Cholesterol gallstone disease (CGD) is a multifactorial and multistep disease. Apart from female gender and increasing age being the documented non-modifiable risk factor for gallstones the pathobiological mechanisms underlying the phenotypic expression of CGD appear to be rather complex, and one or more variations in genes could play critical roles in the diverse pathways further progressing to cholesterol crystal formation. In the present study we performed genotyping score, Multifactor dimensionality reduction (MDR) and Classification and Regression Tree analysis (CART) to identify combinations of alleles among the hormonal, hepatocanalicular transporter and adipogenesis differentiation pathway genes in modifying the risk for CGD.ESR1, ESR2, PGR, ADRB3, ADRA2A, ABCG8, SLCO1B1, PPARγ2, and SREBP2 gene polymorphisms to find out combinations of genetic variants contributing to CGD risk, using multi-analytical approaches .The present case-control study recruited total of 450 subjects, including 230 CGD patients and 220 controls. We analyzed common ESR1 IVS1-397C>T (rs2234693), IVS1-351A>G (rs9340799) PGR ins/del (rs1042838) ADRB3-190 T>C (rs4994) ABCG8 D19H (rs11887534), SLCO1B1 Exon4 C>A (rs11045819) and SREBP2 1784G>C (rs2228314) with CGD risk. However, the MDR and CART analysis revealed ESR1 IVS1-397C>T (rs2234693) ADRB3-190 T>C (rs4994) and ABCG8 D19H (rs11887534) polymorphisms as the best polymorphic signature for discriminating between cases and controls. The overall odds ratio for the applied multi-analytical approaches ranged from 4.33 to 10.05 showing an incremental risk for cholesterol crystal formation. In conclusion, our muti-analytical approach suggests that, ESR1, ADRB3, in addition to ABCG8 genetic variants confer significant risk for cholesterol gallstone disease.Single locus analysis by logistic regression showed association of  Cholesterol Gallstone disease (CGD) corresponds to one of the most recurrent and costly gastroenterological disorder. It is world-wide health problem representing 10% to 15% of the adult population in industrialised countries ESR, PGR and ADR genes have been shown to be associated with susceptibility or progression with various disorders such as myocardial infarction In human physiology, the gender disparity commences with puberty and continues through the childbearing years ABC and SLCO1B1 genes respectively. Mutations in genes encoding these transporters have been implicated in cholesterol gallstones formation owing to their ability to influence bile composition and causing retention of substances normally secreted in bile.Another area of interest is hepatocanalicular transporters namely ATP-binding cassette transporters (ABC transporters) and organic anion transporters both encoded by PPARγ2) orchestrate the adipocyte differentiation process whereas sterol regulatory element binding protein 2 (SREBP-2) is involved in adipocyte differentiation followed by cholesterol homeostasis. Series of previous observations have suggested that regulatory interactions between the SREBPs and PPARγ2 can coordinate cholesterol and fatty acid metabolism. Therefore, sequence variation in these genes may further disrupt the cholesterol homeostasis which in turn may nurture the development of CGD.Peroxisome proliferator-activated receptor γ 2 , IVS1-351A>G (rs9340799), Ex4-122C>G (rs1801132), ESR2 -789 A>C (rs1271572), 1082 G>A (rs1256049) PGR ins/del (rs1042838) ADRB3-190 T>C (rs4994) and ADRA2A (rs1800544)], hepatocanalicular transporter pathway  and adipogenesis differentiation pathway [PPAR γ2 C>G (rs1801282) SREBP2 1784G>C (rs2228314)], avoiding the problem of dimensionality and multiple comparisons.The analysis of such interactions in case-control studies is weighed down by one of the major problems, namely, the curse of dimensionality. Recently, Multifactor-Dimensionality Reduction (MDR) approach, tree-based techniques: classification and regression trees (CART), and genotyping score The demographic profile of gallstone patients with respect to their age and gender matched controls are presented in ESR1 IVS1-397C>T, IVS1-351A>G, Ex4-122C>G, ESR2 -789 A>C, 1082 G>A PGR ins/del ADRB3-190 T>C and ADRA2A -1291 C>G in hormonal pathway, ABCG8 D19H, SLCO1B1 Exon4 C>A, Ex6+40T>C in hepatocanalicular transporter pathway and PPAR γ2 C>G SREBP2 1784G>C in adipogenesis differentiation pathway are shown in The genotypic and allelic distribution of ESR1, ESR2, PGR, and ADR in hormonal pathway. On comparing the genotype frequency distribution of our study groups i.e. gallstone patients with that of healthy subjects (HS), the homozygous variant genotypes of ESR1 IVS1-397C>T, IVS1-351A>G and ADRB3 -190 T>C polymorphism showed statistically significant increased risk for developing gallstone . On the contrary, no significant differences were observed in the distribution of Ex4-122C>G, , ESR2 -789 A>C , Ex6 1082 G>A , ADRA2A -1291 C>G  polymorphisms in selected groups, both at genotypic and allelic levels. The variant-containing genotypes (DI+II) of PGR ins/del showed low risk in gallstone patients which was also significant  of ABCG8 145 G>C and (CA+AA) of SLCO1B1 463 C>A were significantly associated and conferred increased risk of gallstone disease . On the contrary, no significant difference were observed in the distribution of SLCO1B1 521 T>C (rs4149056)  polymorphism, both at genotypic and allelic levels and therefore conferred no risk for developing gallstones.SREBP2 1784 G>C and PPAR γ2 C>G. A borderline statistical significance was observed when the homozygous variant genotypes of SREBP2 1784 G>C (rs2228314) was compared i.e gallstone patients with that of healthy subjects (HS) . Furthermore, no significant difference was observed in the distribution of PPAR γ2 C>G (rs1801282) .ESR1 rs2234693 and rs9340799 were found to be in strong linkage disequilibrium (D’ = 0.575). Haplotypes were constructed for the three polymorphisms in ESR1 gene including IVS1-397C>T, IVS1-351A>G and Ex4-122C>G. The haplotypes comprising the homozygous wild alleles were taken as reference and the difference in the frequencies of haplotypes between patients and controls were tested using chi-square test.On LD analysis, ESR1 revealed that distribution of T,G,C haplotypes was significantly higher in gallstone patients (25.1% v/s 13.7) in comparison to controls and was conferring high risk for gallstone disease . Global haplotypes analysis indicated a statistically significant difference between cases and controls based on the distribution pattern of the ESR1 haplotypes (p = <0.001). Furthermore, none of the ESR2 haplotypes conferred risk for gallstones presenting the global haplotypes association p-value = 0.65 . Haplotypes analysis of these two polymorphisms gave rise to three haplotypes, of which C, T was the most common haplotypes in control population. On comparing the haplotypes frequencies in controls and gallstone cases, A, T haplotypes was more commonly distributed in gallstone patients and was imposing risk for the disease  ( = 2.21) .For each individual, we counted the number of risk-increasing alleles. The number of risk alleles ranged from 1 to 11 in overall 450 subjects . The meaABCG8 145 G>C SNP . The two-factor model of ESR1 IVS1 351A>G and ADRB3 -190 T>C had an improved testing accuracy of 0.578 (permutation p = <0.001) however, the CVC was increased (10/10). The three factor model was the three-factor model including ESR1 IVS1-397C>T ESR1 IVS1 351A>G and ADRB3 -190T>C SNPs, which yielded the testing accuracy of 0.605 and the CVC of 08/10 (permutation p = <0.001). The best four-factor interaction model consisted of ESR1 IVS1-397C>T, ESR1 IVS1 351A>G, ADRB3 -190T>C and ABCG8 145 G>C with highest testing accuracy compared with the one-factor model (CVC = 10/10 permutation p = <0.001).ESR1 IVS1-397C>T, suggesting that this SNP is the strongest risk factor for GSD among the polymorphisms examined. Individuals carrying ESR1 IVS1 -397CC, ADRB3-190 TT, ABCG8 145 GG and ADRA2A GG genotypes had the lowest case rate of 17.2%, considered as reference. Further inspection of the tree structure revealed distinct interaction patterns between individuals carrying the ESR1 IVS1-397 variant and those with the ADRB3 variant and SLCO1B1 463 C>A wild genotypes. Using the terminal node with lowest case rate as reference, individuals carrying the combination of ESR1 IVS1-397TT, SLCO1B1 Exon4CC, ESR2 1082GG, ESR1 IVS1-351AA and ESR1 Ex4-122GG exhibited a significantly higher risk for GSD , whereas individuals with the combined genotypes of ESR1 IVS1-397TT, ABCG8 145 GC+CC, ESR2 1082GG+ ESR1 IVS1-351GG and ADRB3 TC+CC had the highest risk for CGD . (–22.08). .In order to achieve a more comprehensive evaluation of CGD risk, present analysis was performed in order to identify high and low intrinsic risk sets of sequence variants. Of the included 13 polymorphisms, some of them were found to be significantly associated with CGD risk in our previous studies ESR1 IVS1-397C>T, IVS1-351A>G and ADRB3 190 T>C were significantly associated with GSD risk Alu insertion polymorphism of progesterone receptors (PGR) conferred lower risk with