Phosphatase of regenerating liver-3 (PRL-3) is a member of the novel phosphatases of regenerating liver family, characterized by one protein tyrosine phosphatase active domain and a C-terminal prenylation (CCVM) motif. Though widely proposed to facilitate metastasis in many cancer types, PRL-3's cellular localization and the function of its CCVM motif in metastatic process remain unknown.

Polycystin-1 (PC-1), the product of the PKD1 gene, mutated in the majority of cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD), is a very large (approximately 520 kDa) plasma membrane receptor localized in several subcellular compartments including cell-cell/matrix junctions as well as cilia. While heterologous over-expression systems have allowed identification of several of the potential biological roles of this receptor, its precise function remains largely elusive. Studying PC-1 in vivo has been a challenging task due to its complexity and low expression levels. To overcome these limitations and facilitate the study of endogenous PC-1, we have inserted HA- or Myc-tag sequences into the Pkd1 locus by homologous recombination. Here, we show that our approach was successful in generating a fully functional and easily detectable endogenous PC-1. Characterization of PC-1 distribution in vivo showed that it is expressed ubiquitously and is developmentally-regulated in most tissues. Furthermore, our novel tool allowed us to investigate the role of PC-1 in brain, where the protein is abundantly expressed. Subcellular localization of PC-1 revealed strong and specific staining in ciliated ependymal and choroid plexus cells. Consistent with this distribution, we observed hydrocephalus formation both in the ubiquitous knock-out embryos and in newborn mice with conditional inactivation of the Pkd1 gene in the brain. Both choroid plexus and ependymal cilia were morphologically normal in these mice, suggesting a role for PC-1 in ciliary function or signalling in this compartment, rather than in ciliogenesis. We propose that the role of PC-1 in the brain cilia might be to prevent hydrocephalus, a previously unrecognized role for this receptor and one that might have important implications for other genetic or sporadic diseases.

Converting single-stranded viral RNA into double stranded DNA for integration is an essential step in HIV-1 replication. Initial polymerization of minus-strand DNA is primed from a host derived tRNA, whereas subsequent plus-strand synthesis requires viral primers derived from the 3' and central polypurine tracts (3' and cPPTs). The 5' and 3' termini of these conserved RNA sequence elements are precisely cleaved by RT-associated RNase H to generate specific primers that are used to initiate plus-strand DNA synthesis. In this study, siRNA wad used to produce a replicative HIV-1 variant contained G(-1)A and T(-16)A substitutions within/adjacent to the 3'PPT sequence. Introducing either or both mutations into the 3'PPT region or only the G(-1)A substitution in the cPPT region of NL4-3 produced infectious virus with decreased fitness relative to the wild-type virus. In contrast, introducing the T(-16)A or both mutations into the cPPT rendered the virus(es) incapable of replication, most likely due to the F185L integrase mutation produced by this nucleotide substitution. Finally, the effects of G(-1)A and T(-16)A mutations on cleavage of the 3'PPT were examined using an in vitro RNase H cleavage assay. Substrate containing both mutations was mis-cleaved to a greater extent than either wild-type substrate or substrate containing the T(-16)A mutation alone, which is consistent with the observed effects of the equivalent nucleotide substitutions on the replication fitness of NL4-3 virus. In conclusion, siRNA targeting of the HIV-1 3'PPT region can substantially suppress virus replication, and this selective pressure can be used to generate infectious virus containing mutations within or near the HIV-1 PPT. Moreover, in-depth analysis of the resistance mutations demonstrates that although virus containing a G(-1)A mutation within the 3'PPT is capable of replication, this nucleotide substitution shifts the 3'-terminal cleavage site in the 3'PPT by one nucleotide (nt) and significantly reduces viral fitness.

