3Flow cytometric analyses of HCC cells treated with 5-FU. Flow cytometric profiles in cells treated with 5-FU (10μg/ml) for 48 hours. The percentages of positive fractions for the indicated markers are shown as the mean values for three independent analyses.(TIF)Click here for additional data file.Figure S4− cells treated with DSF. (A) Non-adherent sphere formation assay on EpCAM− cells at day 14 of culture. Bright-field images are shown. Scale bar = 200 μm. (B) Number of large spheres generated from 1,000 HCC cells treated with DSF. *Statistically significant (p<0.05). (C) Fluorescence images of EpCAM− HCC cells. The expression of p-p38 (red) was merged with nuclear DAPI staining (blue). Scale bar = 100 μm.In vitro assay of sorted EpCAM(TIF)Click here for additional data file.Figure S5+ cells co-treated with DSF and a p38-specific inhibitor (SB203580). (A) Cell proliferation at 96 hours in culture. *Statistically significant (p<0.05). (B) Quantification of apoptotic cells based on the results of immunostaining for CASP3. *Statistically significant (p<0.05).In vitro assay of sorted EpCAM(TIF)Click here for additional data file.Figure S6+ cells treated with DSF or 5-FU. (A) Log2-fold heat map of genes involved in cell cycle in EpCAM+ cells treated with DSF. (B) Quantitative RT-PCR analyses of cell cycle-related genes. *Statistically significant (p<0.05). (C) Gene set enrichment analysis (GSEA) of the proteasome pathway in EpCAM+ cells treated with DSF or 5-FU. Both the normalized enrichment score (NES) and false discovery rate (FDR) are shown in each enrichment plot. (D) Log2-fold heat map of genes involved in the ROS scavenger pathway in EpCAM+ cells treated with DSF or 5-FU.Gene expression profiles of EpCAM(TIF)Click here for additional data file.Figure S7GPC3 expression and loss-of-function assay of GPC3 in tumor-initiating HCC cells. (A) Quantitative RT-PCR analyses of GPC3 expression in EpCAM+ HCC cells co-treated with DSF and NAC or SB203580. *Statistically significant (p<0.05). (B) Quantitative RT-PCR analyses of GPC3 expression in EpCAM+ HCC cells treated with MG132. (C) Cell proliferation in GPC3-knockdown HCC cells at 96 hours in culture. *Statistically significant (p<0.05). (D) Quantification of apoptosis in cells transduced with indicated the lentiviruses based on the results of immunostaining for CASP3. *Statistically significant (p<0.05). (E) H&E staining and immunocytochemical analysis of EpCAM and AFP in spheres derived from EpCAM+ cells. Scale bar = 20 μm. (F) Quantification of the percentage of EpCAM+ or AFP+ cells. *Statistically significant (p<0.05).Regulatory machinery of (TIF)Click here for additional data file.Figure S8+ cells. (A) Cells transduced with the indicated retroviruses were subjected to Western blotting using anti-GPC3 and anti-tubulin (loading control) antibodies. (B) Proliferation of Huh1 EpCAM+ cells at 96 hours in culture. The percentages of cells are shown. *Statistically significant (p<0.05). (C) Bright–field images of Huh1 EpCAM+ cells in non-adherent sphere formation at day 14 of culture. Scale bar = 100 μm. (D) Number of large spheres derived from 1,000 EpCAM+ cells on day 14 of culture. *Statistically significant (p<0.05). (E) Number of secondary spheres 14 days after replating. *Statistically significant (p<0.05).Gain-of-function assay of GPC3 in Huh1 EpCAM(TIF)Click here for additional data file.Table S1Top five ontology terms with molecular and cellular function of upregulated genes after DSF or 5-FU treatment.(DOC)Click here for additional data file.Table S2Top five ontology terms with molecular and cellular function of downregulated genes after DSF or 5-FU treatment.(DOC)Click here for additional data file.Table S3Primer sequences used for real-time RT-PCR.(DOC)Click here for additional data file.