gallstones. In hepatocanalicular transporter pathway ABCG8 D19H and SLCO1B1 Exon4 C>A conferred increased risk for CGD At haplotypes level, we found that the gallstones subjects who carry ESR1 haplotypes IVS1-397T, IVS1-351G, Ex4-122C and SLCO1B1 haplotypes Exon4A, Ex6+40T conferred increased risk for gallstones.In the single-locus analysis, genetic variants of hormonal pathway, Based on the candidate SNPs in genes involved in the gallstone pathway. We created a consolidated Genotype Score (G-score) from the number of risk alleles as previously reported for risk assessment of cardiovascular events and diabetes ESR1 IVS1-397C>T, ESR1 IVS1 351A>G, ADRB3 -190T>C and ABCG8 145 G>C with highest testing accuracy compared with the one-factor model.For the higher order gene-gene interaction analysis, we employed statistical approaches namely MDR and CART analysis to find out the particular combinations of genetic variants contributing to CGD risk. In MDR analysis, we observed the best four-factor interaction model consisting of ESR1IVS1-397TT, ABCG8GC+CC, ESR1IVS1-351GG and ADRB3 TC+CC polymorphisms are the most important single susceptibility factor for CGD development.In CART analysis, which is a non-parametric statistical approach for conducting regression and classification analyses by recursive partitioning. ESR1 gene is approximately 50 fold higher compared to ESR2. In animal models, 17beta estradiol promoted gallstone formation which further involves the upregulation of hepatic expression of ERalpha but not ERbeta. These studies show that ESR-1 is key player and findings may offer a new approach to treat gallstones by inhibiting hepatic ER activity with a liver-specific, ERalpha-selective antagonists.The association between hormonal receptor gene polymorphisms and risk of gallstones are biologically convincing. It has been assumed that the gallbladder is a female sex hormone responsive organ, and these hormones might be involved in the pathogenesis of gallbladder diseases. Elaborating on estrogen receptor the animal studies have shown that ESRs are present in the hepato-pancreatic-biliary tree ADRB3 confirms that it is localized in the smooth muscles of the vasculature and the muscularis propria of the gallbladder ADRB3 also results in smooth muscle relaxation in the guinea-pig common bile duct, ADRB3 might result in gallstone formation by impairing the relaxation of the gallbladder and probably the biliary tree too, setting the stage for crystal formation.The literature regarding the ABCG8 145G>C conferred increased risk both individually and in combination to hormonal receptors. A genome wide scan carried out by Buch et al., ABCG8) as major susceptibility factor for human gallstone disease. Subsequently, this association has been replicated in various populations In the selected hepatocanalicular transporters The phenomenon that a combination of polymorphisms within genes of unrelated pathways may elevate the risk for CGD could be explained by two hypotheses. One possibility is that some connection between these genes or proteins exists but still remains to be discovered. Another hypothesis, more credible in our opinion, is that the genes influencing risk for CGD may as well comprise a set of alterations located within genes not related to each other.ESR1 IVS1-397 variant, ABCG8 145 variant, ESR1 IVS1-351 variant and ADRB3 190 variant pose a significant risk for developing gallstone. Comparing to the results of single locus analysis the role of SLCO1B1 Exon4 C>A SREBP2 1784 G>C and PGR ins/del was diminished when the overall analysis of 13 selected polymorphisms was performed. It also suggests that ESR1, ADRB3 and ABCG8 have significant incremental risk factors for gallstone disease. Thus, the application of these multi analytical approaches allowed creating a decision that has more sensitivity or specificity and was more accurate with reasonable power as compared to single strategy employed in calculating risk allele for disease prediction.Our multi-analytic approach revealed that the combination of genotypes of respective polymorphisms as Also a prominent significant role of hormonal pathway was elucidated when the means of genotyping scores of selected pathways was calculated separately or all together. Therefore, exhaustive analysis of multi-analytic approaches as MDR, CART and G-scores are well recognized methods in understanding complex traits, such as disease susceptibility and also the etiology of complex diseases.ESR1, ADRB3 and ABCG8 genes were the most important.In summary, this is the first comprehensive study to use a multigenic analysis for cholesterol gallstone disease, and the data suggest that individuals with a higher number of genetic variations in hormonal and hepatocanalicular transporter pathway genes are at an increased risk for cholesterol gallstone disease, confirming the importance of taking a multigenic pathway based approach to risk assessment. The finding also indicates that the development of gallstone involves complex genetic interactions and follows different pathways depending on the specific genetic background of the subjects. The present study provided evidence supporting the cholesterol supersaturation contribution of hormonal, hepatocanalicular transporter and adipogenesis differentiation pathway genes, of which interaction between Thus, our results support the concept that genetic polymorphisms can be used as cholesterol gallstone risk predictors and multiple polymorphisms allow more precise delineation of risk groups and suggest the future direction of association studies. However, the present study included only North Indian individuals, therefore the results need to be replicated in other ethnic groups.The institutional ethical committee of Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS) approved the present study protocol and the authors followed the norms of World’s Association Declaration of Helsinki. All the participants provided written informed consent.The case control study recruited a total of 450 subjects, including 230 cholesterol gallstone patients (GS) and 220 healthy subjects. From the year June 2006 to September 2011 symptomatic cholesterol GS patients attending the Department of Gastro-surgery, Sanjay Gandhi Post Graduate Institute of Medical Sciences and Department of Surgical Oncology Lucknow India, were approached for participation in the present study. All subjects were unrelated and confirmed to North Indian ethnicity.For each individual, ultrasound examinations were conducted at the Department of Radio-diagnosis and Imaging SGPGIMS, Lucknow. Participants were considered as having gallstones when one of the subsequent diagnostic criteria was satisfied: (1) Gallbladder lumen with mobile nodular or dependent layering echoes that exhibited posterior acoustic shadowing, or (2) Gallbladder with hyperechoic shadowing material filling the gallbladder lumen with an appearance of the WES triad  was defined as the cumulative number that counts the total number of risk-increasing alleles in individuals. Genotyping of 13 selected SNPs in candidate genes involved in hormonal, hepatocanalicular and adipogenesis differentiation pathways was performed and G-score was computed from the number of variant alleles. A value of 2, 1 and 0 was allotted to homozygous variant, heterozygous and homozygous wild type genotypes respectively. Variant genotype was considered as risk conferring. Using these 13 SNPs a Genotype Score (G-score) was constructed ranging from 0 to 26 on the basis of the number of risk alleles. For each sample a consolidated G-score was calculated by adding the values from all 13 SNPs together.http://bioinfo.iconcologia.net/SNPstats).Descriptive statistics were presented as mean and standard deviation [SD] for continuous measures while absolute value and percentages were used for categorical measures. Differences in genotype and allele frequencies between study groups were estimated by chi-square test. Unconditional logistic regression was used to estimate odds ratios [ORs] and their 95% confidence intervals [CIs] adjusting for age and sex. A two-tailed p-value of less than 0.05 was considered a statistical significant result. All statistical analyses were performed using SPSS software version 16.0 . Ptrend and Monte Carlo Simulation (MCS) were calculated through Cochrane Armitage trend using XLstats whereas haplotype analysis was performed using SNPstats  using software version 2.0 beta8 and classification regression tree analysis (CRT) using SPSS software version 16.0.Table S1The genes and SNPs investigated.(DOC)Click here for additional data file.Table S2Haplotypes association of ESR1 gene (age and gender adjusted).(DOC)Click here for additional data file.Table S3Haplotypes analysis of ESR2 gene (age and gender adjusted).(DOC)Click here for additional data file.Table S4Haplotypes analysis of SLCO1B1 gene (age and gender adjusted).(DOC)Click here for additional data file.Table S5Odds Ratios and 95% CI for Gallstones in Relation to Polymorphisms of Hormonal Pathway after Subdividing on the Basis of Gender.(DOC)Click here for additional data file.