Gastric cancer is the most common cancer and the most frequent cause of cancer death worldwide. Several studies have identified the role of thymidylate synthase (TS) 5'- and 3'-UTR and gastric cancer susceptibility; however, the results still remain inconclusive. The purpose of this meta-analysis was to reinvestigate this correlation. In the present study, online databases were searched to retrieve relevant articles published between January 2000 and 2016. The odds ratio (OR) and 95% confidence interval (CI) were employed to calculate the strength of association. Overall, a total of 13 articles were screened out, including 2382 gastric cancer patients and 3171 healthy controls. We found that polymorphisms of TS 5'-UTR 2R (double repeats)/3R (triple repeats) of a 28-bp sequence (11 articles) and 3'-UTR del6/ins6 (seven articles) were not significantly associated with increased risk of gastric cancer. Subgroup analysis by ethnicity showed that 2R allele and 2R/2R genotype in TS 5'-UTR were associated with gastric cancer susceptibility in Caucasian and African populations; del6 allele, del6/del6 and del6/ins6 genotypes were correlated with gastric cancer in Caucasian population. In conclusion, our result suggested that TS polymorphisms might be the risk factors for gastric cancer risk in Caucasian population, although this association needs further study, and future large-scale researches are still required.

Substantial evidence suggests that the epithelial-mesenchymal transition (EMT) phenotype is associated with the invasive characteristics of cancer stem cells (CSCs),which possess an EMT phenotype that may predominate in tumor invasion and metastasis. However, the mechanisms for the generation and regulation of these CSCs have not been clearly defined. As hypoxia and EMT-related factors may have important functions in EMT-like CSCs, the aim of this study was to investigate the effects of hypoxia on these cells. CSCs were established from the gastric cancer cell lines MGC-803 and SGC7901, and the relationship between hypoxia and EMT-like CSCs was investigated in gastric cancer. After hypoxia treatment, some gastric CSCs exhibited a marked increase in hypoxia-inducible factor-1α (HIF-1α)expression and increased migration and invasion capabilities compared with the normoxic control. These CSCs were defined by activation of the mesenchymal cell marker Vimentin and by inhibition of the epithelial cell marker E-cadherin. Our analyses also show that HIF-1α was responsible for activating EMT via increased expression of the transcription factor Snail in gastric CSCs. Moreover, inhibition of Snail by shRNA reduced HIF-1α-induced EMT in gastric CSCs. The results demonstrated that hypoxia-induced EMT-like CSCs rely on HIF-1αto activate Snail, which may result in recurrence and metastasis of gastric cancer.

Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms. In particular, the immunogenicity in mice of a recombinant malaria transmission blocking vaccine candidate, the ookinete specific Plasmodium falciparum 25 kDa protein (Pfs25), was increased more than 1000-fold when evaluated as a chemical cross-linked protein-protein conjugate as compared to a formulated monomer. Whether alternative approaches using protein complexes improve the immunogenicity of other recombinant malaria vaccine candidates is worth assessing. In this work, the immunogenicity of the recombinant 42 kDa processed form of the P. falciparum merozoite surface protein 1 (MSP1(42)) was evaluated as a self-associated, non-covalent aggregate and as a chemical cross-linked protein-protein conjugate to ExoProtein A, which is a recombinant detoxified form of Pseudomonas aeruginosa exotoxin A. MSP1(42) conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP1(42) self-associated aggregates, cross-linked chemical conjugates and monomers were compared in BALB/c mice after adsorption to aluminum hydroxide adjuvant, and in one instance in association with the TLR9 agonist CPG7909 with an aluminum hydroxide formulation. Antibody titers were assessed by ELISA. Unlike observations made for Pfs25, no significant enhancement in MSP1(42) specific antibody titers was observed for any conjugate as compared to the formulated monomer or dimer, except for the addition of the TLR9 agonist CPG7909. Clearly, enhancing the immunogenicity of a recombinant protein vaccine candidate by the formation of protein complexes must be established on an empirical basis.