In the crystal, the SrII ions  are connected by two crystallographically distinct benzene-1,2-dicarboxylate ligands and one formate ligand, forming a two-dimensional polymer parallel to (001). The asymmetric unit of the title complex, [Sr DOI: 10.1107/S1600536811044977/lh5360Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Caenorhabditis elegans genome contains 21 T-box genes, and their characterized functions include cell fate specification in a variety of tissues. The C. elegans Tbx1 sub-family member MLS-1 functions during larval development to specify the fate of non-striated uterine muscles; and, in mls-1 mutants, uterine muscles are transformed to a vulval muscle fate. Here we demonstrate that MLS-1 function depends on binding to the Groucho-family co-repressor UNC-37. MLS-1 interacts with UNC-37 via a conserved eh1 motif, and the MLS-1 eh1 motif is necessary for MLS-1 to specify uterine muscle fate. Moreover, unc-37 loss-of-function produces uterine muscle to vulval muscle fate transformation similar to those observed in mls-1 mutants. Based on these results, we conclude that MLS-1 specifies uterine muscle fate by repressing target gene expression, and this function depends on interaction with UNC-37. Moreover, we suggest that MLS-1 shares a common mechanism for transcriptional repression with related T-box factors in other animal phyla.T-box proteins are conserved transcription factors that play crucial roles in development of all metazoans; and, in humans, mutations affecting T-box genes are associated with a variety of congenital diseases and cancers. Despite the importance of this transcription factor family, very little is known regarding how T-box factors regulate gene expression. The  C. elegans to explore the mechanism(s) of the muscle-specifying T-box factor MLS-1. We show that MLS-1 physically interacts with another conserved protein called Groucho to repress gene expression and that this interaction is necessary for normal MLS-1 function. C. elegans MLS-1 is highly related to T-box factors in humans and other organisms, and we suggest Groucho interaction may be a common mechanism for T-box factor function. These insights may lead to therapies that modulate the activity of T-box factors in disease.T-box proteins are a conserved family of transcription factors that play important roles in development of all animals. A variety of human congenital diseases and cancers have been associated with mutations that either decrease or increase T-box factor activity. By learning more about how T-box transcription factors function, we can better understand and treat these disorders. Here we use genetic and molecular techniques in the roundworm  T-box transcription factors play essential roles in the development of all multicellular organisms, where their functions include the specification of primary germ layers and the specification of cell fates during organogenesis Caenorhabditis elegans T-box factors MLS-1 and MAB-9 in vivo.Groucho family (Gro/TLE) proteins are conserved transcriptional co-repressors that interact with distinct Engrailed homology 1 (eh1) or WRPW/Y motifs in a wide variety of transcription factors and, in many cases, recruit histone deacetylases to target gene promoters . Thal cells , and traFSI→ASA::vn173mls-1 and unc-37::vc155 would present a BiFC signal. After a one hour heat shock, transgenic animals co-expressing these proteins displayed no BiFC signal, whereas animals co-expressing wild-type mls-1::vn173 with unc-37::vc155 exhibited abundant BiFC signal  mutants in five independent lines, but at reduced percentages (FSI→ASAmls-1 transgene did not rescue mls-1(cc569) in seven of seven lines  on egl-15::gfp animals. These RNAi affected animals displayed egl-15::gfp expression in the four vm1 cells but also in up to four additional cells (unc-37(RNAi) experiments on animals with an integrated rgs-2::gfp transgene. In wild-type adults rgs-2::gfp is expressed in the pharynx, the ventral nerve cord, lumbar ganglia, and uterine muscles unc-37(RNAi) greatly reduced the number of and intensity of rgs-2::gfp expressing uterine muscles, while having no effect on rgs-2::gfp expression in other tissues  animals we examined had no rgs-2::gfp expressing uterine muscles.Because the MLS-1 eh1 motif mediates interaction with UNC-37 and is necessary for rescue of al cells , Table 2 tissues , Table 2egl-15::gfp expression in two unc-37 mutants: unc-37(e262) and unc-37(wd17wd21). unc-37(e262) is a hypomorphic allele that can be maintained as a homozygous strain, and it encodes a missense mutation affecting a conserved residue in the WD repeat domain that specifically affects UNC-37 function in VA motor neurons unc-37(wd17wd21) is a putative null allele containing a splice acceptor site mutation upstream of the WD repeats unc-37(wd17wd21) mutants exhibit a maternal effect embryonic lethal phenotype, but unc-37(wd17wd21) homozygotes produced from heterozygous mutant hermaphrodites grow to