HNF1A was identified in predominantly European descent populations. A population-specific G319S polymorphism in HNF1A was previously identified in Aboriginal Canadians who have a high prevalence of type 2 diabetes. We aimed to investigate the association of the HNF1A G319S polymorphism with incident type 2 diabetes and to assess whether clinical risk variables for type 2 diabetes influence the association in an Aboriginal population.In a recent report of large-scale association analysis, a type 2 diabetes susceptibility locus near HNF1A G319S polymorphism. Interviewers administered questionnaires on smoking behavior.Of 606 participants who were free of diabetes at baseline in 1993-1995, 540 (89.1%) participated in 10-year follow-up assessments in 2003-2005. Fasting glucose and a 75-g oral glucose tolerance test were obtained to determine incident type 2 diabetes. Participants were genotyped for the HNF1A G319S carrier status was associated with incident type 2 diabetes (odds ratio [OR] 3.78 [95% CI 2.13-6.69]) after adjustment for age, sex, hypertension, triglyceride, HDL cholesterol, and waist circumference. A statistical interaction was observed between HNF1A G319S and baseline active cigarette smoking on the development of type 2 diabetes with similar adjustment (p = 0.006). When participants were stratified by baseline smoking status, HNF1A G319S carriers who were active smokers had increased risk of developing diabetes (OR 6.91 [95% CI 3.38-14.12]), while the association was attenuated to non-significance among non-smokers (1.11 [0.40-3.08]).The incidence rates of type 2 diabetes were 14.2% (55/388) in major allele homozygotes and 31.2% (29/93) in minor allele carriers (p < 0.001). The HNF1A G319S variant is associated with incident type 2 diabetes in Aboriginal Canadians. Furthermore, cigarette smoking appears to amplify incident diabetes risk in carriers of HNF1A G319S.The  HNF1A gene in an Aboriginal population was significantly associated with increased type 2 diabetes prevalence in cross-sectional analysis ), while the association was attenuated and became non-significant among non-smokers (1.11 [0.40-3.08])  with adjustment for age, sex, hypertension, triglyceride, and waist circumference. When participants were stratified by baseline active cigarette smoking status, however, HNF1A G319S was strongly associated with fasting glucose changes among active smokers , while the association was not significant among non-smokers . An interaction between HNF1A G319S and active cigarette smoking at baseline was significant on the outcome of 10-year changes in 2-hour postload glucose (p = 0.027) with adjustment for age, sex, hypertension, triglyceride, and waist circumference. When participants were stratified by baseline active cigarette smoking status, HNF1A G319S was strongly associated with changes in 2-hour postload glucose among active smokers , while the association was not significant among non-smokers . An interaction between HNF1A G319S and baseline pack-years was borderline significant (p = 0.05). Among those who provided both baseline and follow-up smoking behavior information and who were genotyped for HNF1A G319S, 142 of 420 (33.8%) participants changed their smoking behavior during 10-year follow-up (84 stopped smoking and 58 started smoking). Therefore, we performed interaction testing between changes in smoking behavior and the HNF1A G319S polymorphism and determined that it was not statistically significant (p = 0.50). In addition, the S319 carrier distributions among those who changed their smoking behavior and those who did not were not significantly different (p = 0.30).An interaction was present between the HNF1A G319S polymorphism was associated with increased risk of developing type 2 diabetes after adjustment for previously reported baseline clinical risk factors ) ) . In seveobserved ,20. Holmobserved  argued tobserved  would ciHNF1A G319S polymorphism demonstrated reduced in vitro capacity to transactivate insulin response, this disorder was not as severe as that seen in some nonsense mutations in HNF1A that underlie MODY type 3 [HNF1A G319S genomic sequence was reported to give rise to two abnormal transcripts, with altered quantities of the normal splicing products and reduced total HNF1A transcript levels [HNF1A G319S might compromise insulin response through at least two mechanisms: a failure of the mutant protein to transactivate insulin response and decreased translation of normal HNF-1α due to abnormal splicing. Since HNF-1α protein plays a key role in β-cell function [HNF1A G319S polymorphism and fasting insulin on the outcome of incident type 2 diabetes observed in our study.While the t levels . Thus, Hfunction , the redHNF1A G319S. To our knowledge, there is no previously reported relation between smoking and loss of glycemic control in individuals with other polymorphisms in HNF1A or in other MODY genes. A meta-analysis report indicated that active smoking behavior might be generally associated with increased risk for developing type 2 diabetes [HNF1A G319S carriers who were active smokers had increased risk of developing diabetes in the present study. Hence, smokers who carry S319 allele, and therefore are exposed to compromised insulin function [The presence of cigarette smoking appears to amplify the predisposition to the development of type 2 diabetes in carriers of diabetes . In a Eudiabetes . The unddiabetes ,25. In adiabetes . In a dodiabetes  and effediabetes . In the diabetes . HoweverHNF1A and incident type 2 diabetes.Limitations of our study include challenges of conducting investigations in a remote community setting. Specifically, we were unable to collect interim data to analyze the time to the onset of type 2 diabetes. We were also unable to obtain follow-up blood samples from all participants: diabetes outcome assessments of 109 (22.2%) participants were by self-reported clinical diagnosis only. This 22.2% without blood samples might have caused under-reporting of incident diabetes. We did not have a number of cigarettes smoked by former smokers at baseline. Therefore, we excluded 75 former smokers in our smoking dose-response analysis by pack-years . This might have contributed to the attenuated interaction observed between the G319S variant and smoking dose in this study. We also note that our study results, especially the smoking interaction, need to be confirmed in other populations. Nonetheless, the current study was able to retain a high 10-year follow-up rate of 89.1% and was able to investigate the prospective association between the population-specific genetic variation in HNF1A G319S variant was associated with incident type 2 diabetes in Aboriginal Canadians. Our results support that an environmental variable, cigarette smoking, may influence the development of the diabetes phenotype in the HNF1A G319S carriers. This is a particularly important public health concern among Aboriginal populations of Canada who have high prevalence rates of smoking and type 2 diabetes compared to non-Aboriginal populations [The population-specific ulations . In futuThe authors declare that they have no competing interests.All of authors read and approved the final manuscript and contributed in revising the manuscript critically for important intellectual content. SHL contributed to the statistical analysis and interpretation of the data and drafted the manuscript. RAH and HC contributed to the genetic analysis and interpretation of the data. MM contributed to the acquisition of data. RAH, PWH, SBH, JG, RR, BZ and AJH contributed to the conception and design of the study.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/1/prepub


ARL4D, ARL4A, and ARL4C are closely related members of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of GTPases. All three ARL4 proteins contain nuclear localization signals (NLSs) at their C-termini and are primarily found at the plasma membrane, but they are also present in the nucleus and cytoplasm. ARF function and localization depends on their controlled binding and hydrolysis of GTP. Here we show that GTP-binding-defective ARL4D is targeted to the mitochondria, where it affects mitochondrial morphology and function. We found that a portion of endogenous ARL4D and the GTP-binding-defective ARL4D mutant ARL4D(T35N) reside in the mitochondria. The N-terminal myristoylation of ARL4D(T35N) was required for its localization to mitochondria. The localization of ARL4D(T35N) to the mitochondria reduced the mitochondrial membrane potential (ΔΨm) and caused mitochondrial fragmentation. Furthermore, the C-terminal NLS region of ARL4D(T35N) was required for its effect on the mitochondria. This study is the first to demonstrate that the dysfunctional GTP-binding-defective ARL4D is targeted to mitochondria, where it subsequently alters mitochondrial morphology and membrane potential. ADP-ribosylation factors (ARFs), members of the Ras family of small GTPases, are involved in membrane transport, the maintenance of organelle integrity, membrane lipid modification, and cytoskeletal dynamics Three isoforms of ARL4  can be distinguished from the other members of the ARF family by a short basic extension at the C terminus and a short insertion in the loop between the two switch regions A recent study has shown that modifying either terminus of Arf1 through the fusion of a peptide or protein interferes with some, but not all, Arf1 activities and functions In this study, we report that the GTP-binding-defective mutant ARL4D(T35N) localizes to mitochondria and subsequently alters the mitochondrial morphology and membrane potential. We show that a portion of the endogenous ARL4D localizes to mitochondria. N-terminal myristoylation is required for ARL4D(T35N) mitochondrial targeting. The association of ARL4D(T35N) with the mitochondria reduced mitochondrial membrane potential and caused mitochondria fragmentation, but there was no evidence suggesting that ARL4D(T35N) affects cell viability or proliferation. Removing the C-terminal NLS region facilitated the mitochondrial association of ARL4D, but reduced the effects of ARL4D(T35N) on mitochondrial membrane potential. We infer that GTP-unbound ARL4D is targeted to the mitochondria to alter mitochondrial function via its C-terminal NLS region.Both our group and Katayama et al. have previously reported that overexpressed wild-type (WT) and the putative GTP-bound ARL4D mutant (Q80L) localize to the plasma membrane, while the GTP-binding-defective mutant ARL4D(T35N) locates to perinuclear punctate structures The subcellular distribution of exogenous ARL4D(T35N) and ARL4D(Q80L) was also determined by the density gradient centrifugation of postnuclear supernatants . DiffereTo determine the sub-mitochondrial localization of ARL4D(T35N), we treated mitochondria that were isolated from ARL4D(T35N)-expressing COS-7 cells with proteinase K, a non-specific serine protease that cannot penetrate mitochondria . The int2CO3, which efficiently dissociates peripherally associated, but not integral, membrane proteins  remained in the membrane fractions, along with VDAC and Tim23  . Proteinproteins . In contnd Tim23 . Finallynd Tim23 . This reThe ARL4D(T35N) mutant is homologous to ARF1(T31N), in which the conserved Thr residue in the phosphate-binding P-loop has been replaced by Asn. Initially, it was thought that these mutants remain in constitutively GDP-bound states 2CO3 to extract the peripherally associated proteins from the mitochondrial membranes. In contrast to cytochrome c, which is loosely associated with the mitochondrial inner membrane and was extracted to the supernatant after Na2CO3 treatment, endogenous ARL4D remained in the membrane pellets -sensitive fluorescent mitochondrial dye, to monitor the ΔΨm in cells expressing ARL4D mutant proteins. MitoTracker Red CMXRos accumulates only in actively respiring mitochondria that have an intact ΔΨmTo study the significance of the ARL4D(T35N) association with mitochondria, mitochondrial function was examined. We first used MitoTracker Red CMXRos, a mitochondrial membrane potential  Δ16C only slightly affected the ΔΨm. The significant difference in MitoTracker Red and JC-1 staining between the cells expressing ARL4D(T35N) and ARL4D(T35N)Δ16C suggests that the C-terminus of ARL4D not only regulates the mitochondrial localization of ARL4D but also contributes to the disruption of the ΔΨm.Because C-terminally truncated ARL4D(WT), ARL4D(Q80L), and ARL4D(T35N) all localized to the mitochondria, we next measured the ΔΨol cells , the redol cells . To conf mutants  by calcuThe dissipation of the ΔΨm has been shown to initiate cell death signaling, such as the permeability transition signal for apoptosis We also assessed the effect of ARL4D(T35N) on cell viability and proliferation. MTT assays were performed on RD cells at 24, 48 and 96 h after transfection with the wild-type or mutant forms of ARL4D. The viability of cells overexpressing ARL4D(T35N) was similar to that of the control cells or cells expressing wild-type or Q80L mutant ARL4D . To ensuin vitro using recombinant proteins. The nucleotide binding and conformational properties of these mutants would be fine-tuned in vivo when associated with regulatory proteins. Nevertheless, our findings suggest that ARL4D mutants defective in GTP binding localize to the mitochondria.The appropriate function of ADP-ribosylation factors depends on the precise temporal and spatial control of their GTP binding and hydrolysis. The conformational differences that accompany the binding of GDP or GTP can directly account for the changes in their affinities for specific proteins, lipids, and membranes It is known that the subcellular localization of ARL4D depends on its guanine nucleotide-binding state, N-terminal myristoylation status, and C-terminal NLS ARL4 proteins have longer Ras-like interswitches and longer C-terminal regions than do other ARF/ARL proteins We found that overexpressed ARL4D(T35N) either localizes to fragmented mitochondria or is diffusely distributed in the cytoplasm of COS-7 cells. Longer transfection periods seem to enhance the mitochondrial localization of ARL4D(T35N) (data not shown). In addition to the two GTP-binding defective mutants, we also found that endogenous ARL4D shifted to the heavier density gradient fractions after a longer culture time (24 h versus 72 h) (data not shown). These observations suggest that ARL4D might execute different functions in different organelles, depending on the physiological conditions of the cell.Our analysis revealed that ARL4D(T35N) and ARL4D(T52N) are tightly associated with the mitochondrial inner membrane. The mitochondrial respiratory chain generates an electrochemical proton gradient, also known as the mitochondrial membrane potential (ΔΨm), which is important for ATP production and mitochondrial protein transport Mitochondrial permeability transition inhibitors could not rescue these phenotypes, suggesting that the ARL4D(T35N)-or ARL4D(T52N)-induced disruption of ΔΨm may not be caused by a transition in mitochondrial membrane permeability. Other signaling pathways that disrupt the ΔΨm have been shown to lead to mitochondrial swelling, outer membrane rupture, and the release of apoptotic activators such as cytochrome c. Our data show no clear evidence of cytochrome c release in ARL4D(T35N)-expressing cells, and cell viability was not altered in these mutants. Interestingly, we did not observe the same mitochondrial fragmentation or reduction in membrane potential in cells depleted of ARL4D. This finding indicates that the phenotype was caused by the presence of GTP-binding-defective ARL4D rather than the absence of wild-type ARL4D protein. Further studies investigating how ARL4D regulates mitochondrial morphology may provide novel insights into the functional relationship(s) between mitochondrial fission and fusion, mitochondrial movement and transport, and cellular physiology.The uncontrolled temporal and spatial binding of GTP or GDP to small GTPases can cause serious pathologies and disease. A mutation in the ARL6 Thr31 residue, which is important for GTP binding, was reported to cause Bardet-Biedl syndrome (BBS) +/K+ ATPase, Bax , VDAC , cytochrome c, EEA1, Tim23, Lamp-1, Bcl2, calnexin , PARP , mannose 6-phosphate receptor , pyruvate dehydrogenase and cytochrome c oxidase complex IV . The horseradish peroxidase-conjugated sheep anti-rabbit or anti-mouse immunoglobulin (IgG) antibodies were purchased from GE Healthcare , and the Alexa Fluor 594 or 488-conjugated anti-rabbit and anti-mouse antibodies were obtained from Invitrogen.The rabbit anti-ARL4D The cDNAs corresponding to ARL4D, ARL4C, ARL4A and their various mutants were cloned into the mammalian expression vector pSG5 , as previously described. These constructs were used to express untagged ARL4s 2 at 37°C. The cells were transiently transfected using the calcium phosphate method or by using Lipofectin or Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocols. The cells were harvested for analysis at 24–48 h after transfection.COS-7 cells and HeLa cells were obtained from the American Type Culture Collection (ATCC) . Human rhabdomyosarcoma (RD) (ATCC No. CCL-136) cells were a kind gift from Dr. Passion Huang ; the original cell lines were obtained from the ATCC. COS-7 cells, HeLa cells, and RD cells were maintained in DMEM  with 10% FBS (Hyclone) in a humidified incubator containing 5% COSDS-PAGE was performed according to the Laemmli method Cells were processed for indirect immunofluorescence as previously described g) for 6 min at 4°C to remove the nuclei and unbroken cells. Samples (0.6 ml) of the PNS were loaded on top of iodixanol step gradients containing 2%, 6%, 10%, 14%, 18%, 22% and 26% iodixanol  according to the manufacturer’s protocol . After centrifugation , 14 fractions (collected from the top) were concentrated by TCA precipitation, resuspended in SDS sample buffer, separated by SDS-PAGE, and analyzed by Western blotting. The protein levels in each fraction were quantified by densitometry and reported as the percentage of total protein recovered.The subcellular fractionation by differential centrifugation and density gradient centrifugation of the cellular postnuclear supernatant (PNS) was performed as previously described 2, 1 mM EDTA, 1 mM EGTA, 2 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 250 mM sucrose, and a protease inhibitor cocktail. The cells were homogenized at 4°C by passing the buffer through a 26-gauge needle 30 times. The homogenate was centrifuged to separate the PNS (1000×g for 5 min). The PNS fraction was further centrifuged at 10,000×g for 15 min, isolating the crude mitochondrial pellet. The crude mitochondrial pellet was washed once with isotonic mitochondrial buffer  and resuspended in the same buffer. To determine the nature of the ARL4D-membrane association, the mitochondria-enriched fractions were treated with 0.1 M Na2CO3 (pH 12), 0.1% or 1% Triton X-100, or isotonic mitochondrial buffer (Mock) for 1 h on ice and centrifuged at 20,000×g for 20 min. The resulting pellet and supernatant fractions were analyzed.The cells were homogenized and subjected to subfractionation as previously described g for 20 min at 4°C. The supernatant was saved separately. The pellet suspensions were assayed by Western blotting to detect marker protein release from each mitochondrial subcompartment. Mitochondrial preparations that were subjected to the same treatment in the absence of digitonin served as controls.The submitochondrial fractionation with digitonin was performed as previously described, with minor modifications 2, 10 mM KCl, 10 mM Tris-Cl, and 0.25 M sucrose, pH 6.7) with a Dounce homogenizer. The homogenate was then centrifuged  to remove the nuclei and unbroken cells. The crude mitochondrial pellets resulting from the centrifugation  of the PNS fraction were washed once with ice-cold sucrose/Mg2+ medium , resuspended in ice-cold suspension medium , and treated with proteinase K (50 µg/ml) plus or minus 0.1% Triton X-100 for 30 min on ice. The reactions were stopped by the addition of trichloroacetic acid . The proteins that were precipitated by TCA  were washed once with acetone and subjected to immunoblotting.To isolate mitochondria for the proteinase K sensitivity assays, cells were homogenized in ice-cold cell homogenization medium (150 mM MgClThe COS-7 cells that were transfected with ARL4D(T35N)-myc were fixed with 0.1% glutaraldehyde and 4% formaldehyde in sodium phosphate buffer (pH 7.2). Cryosectioning and electron microscopy were performed as previously described m was measured by immunostaining using MitoTracker Red (Invitrogen) or by flow cytometry using JC-1 (Invitrogen). For MitoTracker Red staining, the cells were incubated with prewarmed (37°C) medium containing 100 nM MitoTracker Red CMXRos or 300 nM CM-H2XRos (Invitrogen) for 30 min at 37°C, washed twice with prewarmed medium to remove excess dye, and then fixed in prewarmed growth medium containing 4% formaldehyde for 15 min at 37°C. The quantification of MitoTracker fluorescence was performed using Axiovision 4.6 software (Carl Zeiss). The images were taken under the same conditions, and any intensity-saturated images were excluded from the analysis. The area around each cell was delineated, and the fluorescence intensity was measured in pixels. The background fluorescence was obtained from a cell-free field in each image and subtracted from the actual fluorescence. The cationic dye JC-1 emits a bright red fluorescence after forming aggregates in intact mitochondria. Mitochondrial aggregates do not form when the ΔΨm is disrupted; rather, the dye remains in monomeric form in the cytoplasm and emits green fluorescence. The cells were incubated with 2 µM JC-1 in medium for 30 min at 37°C and harvested by trypsinization. After washing the cells with PBS twice, the green (λem = 527 nm) and red (λem = 590 nm) fluorescence was measured using a FACSCalibur flow cytometer (BD Bioscience) and CellQuest software. The results are presented as the mean ratio of red to green fluorescence intensity. The raw flow cytometry data for the JC-1 red-to-green ratio, representing the mean intensities of 104 cells, included cells that had not been transfected or in which ARL4D was not localized to the mitochondria. Therefore, we calculated the ΔΨm of the mitochondrial ARL4D(T35N)-and ARL4D(T35NΔ16C)-expressing cells by taking the transfection efficiency (∼40% for each plasmid) and the percentage of the different ARL4D mutants localized to the mitochondria (∼60% for ARL4D(T35N) and ∼95% for ARL4D(T35NΔ16C)) into account.The ΔΨThe ARL4D gene was silenced using synthetic 21-nt RNA duplexes according to previously described protocols 4 cells/well in 96-well plates 24 h after transfection and incubated in medium (100 µl) for 24 or 48 h. After incubation with 100 µl of medium containing 0.5 mg/ml MTT for an additional 3 h at 37°C, the accumulated formazan was dissolved in 100 µl of DMSO, and the absorbance of this solution was measured at 570 nm. For the colony formation assay, equal numbers of transfected cells were seeded in 100-mm culture dishes and cultured under G418 selection (800 µg/ml). After two weeks, the colonies were stained with 0.1% crystal violet in 50% methanol and photographed.Cell viability was determined using the MTT  assay. In brief, the cells were plated at 2×102, and 5 mM DTT. The mixtures were incubated at 4°C for 16 h and transferred into a 96-well black plate. Fluorescence was evaluated using a SpectraMax M5 (Molecular Devices) spectrofluorimeter excited at 355 nm and emissions were monitored between 400 and 600 nm and fluorescence intensities were measured at 448 nm.Preparation and purification of recombinant proteins were carried out as described previously Figure S1ARL4D(T35N) localizes to the mitochondria. COS-7 cells were transfected with a plasmid encoding untagged ARL4D(T35N) and incubated with antibodies against ARL4D, βCOP, calnexin, EEA1, transferrin, mannose 6-phosphate receptor (M6PR) or Lamp-1. The cells were then examined by confocal microscopy. The nuclei were stained with Hoechst 33258. Bar, 10 µm.(PDF)Click here for additional data file.Figure S2Localization of the mutant ARL4 family proteins and their effects on mitochondrial membrane potential. (A) COS-7 cells were transfected with plasmids encoding untagged ARL4D(T52N), ARL4A(T34N), or ARL4C(T27N) and incubated with antibodies against ARL4D, ARL4A, or ARL4C plus cytochrome c, as indicated. (B) COS-7 cells were transfected with plasmids encoding untagged ARL4D(T52N), ARL4A(T34N), or ARL4C(T27N) for 48 h and stained with MitoTracker CMXRos before fixation and immunostaining with antibodies against ARL4D, ARL4A, or ARL4C. Bars, 10 µm.(PDF)Click here for additional data file.Figure S3T35N and T52N mutants of ARL4D bind to N-methylanthraniloyl-GDP (mGDP). Fluorescence intensity of mGTP or mGDP was measured  in the absence or after 16 h incubation with (A) ARL4D(T35N) or (B) ARL4D (T52N) at 4°C. Green and purple curves indicate fluorescence intensity from mGDP alone or mGDP-ARL4D complexes. Dark blue and red curves indicate fluorescence intensity from mGTP alone or mGTP-ARL4D complexes. ARL4D(T35N)-mGDP and ARL4D(T35N)-mGTP showed slight increase  in fluorescence intensity compare to nucleotides alone. ARL4D(T52N)-mGDP complex showed a sixfold increase in fluorescence intensity while ARL4D(T52N)-mGTP complex showed no increase. Guanine nucleotide binding assay was carried out as described in (PDF)Click here for additional data file.