adulthood and exhibit uncoordinated and protruding vulva phenotypes. Most unc-37(e262) mutants exhibited a wild-type pattern of egl-15::gfp expression, but approximately 5% of these animals expressed egl-15::gfp in extra cells. A much higher percentage of unc-37(wd17wd21) adult animals  contained supernumerary egl-15::gfp expressing vulval muscles. In both cases GFP-expressing cells were near the vulva similar to mls-1(cc569) animals (egl-15::gfp expressing cells has previously been observed in mls-2 mutants unc-37(e262) may affect the activity of both MLS-1 and MLS-2 in specifying cell fates in the M lineage.We next examined  animals , but in  animals . A similC. elegans depends on this interaction. These results indicate that MLS-1 specifies uterine muscle fate at least in part by repressing target gene transcription.MLS-1 is a selector gene that is necessary and sufficient to specify uterine muscle development in the M lineage mls-1 loss-of-function results in a transformation of uterine muscle to vulval muscle, whereas ectopic mls-1 expression results in production of ectopic uterine muscles mls-1 in the M lineage can convert many M lineage cells to a uterine muscle fate egl-15 promoter is active in the supernumerary vulval muscles in mls-1 mutants, and we asked if it might be directly repressed by MLS-1. However mutation of predicted T-box binding sites in this promoter did not lead to expanded expression egl-15::gfp reporter , suggesting MLS-1 regulation of the egl-15 promoter is indirect.What types of genes does MLS-1 regulate? We suggest that MLS-1 represses expression of regulatory genes that themselves encode inhibitors of uterine muscle development and activators of vulval muscle development. This model is consistent with previous observations that mls-1 in these cell types is unknown MLS-1 may also function with UNC-37/Groucho in other MLS-1 expressing cells. When expressed with its own promoter, MLS-1 interacts with UNC-37 in BiFC assays in the vm2 vulval muscles and one to two intestinal/enteric muscles. The function of unc-37(RNAi) and some mutant alleles produce a protruding vulva (Pvl) phenotype that is not observed in mls-1 mutants egl-15::gfp expressing cells that looked like body wall muscles in unc-37(e262) and more frequently in unc-37(wd17wd21) mutants, while we never observed this phenotype in mls-1 mutants. At least two other transcription factors expressed in the M lineage contain high scoring eh1 motifs  I; ayIs2 [egl-15::gfp] IVlin-15(n765ts); vsIs5 unc-37(e262) Iunc-37(wd17wd21)/dpy-14(e188) Iunc-37(e262) I; ayIs2 [egl-15::gfp] IV, and OK0787 unc-37(wd17wd21)/dpy-14(e188) I; ayIs2 [egl-15::gfp] IV. The following transgenic strains were constructed for this work.mls-1(+) genomic fragment  or FSI→ASAmls-1 (pOK257.02) at 2.5 ng/µl and pRF4 at 100 ng/µl mls-1(cc569); ayIs2; cuEx575[mls-1(+)], OK0721 mls-1(cc569); ayIs2; cuEx576[mls-1(+)], OK0722 mls-1(cc569); ayIs2; cuEx577[mls-1(+)], OK0723 mls-1(cc569); ayIs2; cuEx578[mls-1(+)], OK0724 mls-1(cc569); ayIs2; cuEx579[mls-1(+)], OK0725 mls-1(cc569); ayIs2; cuEx580[mls-1(+)], OK0683 FSI→ASA]mls-1(cc569); ayIs2; cuEx568mls-1(cc569); ayIs2; cuEx596mls-1(cc569); ayIs2; cuEx597mls-1(cc569); ayIs2; cuEx598mls-1(cc569); ayIs2; cuEx599mls-1(cc569); ayIs2; cuEx600mls-1(cc569); ayIs2; cuEx601, OK0689 cuEx573[mls-1::vn173+unc-37::vc155], OK0690 cuEx574[mls-1::vn173+unc-37::vc155], OK0691 cuEx575[mls-1::vn173+unc-37::vc155], OK0709 FSI→ASA::VN173+unc-37::vc155]cuEx567cuEx568, OK0743 cuEx594[genomic mls-1::VN173+genomic unc-37::VC155], OK0744 cuEx595[genomic mls-1::VN173+genomic unc-37::VC155].Transgenic lines were constructed by injecting N2 with pRF4 at 100 ng/µl and the BiFC plasmids pCE-BiFC-VN173, pCE-BiFC-VC155, pOK257.05 (mls-1 (Open Biosystems) and unc-37  cDNA were cloned into pCE-BiFC-VN173 and pCE-BiFC-VC155 plasmids  to generate pOK257.05 (mls-1::vn173) and pOK257.06 (unc-37::vc155). FSI→ASAmls-1 cDNA was generated by site directed mutagenesis of mls-1 cDNA , and cloned into pCE-BiFC-VN173 producing pOK258.06 (FSI→ASA::vn173mls-1). Transgenic adults were picked to OP50 seeded plates, heat shocked for one hour at 33°C as described in Results, allowed to recover for one hour at 20°C, and examined for BiFC signal at 63× magnification.mls-1::vn173 and unc-37::vc155 constructs, with gene expression under the control of their respective endogenous promoters, were