The induction of a broadly neutralizing antibody (BNAb) response against HIV-1 would be a desirable feature of a protective vaccine. Vaccine strategies thus far have failed to elicit broadly neutralizing antibody responses; however a minority of HIV-infected patients do develop circulating BNAbs, from which several potent broadly neutralizing monoclonal antibodies (mAbs) have been isolated. The findings that several BNmAbs exhibit autoreactivity and that autoreactive serum antibodies are observed in some HIV patients have advanced the possibility that enforcement of self-tolerance may contribute to the rarity of BNAbs. To examine the possible breakdown of tolerance in HIV patients, we utilized the 9G4 anti-idiotype antibody system, enabling resolution of both autoreactive VH4-34 gene-expressing B cells and serum antibodies. Compared with healthy controls, HIV patients had significantly elevated 9G4+ serum IgG antibody concentrations and frequencies of 9G4+ B cells, a finding characteristic of systemic lupus erythematosus (SLE) patients, both of which positively correlated with HIV viral load. Compared to the global 9G4−IgD− memory B cell population, the 9G4+IgD− memory fraction in HIV patients was dominated by isotype switched IgG+ B cells, but had a more prominent bias toward “IgM only" memory. HIV envelope reactivity was observed both in the 9G4+ serum antibody and 9G4+ B cell population. 9G4+ IgG serum antibody levels positively correlated  with the serum HIV BNAbs. Interestingly, other serum autoantibodies commonly found in SLE  did not correlate with serum HIV BNAbs. 9G4-associated autoreactivity is preferentially expanded in chronic HIV infection as compared to other SLE autoreactivities. Therefore, the 9G4 system provides an effective tool to examine autoreactivity in HIV patients. Our results suggest that the development of HIV BNAbs is not merely a consequence of a general breakdown in tolerance, but rather a more intricate expansion of selective autoreactive B cells and antibodies. HIV infection is a major global health issue, and there is a critical need for a protective vaccine. The primary focus for humoral-mediated protection is the induction of neutralizing antibodies that recognize the HIV Envelope glycoprotein (Env). Although antibodies that recognize Env readily develop in HIV-1 -infected patients and can be induced by vaccination, these antibodies primarily recognize immunodominant, highly variable domains The limited incidence of persons producing HIV-reactive BNAbs in response to infection may in part result from proper enforcement of immunological tolerance for cross-reactive self-antigens. A relationship between autoreactive antibody and HIV BNAb development has been highlighted by several observations. In HIV patients, anti-CL serum antibodies correlate with increased HIV neutralization breadth Our group and others previously described an approach to monitor the development of autoreactive B cells and antibodies in SLE using the 9G4 anti-idiotype antibody In this study, we utilized the 9G4 system to examine both autoreactive 9G4+ serum antibodies and B cells in HIV patients, and their relationship with clinical characteristics, HIV reactivity, and the occurrence of HIV BNAbs.Peripheral blood samples were obtained from HIV-1 -infected patients at the University of Rochester Medical Center and the University of Washington HIV clinics between 2004 and 2010. Samples from healthy control (HC) subjects were obtained at the University of Rochester. All subjects provided signed written informed consent. Isolated peripheral blood mononuclear cells and serum were cryopreserved before subsequent analysis. All procedures and methods were approved by the University of Rochester Research Subjects Review Board and the University of Washington Institutional Review Board.°C or 4°C for 30 minutes in complete media , and washed once with PBS, prior to staining with fluorochrome-conjugated antibodies at 4°C for 60 minutes. One-to-two million total events per sample were collected on an LSRII instrument (BD Biosciences) and analysis performed in a blinded manner using FlowJo software . Total PBMC were gated on lymphocytes using FSC and SSC. Live/Dead stain and anti-CD3 were used to exclude dead cells and T cells, respectively.For global B cell phenotypic analysis peripheral blood mononuclear cells (PBMCs) were stained with anti-CD19-APC-Cy7 , anti-CD20-AlexaFluor 700 , anti-CD3-PerCP-Cy5.5 , anti-IgD-FITC , anti-IgG-PE , anti-IgM-PE-Cy5 , anti-CD27-Qdot655 , 9G4-Pacific Blue, and Live/Dead fixable aqua dead cell stain (Invitrogen). The 9G4 mAb was kindly provided by F.K. Stevenson , and recognizes a framework 1 region–encoded idiotype that is expressed by all unmutated, and close to 90% of mutated VH4-34 B cells present in the normal repertoire °C to reduce 9G4 painting, then stained with purified oligomeric HIV-1 SF162 (clade B) and KNH1144 (clade A) gp140 directly conjugated to AlexaFluor660 and AlexaFluor 647, respectively and HIV-1 p24 (NIH AIDS Research and Reference Reagent Program) directly conjugated to AlexaFluor488, in addition to anti-CD19-PE-Cy7, anti-CD20-APC-Cy7, anti-IgD-PE, anti-IgM-PerCP-Cy5.5, anti-CD3-PE-Cy5, anti-CD14-PE-Cy5, 7AAD for dead cell exclusion, and biotinylated 9G4 mAb/streptavidin Qdot800 (Invitrogen) at 4°C for 60 minutes. At least one million total CD19+ B cells  were analyzed per sample. p24+ gp140+ double-positive cells, a very rare population, were excluded from the analysis because they represent B cells that non-specifically react with gp140.To measure gp140-specific B cells, PBMC were first incubated for 30 minutes at 37°C, then washed with 0.1% Tween 20 in PBS. Samples were serially diluted in triplicate in PBS containing 0.01% Tween 20 and 0.5% BSA, and incubated for 90 minutes at 37°C. Plates were washed, and binding was detected using biotinylated 9G4 mAb and streptavidin alkaline phosphatase. A human VH4-34 9G4-reactive mAb, 75D9, was used as a standard. For detection of gp140-reactive IgG and 9G4+ Abs, ELISA plates were coated with purified oligomeric HIV-1 YU2 gp140 The detection of IgG 9G4+ Abs was performed as previously described The neutralizing activity of sera was determined as previously described Two-tailed t test or Mann-Whitney test were used as appropriate to compare HC and HIV groups. Spearman two-tailed correlation co-efficient was used to measure the correlation of two variables. Statistical analyses were performed using Prism 5.0 software  and significance was taken as p<0.05.Peripheral blood lymphocytes and serum samples were obtained from a diverse group of 90 HIV-1 seropositive patients from the University of Rochester and the University of Washington Center for AIDS Research (CFAR) cohorts. Most HIV patients (84%) had no history of anti-retroviral treatment (ART naïve) and the remainder were not receiving ART at time of sampling as per standard of care Table 1.Increased autoreactive antibodies are frequently observed in HIV patients, and therefore we asked if this breakdown in tolerance could be monitored using the 9G4 system as we and others have previously used for SLE +D), although only reached significance for 9G4+ B cells. HIV VL was significantly positively correlated with both 9G4+ IgG serum antibody  and 9G4+ B cells  (+F). Interestingly, HIV VL did not correlate with the abundance of other autoreactive serum antibodies including anti-dsDNA, ANA, or anti-CL , and that soluble autoreactive 9G4+ serum antibody is bound to a substantial proportion of the total B cells. Interestingly, limited longitudinal sampling (HIV055 and HIV056) indicated that 9G4 painting may substantially fluctuate over time thereby suggesting episodic stimulation of 9G4 antibodies rather than steady production through long-lived plasma cells or continuous B cell stimulation . However, the amount of Env-reactive 9G4+ serum antibody may be underestimated by this approach, because binding to Env may be mediated in part through the specific VH4-34 FR1 idiotope recognized by the 9G4 monoclonal antibody, thus blocking its detection.Observing the positive correlation of HIV VL and 9G4+ serum IgG, and considering the autoreactivity of several well-characterized HIV Env-specific monoclonal antibodies, we next sought to determine if 9G4+ serum antibodies are reactive to HIV Env. YU2gp140-reactive 9G4+ serum antibodies were detected in many HIV patients Fig. 3A,We also conducted Env-specific flow cytometry to identify Env-reactive 9G4+ memory B cells in HIV patients. 