produced by PCR of promoter+gene fragments using pSAK244.13 and N2 genomic DNA templates, respectively. hsp-16.41 promoter fragments were removed from pCE-BiFC-VN173 and pCE-BiFC-VC155 and replaced with these mls-1 and unc-37 promoter+gene cassettes generating pOK263.03 and pOK266.02. Transgenic animals from various stages (L1 to Adult) were examined at 63× for BiFC signal.Genomic mls-1 bait plasmid (pOK248.03) was constructed by inserting the full-length mls-1 cDNA in to pLexA-NLS, and the unc-37 prey plasmid was previously isolated from the pACT-RB1 cDNA library (provided by R. Barstead).Yeast two-hybrid assays were performed as previously described mls-1(cc569); ayIs2 strains with mls-1(+) or FSI→ASAmls-1 extrachromosomal arrays were generated as above. Adult transgenic animals from these lines and animals from NH2447 and PD4285 were examined and scored at 40× and 63× magnification for the number of cells expressing egl-15::gfp.unc-37 cDNA was cloned into the L4440 (double T7 promoter) vector and the resulting plasmid (pOK247.03) was used to transform HT115(DE3) E. coli cells. unc-37 “feeding” RNAi was performed as before with some modifications ayIs2 [egl-15::gfp] IV (NH2447) or lin-15(n765ts) X; vsIs5 [rgs-2::gfp] (LX354)  adults and embryos were bleached and embryos were transferred to unseeded NGM plates and placed at 25°C overnight to allow embryos to hatch and synchronize as L1s. The next day synchronized L1s were transferred to unc-37(RNAi) seeded plates and placed at 20°C for 48 hours to 72 hours. Adult animals were examined and scored at 40× and 63× magnification for egl-15::gfp or rgs-2::gfp (mis)expression. Note that it is only possible to qualitatively evaluate rgs-2::gfp expression due to the unusual morphology of uterine muscles.Figure S1pmls-1::Venus expression in enteric and sex muscles of L4s and adults. A) L4 animal showing expression of pmls-1::Venus in left intestinal (Int) and anal depressor (AnDep) muscles. B) L4 animal (top) showing expression of pmls-1::Venus in vulval muscles (VM). A young adult (bottom) displays pmls-1::Venus expression in expanded uterine muscles. The pmls-1::Venus plasmid contains bp 1–1308 of cosmid H14A12 (Accession AF025459) cloned into pPD95.79-Venus (kindly provided by D. Byrd and J. Kimble).(TIF)Click here for additional data file.Figure S2egl-15::gfp in body wall muscle cells of unc-37(e262) animals. Image of two unc-37(e262); ayIs2[egl-15::gfp] adults shown at 40× and 63× magnification. Arrowheads point to supernumerary vulval muscles. Arrows point to cells with body wall muscle morphology that ectopically express egl-15::gfp.Ectopic expression of (TIF)Click here for additional data file.


In such cells, we unravel the generalized involvement of the beta-catenin-driven machinery in the stabilization of EGF-induced mRNAs, including the cancer stem cell regulator IL6. Our study highlights the crucial role of post-transcriptional mechanisms in the maintenance/acquisition of cancer stem cell features and suggests that the hindrance of cytoplasmic beta-catenin function may represent an unprecedented strategy for targeting breast cancer stem/basal-like cells.Hypoxia has been long-time acknowledged as major cancer-promoting microenvironment. In such an energy-restrictive condition, post-transcriptional mechanisms gain importance over the energy-expensive gene transcription machinery. Here we show that the onset of hypoxia-induced cancer stem cell features requires the beta-catenin-dependent post-transcriptional up-regulation of CA9 and SNAI2 gene expression. In response to hypoxia, beta-catenin moves from the plasma membrane to the cytoplasm where it binds and stabilizes SNAI2 and CA9 mRNAs, in cooperation with the mRNA stabilizing protein HuR. We also provide evidence that the post-transcriptional activity of cytoplasmic beta-catenin operates under normoxia in basal-like/triple-negative breast cancer cells, where the beta-catenin knockdown suppresses the stem cell phenotype  Stem cells are harbored in specialized niches where low oxygen tension (hypoxia) contributes to the regulation of self-renewal and differentiation –3. In faBeta-catenin is a crucial regulator of normal and cancer stem cell self-renewal ,7. In rePhysical interaction between HIF-1alpha, the major player in hypoxia response, and beta-catenin has been described . Moreovede novo transcription and promotes the