9G4+ Env-reactive IgD−IgM− memory B cells were present in HIV patients Fig. 4. Table S1A). Multi-clade neutralization was assessed on select samples (Table S1B). To facilitate regression analysis, we used a composite broadly neutralizing activity measure, the geometric mean ID50 of the serum Tier 2 clade B neutralizing activity, similar to that previously described Given the auto- and Env-reactive properties of 9G4+ antibodies and B cells, and the rationale that impaired tolerance and autoreactivity may be advantageous for developing a broadly neutralizing response against HIV, we assessed the magnitude and breadth of serum neutralizing activity against a panel of Tier 1 and Tier 2 clade B isolates  was observed between 9G4+ IgG serum antibody and HIV neutralizing activity Fig. 5A.in vivo autoreactivity of these 9G4 antibodies as indicated by their binding to B cells (+B). This increase was positively correlated with HIV VL (+D). A fraction of the 9G4+ serum antibodies and B cells were reactive to HIV Env (+4), and the abundance of 9G4+ serum antibodies and B cells positively correlated with serum HIV neutralizing activity  compartment compared to 9G4− B cells Fig. 2D.The observation of a higher frequency of 9G4+ cells in the Env-specific memory B cell population compared with the total memory population suggests in some HIV patients antigen-specific 9G4+ expansion may be occurring. This observation warrants further investigation to determine if such patients have unique features, and to address the possible mechanism of 9G4+ Env-specific memory B cell expansion. The positive correlation observed between serum HIV neutralizing activity and 9G4+ IgG may reflect a direct contribution of 9G4+ IgG to HIV neutralizing activity, or it might reflect an immunological environment, in which HIV infection coincidently favors the expansion of both species. It is evident that 9G4+ cells and serum antibodies represent only a minority of the Env-specific repertoire, but it remains to be determined if 9G4+ Env-specific cells and antibodies have any distinct properties beyond inherent autoreactivity that may uniquely impact HIV. Although numerous HIV broadly neutralizing monoclonal antibodies have been described to date, none are VH4-34-encoded. This does not preclude their existence or the potential of 9G4+ B cells and antibodies to contribute substantially to a protective immune response to HIV. Indeed, given the reactivity 9G4+ antibodies exhibit for glycoproteins, including an N-linked N acetyllactosamine determinant on CD45R/B2220 This work demonstrates the utility of the 9G4 system in interrogating autoreactivity at the cellular level in HIV patients, enabling insight into the development and expansion of autoreactive B cells and their relationship to the serum antibody repertoire. Important future questions include whether autoreactive antibodies are a prerequisite for the development of a protective HIV humoral response. There are clear examples of individual BNAbs against HIV that have minimal autoreactivity (e.g. VRC01) The association of autoreactivity and BNAbs also has important implications for HIV vaccine development – especially if autoreactivity is indeed a prerequisite for the development of a subset of immunoglobulin specificities that have the potential to give rise to BNAbs. Although it may not be feasible to completely uncouple autoreactivity from BNAb development in a vaccine setting, it is important to note that autoreactivity does not necessarily equate to manifestations of chronic autoimmune pathology, or a definitive pre-disposition to autoimmune disease development. Several observations support this, including the development of “non-pathological" autoreactive antibodies during various viral infections including CMV, HCV, and RSV Figure S1Common autoantibodies do not correlate with HIV viral load. Serum samples from ART-negative HIV patients were assessed for IgG antibody reactive to ANA, dsDNA, and CL by ELISA. Plasma VL was determined by PCR at the same timepoint. Dotted line represents positive/negative cut-off value. Spearmann correlation indicated, each symbol represents a unique patient.(TIFF)Click here for additional data file.Figure S2Rarity of 9G4+ B cells in the IgD− compartment of healthy control subjects. PBMC were analyzed ex vivo by flow cytometry. A. Representative plots from a HC and HIV patient gated on total CD19+ B cells. B. The frequency of 9G4+ B cells within the IgD− compartment. Each symbol represents a unique patient. * p<0.05 (Mann Whitney test).(TIF)Click here for additional data file.Figure S3Detection of 9G4+ IgG+ gp140 reactive memory B cells. PBMC were obtained from three HIV patients and IgG+ B cells were isolated and cultured with CpG+IL-2 for 4 days for the generation of antibody-secreting cells. EliSpots were performed to identify total IgG, total 9G4, total IgG gp140, and 9G4+ gp140 specific antibody-secreting cells. EliSpot coating/detection antibody combinations indicated.(TIF)Click here for additional data file.Figure S4Correlation of HIV serum broadly neutralizing activity and viral load. HIV neutralizing activity of serum against a panel of five Tier II isolates was determined by TZMbl assay and geometric mean ID50 presented with HIV VL. Spearmann correlation indicated, each symbol represents a unique patient.(TIFF)Click here for additional data file.Table S1HIV serum neutralizing activity.A. Clade B serum neutralizing activity B. Multi-clade serum neutralizing activity.(PDF)Click here for additional data file.


Previously, we have reported that the human papillomavirus (HPV) type 16 E6 D25E is the most prevalent variant in Korean women at high risk for cervical cancers. Several studies have identified an association between the increased frequency of this variant and the elevated risk of cervical intraepithelial neoplasia and invasive cervical carcinoma. To investigate whether the HPV-16 E6 D25E variant might influence cervical cancer progression, we used an oligonucleotide microarray approach to identify transcriptionally altered gene expression patterns in recombinant wild-type E6 or E6 D25E variant-expressing HPV-negative cancer cells. We found that 211 genes were significantly up- or down-regulated . We identified 14 genes, nine down-regulated and five up-regulated upon E6 D25E expression, compared with wild-type E6 expression. These results further emphasize the unique biological activity of the HPV-16 E6 D25E variant. High-risk types of the human papillomavirus (HPV) are causative agents in most cases of cervical carcinoma. Much of the transforming ability of this virus can be attributed to the activities of its E6 and E7 oncoproteins . The genHPV-16 is the most frequent type found in invasive cervical carcinoma (ICC) as well as in cervical intraepithelial neoplasia (CIN) in Asian women. Accumulated epidemiological studies suggest that certain HPV-16 E6 variants could influence the persistence of the infection ,8, and a2.The HPV-negative cervical cancer cell line C33A was purchased from the American Type Culture Collection  and maintained in modified Eagle's medium supplemented with 10% fetal bovine serum , penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37 °C in a humidified incubator with 5% COThe wild-type E6 and D25E variant genes were amplified from HPV-16-infected cervical swabs  and inse[(Ct/Actin - Ct/gene of interest)]. All data are presented as the mean ± standard deviation (SD) of three separate experiments. The following primers were used for the quantitative real time RT-PCR of survivin and actin genes: survivin forward primer 5"-GCAATGTTTCAGGACCCACA-3" and reverse primer 5"-AGGAACAGCCGAGATGACCT-3" and actin forward primer 5"-ATCTGGCACCACACCTTCTA-3" and reverse primer 5"-GGATAGCACAGCCTGGATAC-3". For microarray analysis, the synthesis of target cRNA probes and hybridization were performed from control and test RNAs using Agilent's Low RNA Input Linear Amplification kit  according to the manufacturer's instructions. The arrays were hybridized at 65°C for 17 h using an Agilent hybridization oven. The hybridized microarrays were washed according to the manufacturer's washing protocol. The hybridized images were scanned using Agilent's DNA microarray scanner and quantified with feature extraction software. All data normalization and selection of changed genes were performed using GeneSpringGX 7.3 (Agilent Technology). Functional annotation of genes was performed according to the Gene Ontology™ Consortium (http://www.geneontology.org/index.shtml) using GeneSpringGX 7.3. Gene classification was based on searches in BioCarta (http://cgap.nci.nih.gov/Pathways/BioCarta_Pathways), GenMAPP (http://www.genmapp.org/), DAVID (http://david.abcc.ncifcrf.gov/) and Medline databases (http://www.ncbi.nlm.nih.gov/).To confirm the expression of the E6 genes, we performed reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence. Total cellular RNAs were isolated using RNeasy kits  and RT-PCR reactions were performed with OneStep RT-PCR kits (QIAGEN) according to the manufacturer's instructions. Primers used for amplifying the gene sequence for E6 were: forward 5"-GCAATGTTTCAGGACCCACA-3" and reverse 5"-ACAGCATATGGATTCCCATCTC-3". Primers for the GAPDH gene  were: forward 5"-CCACAGTCCATGCCATC-3" and reverse 5"-TCCACCACCCTGTTGCTGT-3". The temperature profile comprising the RT round was 50 °C for 30 min. The PCR rounds involved denaturing at 95 °C for 15 min followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s and one cycle at 72 °C for 10 min. For the immunofluorescence, cells were seeded in six-well plates, fixed at room temperature for 15 min with BD Cytofix/Cytoperm  after twenty-four hours and treated for 30 min with Image-iT FX signal enhancer (Invitrogen). Following blocking with 3% bovine serum albumin in phosphate-buffered saline for 10 min, cells were incubated with the primary antibody overnight at 4°C and with the secondary antibody for 1 h at room temperature. The primary antibody was anti-E6  and the secondary antibody was Alexa Fluor 546-labeled mouse immunoglobulin G  (both at 1:100 dilution). Confocal imaging was performed using an LSM 510 META microscope . Messenger RNA expression levels for the survivin gene were analyzed via quantitative real time reverse transcription polymerase chain reaction (RT-PCR) using a Bio-Rad iCycler system . Total RNA was reverse-transcribed into cDNA using an Omniscript RT kit (Qiagen). Primer specificity was tested by running a regular PCR for 40 cycles (95°C for 20 s and 60°C for 1 min), followed by agarose gel electrophoresis. Real time RT-PCR was performed using a SYBR supermix kit (Bio-Rad). Samples were subjected to 45 cycles of 95°C for 20 s and 60°C for 1 min. PCR efficiency was determined by running serial dilutions of template cDNA, and melting curve data were collected to assure PCR specificity. Each cDNA sample was analyzed in triplicate, and the corresponding no-RT mRNA sample was included as a negative control. A GAPDH primer was included in every plate as an internal loading control. The mRNA level of each sample for survivin gene was normalized against that of GAPDH mRNA. The relative mRNA level was determined as 2To investigate the role of individual viral oncoproteins of HPV-16 wild-type E6 and the D25E variant on cervical cancer progression, we stably transduced the E6 expression vector into C33A cells. Cells expressing the E6 protein were selected and maintained in medium containing blasticidin. The expression levels of E6 in C33A cells were detected by RT-PCR and immunofluorescence. As shown in Figure http://david.abcc.ncifcrf.gov/). The known annotations for 211 genes were submitted for level 2 biological process analyses  in Korean women at high risk for cervical cancers . EpidemiZMZ1, RPL23, MAPK4, RPL31, RARS, LAMB3, HSPA14, AIFM2, IFRD1) were down-regulated and five genes  were up-regulated in E6 D25E expressing cells. The specific functions of these genes in cervical oncogenesis remain to be elucidated. However, it is conceivable that genes such as AIFM2, RPL23, and ROCK2 could influence disease progression and promote potential carcinogenicity. In the present study, AIFM2 was reduced in E6 D25E oncoprotein expressing cell lines. It has been reported that AIFM2 has a pro-apoptotic function and that its expression is down-regulated in cancer cell lines by comparison with normal cell lines [Until now, there have been no experimental data on the impact of this single amino acid 25 change in prototype E6, the dominant variant in Asia region, on progression to invasive carcinomas. This study aimed at identifying genes affected by the HPV-16 E6 D25E variant in comparison with the wild-type E6 in the C33A HPV-negative cervical cancer cell line using microarrays. A genomic approach using oligonucleotide microarrays showed that 14 genes involved in interspecies interactions between organisms (including the immune system) and stress response were modulated by the E6 D25E oncogene at an mRNA level. Nine genes  genes HLA-A and HLA-B were elevated in E6 D25E-expressing cells. Down-regulation of HLA Class I genes  is associated with cervical neoplastic lesions and HLA polymorphisms might influence oncogenicity by HPV infection, most likely through an immunological mechanism . These fAIFM2, RPL23, and ROCK2 might be involved in cervical tumorigenicity via E6 D25E. Our findings suggest that E6 D25E might have a unique oncogenic role in cancerous transformation but further studies are needed to define the molecular mechanisms and carcinogenic potential of the HPV-16 D25E variant.This is the first study to identify functional differences in the naturally occurring HPV-16 E6 D25E variant in C33A cell lines and provides a basis for future mechanistic studies. We observed that the HPV-16 E6 D25E-expressing C33A cells modulated several functional genes compared with wild-type E6-expressing cells. This result contributes new and useful information to support the epidemiological data showing that there is more frequent detection of the D25E variant in patients with cervical cancer, and that several genes such as HPV: human papillomavirus; CIN: cervical intraepithelial neoplasia; RT-PCR: reverse transcription polymerase chain reactionThe authors have no competing interests to declare.DH supported performing experiments. M and JE performed the experiments and wrote and edited the manuscript. SS designed the research and edited the manuscript. All authors read and approved the final manuscript.The list of genes differentially changed in C33A cells expressing E6 proteins. The list shows genes that were significantly altered  in E6-expressing cell lines compared with controls. The fold change of these genes is mean of 2 independent experiments.Click here for file


Escherichia coli serotype O157:H7 was isolated and found to be a member of the family Myoviridae. The genomic sequence of phage EC6 was composed of 86,231 bp with a G+C content of 38.9% and is very similar to the sequences of Escherichia phage wV8 and Salmonella phage Felix O1.A bacteriophage, EC6, capable of lysing  Escherichia coli serotype O157:H7 is a major pathogen that causes diarrhea, colitis, and hemolytic-uremic syndrome (E. coli O157:H7 bacteriophage (phage), EC6, from a sewage sample. Several clinical isolates of E. coli were tested to determine the phage’s host spectrum. Phage EC6 might form lytic plaques against sorbitol-nonfermenting E. coli but not against sorbitol-fermenting strains. Transmission electron microscopic images revealed that the EC6 phage belongs to the family Myoviridae. The sequencing of the phage EC6 genome was performed by combining the 454 and Illumina sequencing platforms using Genome Sequencer (GS) assembler 2.6  and CLC Genomics workbench 5.5 , respectively. The resulting contigs were assembled through manual curation using CodonCode aligner v3.7.1 . The final contigs were remapped with raw reads to correct errors, and dubious regions were masked as gaps. Genes, tRNA, and rRNA were identified using Glimmer, tRNAscan-SE, and HMMER programs along with EzTaxon-e rRNA profiles   (syndrome . We isolprofiles . The genE. coli O157:H7-specific phage EC6 is a member of the Myoviridae family and is closely related to the Escherichia phage wV8 and Salmonella phage Felix O1.In conclusion, JX560968.The complete genome sequence of phage EC6 has been stored in GenBank under the accession no.


Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, accounting for about 3% of all childhood tumors PAX3- or PAX7-FOXO1, respectively TP53NRAS, KRAS, HRASPIK3CAFGFR4RMS tumors typically arise from skeletal muscle and are categorized as either of the alveolar (ARMS) or embryonal (ERMS) subtype based on their histology. ARMS tumors are driven by a translocation involving chromosome 2 or 1 with chromosome 13, resulting in the production of the fusion oncogene FGFR4FGFR4 is a direct transcriptional target of the PAX3-FOXO1 fusion protein FGFR4 in 7.5% of RMS tumors. These mutations occur at amino acid 535 and 550 of the kinase domain and promote tumor growth and metastasis in vivo by constitutively activating FGFR4 Fibroblast Growth Factor Receptor 4 (FGFR4), a FGF receptor family member, is a receptor tyrosine kinase that is implicated in the differentiation of myoblasts into skeletal muscle 50 of less than 40 nM FGFR1 or FGFR2 gene in vivo. Therefore, our results indicate that ponatinib is an effective FDA-approved drug which has the potential to treat RMS with overexpressed or mutated FGFR4.Ponatinib is an orally administered tyrosine kinase inhibitor that was initially developed as an inhibitor for native and mutant forms of BCR-ABL This study is compliant with the animal care and experimental procedures which were approved by the National Institutes of Health Animal Care and Use Committee .All RMS772 transfected cell lines were established and maintained as previously described We measured relative cell number with the CellTiter-Glo assay (Promega) or relative percent confluency with the IncuCyte (Essen Bioscience). In brief, cells were seeded overnight in 180 µL of culture medium per well in 96-well plates so that they would reach 80% confluency by the end of the assay. After overnight incubation, 20 µL of culture medium containing the various inhibitors was added. Relative cell number was measured at 24 hour intervals after the addition of drug. Prism (GraphPad Software) was used for curve fitting and calculating IC50s.RNA was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen). RNA integrity number (RIN) was calculated using the RNA 6000 Nano Kit (Agilent) and all were greater than 9.0. Quantitative RT-PCR using Taqman assays (FGFR4: Hs00242558_m1 and GAPDH: Hs99999905_m1) on a Fluidigm system was previously described The FITC BrdU Flow Kit (BD Biosciences) was used for cell cycle analysis. In a T25 flask, 1 million cells were seeded and incubated overnight. Cells were treated with ponatinib at a final concentration of 0, 0.625, 1.25, or 2.5 µM for 24 hours. Then the cells were pulsed with 1 mM BrdU for 30 minutes and stained with an anti-BrdU antibody, followed by 7-AAD staining per the manufacturer's guidelines. FACS data was analyzed using CellQuest software (BD Biosciences).In an opaque, flat-bottom 96-well plate, 5,000 cells were seeded in each well with 80 µL of culture medium. After overnight incubation, 20 µL of culture medium containing ponatinib was added to reach final concentrations of 0, 1.25, 2.5, and 5 µM. After 6 hours, ApoLive-Glo (Promega) was used to measure caspase-3/7 activity per