energy-saving post-transcriptional regulation of pre-existing mRNAs [ Hypoxia is an energy restrictive condition, which markedly decreases total ng mRNAs –12. Receng mRNAs –17. Thesng mRNAs –20. CA9 ng mRNAs ,22. Therng mRNAs ,24. SNAIng mRNAs ,26. in vitro and the xenograft-establishing capability in vivo. Moreover, beta-catenin beta-catenin was able to regulate the mRNA stability of several EGF-induced mRNAs, among which the pro-inflammatory cytokine Interleukin 6 (IL6), an acknowledged cancer stem growth factor [We here report that the cytoplasmic accumulation of beta-catenin in response to hypoxia activates a post-transcriptional de-differentiation and survival program, which enhances stem cell features in breast cancer cells. The phenomenon relies upon the ability of cytoplasmic beta-catenin to bind and stabilize SNAI2 and CA9 mRNAs. We also provide evidence that the post-transcriptional activity of cytoplasmic beta-catenin operates under normoxia in basal-like/triple-negative breast cancer cells. The basal-like/triple-negative breast cancer is a poorly differentiated and aggressive breast cancer subtype, characterized by the expression of a stem cell-like gene profile ,28, by th factor ,34. The 2-humidified atmosphere. Hypoxia  was obtained in an invivo300 hypoxia cabinet  for 48 h. Cell death was evaluated by Trypan blue staining.We used MCF7 cells as a model of well-differentiated luminal breast carcinoma and MDA-MB-468 and MDA-MB-231 cells as a model of poorly differentiated basal-like breast carcinoma . All the2 low attachment plates. Secondary MS generation was obtained by incubating primary or consecutive MS in 1× Trypsin-EDTA solution (Cambrex) for 3 minutes, filtration throughout a 40-μm nylon mesh and single cell re-suspension in complete MEGM. MS were scored at day 7. Clearance was obtained from the S.Orsola-Malpighi Hospital ethical committee, University of Bologna (Prot. n. 75/2011 to LM and MB and MT). Written informed consent was obtained from patients whose tissues were used in the study. MCF7-MS were generated by seeding 5000 MCF7 cells in low attachment 24 well and scored at day 5.MS from human mammary gland tissues were obtained as previously described . BrieflyGAGCCTCTATACCACCCAC) by a PCR-based cloning strategy, as previously described for shSNAI2 vector [Stable beta-catenin knockdown in MCF7, MDA-MB-468 and MDA-MB-231 cells was achieved by transducing the pCtoGMB-GFP retroviral vector carrying human beta-catenin specific 19nt coding sequence . Cells were fixed with 2% paraformaldehyde and permeabilized with 0.2% Triton X-100. Cells were incubated with anti-beta-catenin primary antibody  and secondary anti-mouse FITC conjugated antibody . Nuclei were counter-stained with propidium iodide and mounted in the anti-fade Pro-long reagent mounting medium . Images were captured using a Zeiss LSM 710 on a Zeiss Observer Z1 or a Leica DMI 6000B inverted microscope . Cultured cells were seeded onto glass coverslips at 60% confluence, while MS were cultured in BD reduced MatrigelTM technology) with a matched GC content were purchased from Invitrogen . Plasmids encoding wild type beta-catenin , dominant negative pcDNA4-TCF4DN (TCF4DN), and pTER+shBeta-catenin (shBeta) encoding vector were obtained from Dr. Marc Van De Wetering  and Bert Vogelstein . Plasmid encoding HIF1-alpha was obtained from Eric Huang . Wild type EGFR encoding vector was obtained from Pier Paolo di Fiore ; siRNAs or plasmids were transfected to MCF7 cells (105 cells in a 3 cm2 well) at a concentration of 1 µg/well for 72h or 24 h respectively using lipofectamine 2000 , or Jet-Pei  in the case of MS. MCF7 cells stably transduced with a retroviral vector encoding a p53 dominant inactivating mini-protein (p53D) were previously described [CA9-, HuR-, and SNAI2-specific siRNAs and non-specific siRNA control (siSCR) oligonucleotides , was kindly provided by Dott. Jaromir Pastorek; HIF1alpha responding luciferase plasmid, HRE-Luc, was kindly provided by Dr. Giovanni Melillo . SNAI2-Luc plasmid was kindly provided by Dr. Togo Ikuta ; TopFlash, was a gift of Dr. Rolf Kemler ; Estrogen Response Element (ERE-Luc) reporter was provided by Rakesh Kumar ; Thymidine Kinase Renilla luciferase reporter plasmid  was used as control in luciferase assay after 24 hours using the Dual-Luciferase® Reporter Assay System (Promega), according to the manufacturer’s instructions. Data are expressed as fold changes of firefly over renilla luciferase activity. CA9 and SNAI2 3’UTR luciferase assay constructs were obtained from Genecopoeia . Each cell line was transfected with 3’UTR-CA9/SNAI2 vector or with control pEZX-MT01 empty vector. Data are presented as ratio of 3’UTR-CA9/SNAI2 over control vector, according to manufacturer instructions.Total RNA was extracted from cells using TRIzol® Reagent according to the manufacturer’s protocol . Primers and PCR conditions are reported in Ct target mRNA- ΔCt control)− . Each sample was analyzed in triplicate. Sets of primers and fluorogenic probes specific for CA9, SNAI2, ESR1, IL6 and CD44 mRNAs were purchased from Applied Biosystems. The reactions were incubated at 50° for 2 min; 95°C for 15 min followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. The relative amount of the target mRNA was calculated equal to 2 RNA was isolated using the PerfectPure RNA Cultured Cell Kit with DNAse-1 digestion . cDNA was synthesized using the SuperScriptII first-strand synthesis kit . For qPCR of pre-mRNA and mRNA, respectively, forward primers were positioned in the second intron and exon, and a shared reverse primer  was positioned in the third constitutive exon. For genes in which universal primers sequences were not available, 2 couples of primers were designed in order to amplify respectively intronic and exonic sequences. Each cDNA sample was mixed with the pool of primers for a pre-amplification reaction of 14 cycles with Reagent (Fluidigm PN 85000735) and TaqMan PreAmp Master Mix, according to the manufacturer’s instructions. The Preamplified cDNA was diluted 1:100. The modified 2x TaqMan universal Master Mix was added to the diluted cDNA in order to obtain a final concentration of Master Mix 1x in the samples. The chip was primed in the NanoFlexTM 4-IFC Controller prior to loading the samples and assay reagents into the inlets. Data were analyzed by using Ct values and ΔΔCt values. Each sample was in triplicates and normalized either on GAPDH, B2M and TBP. Primer sequences are listed in Total proteins were extracted with RIPA Buffer  added with Protease Inhibitor Cocktail . Western blot analysis was performed using the following antibodies: Laminin, Beta-tubulin, anti-beta-catenin (E5), anti-Actin (C4) purchased from Santa Cruz Biotechnology, ; anti-HuR  purchased from molecular probes; anti-CA9 (AF2188) purchased from R&D  and clone M75 kindly provided by Prof J. Pastorek . Co-immunoprecipitation assay was performed in CO-IP buffer  added with protease inhibitors (Roche).2, 10mM Hepes, pH 7.0, 0.5% Nonidet P-40) supplemented with RNase and protease inhibitors. Proteins were immunoprecipitated using protein A beads (Santa Cruz) and either anti-Beta-catenin  or anti-HuR (Molecular probes), or normal IgG  mouse antibodies, in NT-2 buffer , supplemented with RNAase inhibitor (40U/ul), DTT (1mM). Immunoprecipitates were lysed in Trizol® reagent (Life technologies). cDNAs were analyzed by RT-PCR  or by Real Time PCR (T-MS). Data are presented as fold increase of each mRNA bound to the specific antibody over the control IgG [mRNA immunoprecipitation assay was performed following standard protocol which preserves mRNA/protein interaction using the Polysome lysis buffer . In brietrol IgG . Stability assay for mRNA was performed by exposing cells to Actinomycin D at 100ng/ml and assessing level of each specific mRNA at different time points (0 to 6 h): mRNA level at first hour after Actinomycin exposure was taken as reference point (time 0).For isolations of ribosomal fractions two 10-cm plates of 80% confluent cells were lysed in lysis buffer . Organelle-free cytoplasm was obtained by saving supernatants after centrifugation at 14000 × g for 5 min at 4°C. Ribosomal fractions were then separated by centrifugation of cytoplasmic lysate at 100.000 g (36.000 rpm) in a SW41Ti rotor (Beckman) onto a 15-50% sucrose gradient added with RNase inhibitor and cycloheximide. Fractions corresponding to 40S ribosome subunit or low-density pre-ribosomal cytoplasm were used for RNA and protein extraction . RNA ext6 cells/100μl of wash buffer. Combinations of fluorochrome-conjugated monoclonal antibodies obtained from BD Bioscience Pharmigen  against human CD44  and CD24  or their respective isotype controls were added to the cell suspension at concentrations recommended