the manufacturer's protocol.RH4, RH5, CTR, RH41, RMS772/FGFR4 (N535K), and RMS772/FGFR4 (V550E) cell lines were treated with ponatinib at 0, 200, and 800 nM for 8 hours. Cells were lysed in RIPA buffer  with 1% Halt Protease and Phosphatase Inhibitor Cocktail (Thermo). For FGFR4 autophosphorylation immunoblots, 200–500 µg of protein lysate, as determined by BCA protein assay (Pierce), was first immunoprecipitated with a FGFR4 antibody  and then incubated overnight with protein A/G agarose beads. Proteins were resolved by SDS-PAGE in a 4–12% Bis-Tris Gel (Invitrogen) and transferred to a nitrocellulose membrane by the iBlot (Invitrogen). Membranes were blocked with 5% nonfat dry milk in PBS with 0.1% Tween-20 (PBST) for one hour and probed overnight with anti-phosphotyrosine , FGFR4, STAT3 , and phospho-STAT3  antibodies. HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Thermo) were used to detect the primary antibodies. Finally, ECL (GE Biosciences) or SuperSignal (Thermo) was added and signal was detected on Biomax MR X-ray film (Kodak).in vivo tumor growth. Approximately 1 million cells were injected subcutaneously into the right flank of each mouse. Mice were monitored every other day. Tumor volume measurements were also performed every other day by caliper and the following formula was used to calculate tumor size: (long axis x short axis2)/2. Daily oral administration by gavage feeding of ponatinib at 30 mg/kg started when the tumor volume exceeded 100 mm3. Mice were euthanized when tumors reached 1,500 mm3.Animal studies were conducted with 6- to 8-week-old nude female, athymic NCr-nu/nu mice . RMS772 transductants were used to assess pDonr253 is a Gateway Donor vector modified from pDonr201 (Life Technologies). pDonr253 replaces the kanamycin resistance gene with a gene encoding spectinomycin resistance, and contains several sequencing primer sites to aid in sequence verification of Entry clones. The following oligonucleotides (Eurofins MWG Operon) were used in this study:7464: 5′- atgtctgagactcctgctcagtg7465: 5′- ggagcggtgcaacagttcaatgg7466: 5′- ccattgaactgttgcaccgctccCCCGCCTTGCTCGCCGGCCTCGTGAG7467: 5′- tgtctgcaccccagacccgaagggg7468: 5′- GGGGACAACTTTGTACAAAAAAGTTGGCACCATGtctgagactcctgctcagtg7469: 5′- GGGGACAACTTTGTACAAGAAAGTTGATTAttatgtctgcaccccagacccgaaggggThe TEL-FGFR4 gene was constructed by fusing the kinase domain of FGFR4 in frame with the extracellular domain of TEL .The TEL-pMSCV-hyg (Clontech) was digested with HpaI and a Gateway reading frame cassette (Life Technologies) was introduced. Proper clones were selected for using ampicillin and chloramphenicol and sequence validated for directionality of the insert and proper sequence at the junctions. The modified Gateway Destination vector was called pDest-450 and was used for subcloning of the TEL-FGFR4 Entry clone via Gateway LR recombination (Life Technologies). Final clones were then transformed into E. coli STBL3 cells (Life Technologies) and final expression constructs were validated by agarose gel electrophoresis and restriction mapping.Retrovirus containing the TEL-FGFR4 fusion gene was transfected into the PT67 packaging cells (Invitrogen) using Fugene 6 Transfection Reagent (Roche). Two days later, the cell culture supernatant containing the virus was collected, centrifuged, and filtered. Polybrene (Sigma-Aldrich) was added to the viral supernatant at a final concentration of 5 µg/mL. The resulting supernatant was then used to infect the Ba/F3 cells for eight hours. The stable, transfected cells were established by selecting cells in full media containing 1 mg/ml of hygromycin.CCGACAGCAGCTCTGCCTAC and reverse primer ATGAACTTGCTGTGTAGGGACAG to amplify the fusion gene. PCR product was analyzed using the DNA 1000 kit (Agilent) to check for a 172 bp band.PAX3/7-FOXO1 fusion gene status was determined by RT-PCR. The same cDNA used in the quantitative RT-PCR was also used for this PCR . We used the forward primer GATTCAGCCCTAGACCTACG; exon 12 reverse: CACTCCACGATCACGTAC; exon 13 forward: CAACCTGCTTGGTGTCTG; and exon 13 reverse: GGAAAGCGTGAATGCCTG. Cell line DNA was amplified by PCR and the product was confirmed by gel electrophoresis. PCR DNA was purified using the AMPure XP kit (Agencourt) and then sequenced by Sanger sequencing. Sequencing results were analyzed with Sequencher (Gene Codes).We used the following PCR Primers for FGFR4 protein-coding exons 12 and 13—exon 12 forward: 50 of 72.2 nM  compared to wild-type FGFR4 , suggesting that the activating FGFR4 mutations make cells more sensitive to ponatinib than wild-type FGFR4. Furthermore, we found in a kinetic study that the effect of ponatinib on cell confluency was rapid, within 6 hours, in RMS772 cell lines harboring the N535K or V550E mutations . Thus far, at least three mechanisms have been reported to result in the gain of function of FGFR4 in RMS. First, elevated FGFR4 expression in RMS tumors can be a direct result of the In our previous study, we have shown that RMS772 cell lines expressing the FGFR4 V550E or N535K mutation were sensitive to PD173074, a FGFR inhibitor, in the micromolar range 50 values in the nanomolar range. Our data confirmed that FGFR4 mRNA expression was significantly higher in fusion-positive cell lines than fusion-negative cell lines  The TEL-FGFR4 construct was created by fusing the extracellular PNT domain of ETV6/TEL in frame with the intracellular kinase domain of FGFR4. ETV6/TEL consists of a pointed (PNT) domain, which polymerizes, and an ETS domain that binds to DNA. FGFR4 contains three extracellular immunoglobulin (IG) domains, a transmembrane domain (unlabeled), and an intercellular tyrosine kinase (TK) domain. Numbers represent start and end sites of domains along the amino acid sequence. (B) Expression and autophosphorylation of FGFR4 is present in the Ba/F3 TEL-FGFR4 model system as shown by immunoprecipitation of FGFR4 and then western blotting against phosphotyrosine. Expression and autophosphorylation of FGFR4 is not present in Ba/F3 cells expressing the empty vector. β-actin was probed as well to ensure equal loading of protein. (C) Ba/F3 cells that were retrovirally transfected with the TEL-FGFR4 construct survived independently of IL-3 over 72 hours. However, Ba/F3 cells expressing the vector control only survived with IL-3 supplementation.(PPTX)Click here for additional data file.Figure S2Ponatinib is the most potent FGFR4 inhibitor and inhibits wild-type FGFR4 phosphorylation. (A) FGFR inhibitor screen with the Ba/F3 TEL-FGFR4 model system shows ponatinib (AP24534) to be the most potent inhibitor among the four other FGFR inhibitors, TKI258, BIBF1120, PHA739358, and AZD2172, in addition to the MET inhibitor, PHA665752, as a control. (B) Immunoprecipitation of FGFR4 and immunoblotting against phosphotyrosine shows a dose-dependent inhibition of FGFR4 phosphorylation with ponatinib using the Ba/F3 TEL-FGFR4 model system.(PPTX)Click here for additional data file.Figure S3PAX3/7-FOXO1 fusion status of cell lines and fusion-positive RMS cell lines express higher levels of FGFR4 mRNA. (A) RT-PCR with a PAX3/7-FOXO1 primer (forward: CCGACAGCAGCTCTGCCTAC and reverse: ATGAACTTGCTGTGTAGGGACAG) shows cell lines RH5, RH4, JR, RH41, RH28, and RH30 to be fusion-positive while cell lines RH18, CTR, BIRCH, RD, TTC-516, CT-10, TTC-442, and RH36 to be fusion-negative. The PAX3/7-FOXO1 band appears at 172 bp. (B) Comparison of FGFR4 mRNA expression levels between fusion-positive (FP) and fusion-negative (FN) cell lines reveals fusion-positive cell lines to express FGFR4 at higher levels than fusion-negative cell lines (p = 0.0005). FGFR4 expression was normalized to GAPDH expression.(PPTX)Click here for additional data file.Figure S4Kinetic analysis of ponatinib-induced growth inhibition for the RMS772 transductants as measured by confluency. (A) Growth curves for the RMS772 transductants illustrate differential sensitivity to ponatinib. Arrow indicates when ponatinib was added. (B) IC50 calculation at 6, 12, and 24 hours after the addition of ponatinib for RMS772 transductants shows a decrease in IC50 as time increases.(PPTX)Click here for additional data file.Figure S51 phase and decreases time in S phase when RH4, RH5, and the two RMS772 cell lines expressing FGFR4 mutations (N535K and V550E) are treated with 0.625 and 1.25 µM ponatinib for 24 hours.Ponatinib (AP24534) holds cell cycling at sub G(PPTX)Click here for additional data file.Figure S6Dot plot of cell cycle flow cytometry data showing the boxes used to determine cell cycle fraction for  and  Each box represents a different cell cycle phase, starting with the top box and going clockwise: S phase, G2 phase, G1 phase, and subG1 phase.(PPTX)Click here for additional data file.Figure S7Treatment of cell lines RH4, RH5, and the two RMS772 cell lines expressing mutated FGFR4 with 0, 1.25, 2.5, and 5 µM ponatinib for 6 hours increases caspase 3/7 levels across all four cell lines.(PPTX)Click here for additional data file.Figure S850s for cell lines (A) 7250 LNCX NILC, (B) U2-OS, and (C) A4573 after 72 hour treatment with ponatinib.Fitted drug-dose response curves and calculated IC(PPTX)Click here for additional data file.Table S1FGFR inhibition profiles for tested inhibitors.(XLSX)Click here for additional data file.Table S2Kinase inhibition profile of tested FGFR inhibitors (XLSX)Click here for additional data file.