by the manufacturer and incubated at 4°C in the dark for 30 min. The labeled cells were washed in the wash buffer and then analyzed on a LSR II Flow Cytometer (BD Biosciences).Cells were washed once with phosphate-buffered saline (PBS) and then harvested with Cell Dissociation Solution Non-enzymatic 1x (Sigma). Detached cells were washed with PBS containing 5% FCS and 0.1% sodium azide (wash buffer) and re-suspended at the concentration of 0.5*10th.To test the ability of selected cell lines to form foci, cells were plated into six-well-plates, maintained at conﬂuence, replacing the medium each 3-4 days. Foci were scored at day 146 MDA-M6B-468 cells were injected in the mammary fat-pad of female nude mice. Tumor growth was monitored weekly and then removed after 10 weeks . 1*106 MDA-MB-231 cells were subcutaneously injected in the flank of nude mice. Tumor growth was monitored weekly and then removed after 4 weeks. . Formalin-Fixed/Paraffine embedded tissues were stained with Hematoxylin-Eosin, and assessed by immunonistochemistry, with the following antibodies: anti-ESR1 ; anti-CDH1 , anti-SNAI2 , anti-CA9 .2*10t test are reported, unless otherwise specified (n=3).Bioinformatics analysis on AU-Rich element-containing mRNA was performed consulting the online database AREsite   [2) increased the MS forming ability of MCF7 cells (de novo mRNA production (res (MS) . Our invF7 cells , and of F7 cells . We thenF7 cells , via de oduction . Importaoduction . Consistoduction . Moreoveoduction  and the oduction , a markeoduction . These doduction .high/CD24low sub-population [high/CD24low cells in normoxia, and blunted CD44high/CD24low population expansion under hypoxia  displayed a dramatic reduction of SNAI2 and CA9 protein expression , coupledpulation . Consist hypoxia . In long hypoxia . Moreove hypoxia , this la of ESR1 , reveali of ESR1 . Interes of ESR1 . These dde novo mRNA transcription. In line with our hypothesis, stable beta-catenin silencing shortened CA9 and SNAI2 mRNA half-life in hypoxia exposed MCF7 cells  transcriptional targets ,20. ReceF7 cells . These dF7 cells . high/CD24low stem/progenitor cell population  mRNA by binding the mRNA 3’ un-translated regions (3’-UTRs), in co-operation with the mRNA binding protein HuR ,15,17. To evaluate whether the beta-catenin dependent increase in CA9 and SNAI2 mRNA stability relies on their 3’-UTRs, we examined luciferase encoding vectors carrying either CA9 or SNAI2 3’UTR sequences inserted between the luciferase coding sequence and the poly-adenylation site. Following this approach, we observed hypoxia-induced increase in the CA9 and SNAI2 3’UTR luciferase reporters activity, which was significantly reduced in normoxic and hypoxic MCF7-shBeta cells, compared to controls . These dHuR ensures timely engagement of mRNA with ribosomes, a function which remains poorly characterized . The anaA peculiar feature of basal-like/triple-negative tumors is the over-expression of epidermal growth factor receptor (EGFR) ,46, a reComprehension of the post-transcriptional mechanisms that steers stem cell features and cancer malignancy represents cutting edge cancer research. Recent studies described a new role of beta-catenin in the post-transcriptional regulation of several cytoplasmic mRNAs –17, in aAt the transcriptional level, our data support the notion that hypoxia and its mediator HIF-1alpha do not cooperate with beta-catenin transcriptional activity. These data are in line with previous observations in colon and lung cancer cells ,56, but  Cytoplasmic beta-catenin stabilizes Cox-2 mRNA through physical interaction with its 3’-UTRs ,15,17. Iin vivo tumor growth of basal-like/triple-negative breast cancer cells [ Basal-like/triple-negative tumors are aggressive breast cancers characterized by the expression of a stem cell gene profile ,28. Inteer cells   assay in Nor/1%pO2 MCF7 cells and T-MS; B, T-MS formation upon ctrl/SNAI2-specific siRNA(siSNAI2) transfection of breast cancer primary cells exposed to 1%pO2; C, WB analysis of e-cadherin protein level in SNAI2-specific shRNA retroviral vector (shSNAI2) MCF7 cells upon Nor/1%pO2 conditions.(TIF)Click here for additional data file.Figure S2beta-catenin knockdown reduces focus forming capability in hypoxia-exposed MCF7 cells.A, Focus assay in 1%pO2 long-term-exposed (2 weeks) ctrl/shBeta MCF7 cells.(TIF)Click here for additional data file.Figure S3beta-catenin role in breast cancer cell proliferation, colony-forming ability in soft agar and in CA9 and Snai2 transcriptional and post-transcriptional regulation. A, soft agar assay in ctrl/shBeta MDA-MB-231 cells; B, RT-PCR analysis of CA9 and SNAI2 mRNA level in 1%pO2-exposed MCF7 cells transfected with ctrl/TCF4DN; C, SNAI2-3’UTR-Luc assay in ctrl/shBeta MDA-MB-231 cells; D, quantitative CA9 and SNAI2 mRNA immunoprecipitation assay by control IgG/anti-beta-catenin antibody.(TIF)Click here for additional data file.Figure S4HuR binds and stabilizes CA9 and SNAI2 mRNAs in hypoxic luminal and normoxic basal-like breast cancer cells.A, schematic representation of CA9 and SNAI2 mRNA 3’-UTRs HuR binding sites as predicted by bio-informatics analysis; B, quantitative CA9 and SNAI2 mRNA immunoprecipitation assay by control IgG/beta-catenin antibody; C, Real Time PCR analysis of SNAI2 and CA9 mRNA levels in Ctrl siHuR-transfected/1%pO2-exposed MCF7 cells and normoxic MDA-MB-468 cells.(TIF)Click here for additional data file.Figure S5beta-catenin knock-down reduces HuR expression and localization to the ribosomal compartment.A, WB analysis of beta-catenin protein levels in MCF7 cells exposed to 1%pO2; B, WB analysis of beta- catenin protein levels in PRF and 40S cytoplasmic fractions of ctrl/shBeta MDA-MB-468 and MDA-MB-231 cells; C, Real Time PCR analysis of SNAI2 mRNA levels in PRF/40S cytoplasmic fractions of ctrl/shBeta 1%pO2 MCF7 and MDA-MB-231 cells; D, WB analysis of HuR protein levels in total cell lysates and in PRF/40S cytoplasmic fractions of ctrl/shBeta MDA-MB-468 and MDA-MB-231 cells; note that actin protein levels of ctrl/shBeta MDA-MB-468 cells refers to Figure 5D.(TIF)Click here for additional data file.Figure S6EGFr overexpression and activation promotes the cytoplasmic localization of beta-catenin and the beta-catenin dependent increase in SNAI2 and CA9 mRNA expression.A, IF analysis of beta-catenin in MCF7 cells stably-transfected with empty (MCF7-ctrl) or wild-type EGFR (MCF7-EGFr) vector, in presence/absence of EGF ; B, RT-PCR analysis of CA9 and SNAI2 mRNA expression levels in MCF7-ctrl/MCF7-EGFR and in MCF7-EGFR cells, transiently transduced with ctrl/shBeta encoding vectors; C, IF analysis of beta-catenin in p53-dominant-negative (p53D) stably transfected MCF7 cells; note that IF of MCF7-ctrl cells refers to panel A; D, WB analysis of EGFR protein levels in MCF7-ctrl/MCF7-p53D.(TIF)Click here for additional data file.Figure S7HuR binds and stabilizes IL6 mRNA.A, schematic representation of IL6 3’-UTR HuR binding sites as predicted by bioinformatics analysis; B, RT-PCR analysis of IL6 mRNA levels in 1%pO2 MCF7 cells, transiently transfected with Ctrl/siHuR; note that the loading control  of ctrl/siHuR 1%pO2 MCF7 cells refers to Figure S4C.(TIF)Click here for additional data file.Figure S8Beta-catenin/HuR physically interacts.A, Co-immunoprecipitation assay of beta-catenin and HuR proteins in MCF7, MCF7-MS and MDA-MB-231 cells.(TIF)Click here for additional data file.Figure S9Beta-catenin/HuR post-transcriptional machinery stabilizes CD44 mRNA.A, CD44 mRNA stability assay  in ctrl/shBeta MDA-MB-468 and MDA-MB-231 cells; B, Real Time PCR analysis of CD44 mRNA levels in PRF/40S cytoplasmic fractions of ctrl/shBeta 1%pO2 MCF7, MDA-MB-468 and MDA-MB-231 cells; C, quantitative CD44 mRNA immunoprecipitation assay with control IgG/anti-HuR/anti-beta-catenin antibody in 1%pO2 MCF7 cells and MDA-MB-468 cells; D, RT-PCR analysis of CD44 mRNA levels in scr/siHuR transfected MCF7 cells, exposed to 1%pO2; note that the loading control  of ctrl/shBeta 1%pO2 MCF7 cells refers to Figure S4C.(TIF)Click here for additional data file.Material S1Cytoplasmic pre-ribosomal and ribosome fractionation.A, profile of cytoplasmic fractions obtained after centrifugation of cytoplasmic lysates; B, fractions corresponding to low density pre-ribosomal cytoplasm (PRF), 40S, 60-80S and polysomes were pooled and examined in 1% agarose gel and western blot to verify the presence of the rRNA 18S, 28S and of the ribosomal protein S6, a component of the 40S ribosomal subunit.(TIF)Click here for additional data file.Table S1List of primer sequences and PCR conditions.(DOC)Click here for additional data file.Table S2List of primer sequences used for Real Time PCR in a Microfluidic Dynamic Array .(DOC)Click here for additional data file.