The process of peritoneal metastasis involves the diapedesis of intra-abdominal exfoliated gastric cancer cells through the mesothelial cell monolayers; however, the related molecular mechanisms for this process are still unclear. Heterocellular gap-junctional intercellular communication (GJIC) between gastric cancer cells and mesothelial cells may play an active role during diapedesis. In this study we detected the expression of connexin 43 (Cx43) in primary gastric cancer tissues, intra-abdominal exfoliated cancer cells, and matched metastatic peritoneal tissues. We found that the expression of Cx43 in primary gastric cancer tissues was significantly decreased; the intra-abdominal exfoliated cancer cells and matched metastatic peritoneal tissues exhibited increasing expression compared with primary gastric cancer tissues. BGC-823 and SGC-7901 human gastric cancer cells were engineered to express Cx43 or Cx43T154A (a mutant protein that only couples gap junctions but provides no intercellular communication) and were co-cultured with human peritoneal mesothelial cells (HPMCs). Heterocellular GJIC and diapedesis through HPMC monolayers on matrigel-coated coverslips were investigated. We found that BGC-823 and SGC-7901 gastric cancer cells expressing Cx43 formed functional heterocellular gap junctions with HPMC monolayers within one hour. A significant increase in diapedesis was observed in engineered Cx43-expressing cells compared with Cx43T154A and control group cells, which suggested that the observed upregulation of diapedesis in Cx43-expressing cells required heterocellular GJIC. Further study revealed that the gastric cancer cells transmigrated through the intercellular space between the mesothelial cells via a paracellular route. Our results suggest that the abnormal expression of Cx43 plays an essential role in peritoneal metastasis and that Cx43-mediated heterocellular GJIC between gastric cancer cells and mesothelial cells may be an important regulatory step during metastasis. Finally, we observed that the diapedesis of exfoliated gastric cancer cells through mesothelial barriers is a viable route of paracellular migration.

The West Nile virus (WNV) is an emerging infection of biodefense concern and there are no available treatments or vaccines. Here we used a high-throughput method based on a novel gene expression analysis, RNA-Seq, to give a global picture of differential gene expression by primary human macrophages of 10 healthy donors infected in vitro with WNV. From a total of 28 million reads per sample, we identified 1,514 transcripts that were differentially expressed after infection. Both predicted and novel gene changes were detected, as were gene isoforms, and while many of the genes were expressed by all donors, some were unique. Knock-down of genes not previously known to be associated with WNV resistance identified their critical role in control of viral infection. Our study distinguishes both common gene pathways as well as novel cellular responses. Such analyses will be valuable for translational studies of susceptible and resistant individuals--and for targeting therapeutics--in multiple biological settings.

Hepatocellular carcinoma (HCC) is a liver cancer that could be induced by hepatitis C virus genotype 2a Japanese fulminant hepatitis-1 (JFH-1) strain. The aim of this study was to investigate the molecular mechanisms of HCC. The microarray data GSE20948 includes 14 JFH-1- and 14 mock (equal volume of medium [control])-infected Huh7 samples. The data were downloaded from the Gene Expression Omnibus. After data processing, soft cluster analyses were performed to identify co-regulated genes with similar temporal expression patterns. Functional and pathway enrichment analyses, as well as functional annotation analysis, were performed. Subsequently, combined networks of protein-protein interaction network, microRNA regulatory network, and transcriptional regulatory network were constructed. Hub nodes, modules, and five clusters of co-regulated genes were also identified. In total, 173 up and 207 down co-regulated genes were separately identified in JFH-1-infected Huh7 cells compared with those of control cells. Functional enrichment analysis indicated that up co-regulated genes were related to skeletal system morphogenesis and neuron differentiation and down co-regulated genes were related to steroid/cholesterol/sterol metabolisms. Hub genes (such as IRF1, GBP1, ICAM1, Foxa1, DHCR7, HMGCS2, and MSMO1) were identified. Transcription factors IRF1 and Foxa1 were the targets of miR-130a, miR-17-5p, and miR-20a. PPARGC1A was targeted by miR-29 family, and MSMO1 was the target of miR-23 family. Hub nodes (such as IRF1, GBP1, ICAM1, Foxa1, DHCR7, HMGCS2, and MSMO1) and microRNAs might be used as candidate biomarkers of JFH-1-infected HCC.

Alterations in myocardial wall texture stand out among ADPKD cardiovascular manifestations in hypertensive and normotensive patients. To elucidate their pathogenesis, we analyzed the cardiac phenotype in Pkd1(cond/cond)Nestin(cre) (CYG+) cystic mice exposed to increased blood pressure, at 5 to 6 and 20 to 24 weeks of age, and Pkd1(+/-) (HTG+) noncystic mice at 5-6 and 10-13 weeks. Echocardiographic analyses revealed decreased myocardial deformation and systolic function in CYG+ and HTG+ mice, as well as diastolic dysfunction in older CYG+ mice, compared to their Pkd1(cond/cond) and Pkd1(+/+) controls. Hearts from CYG+ and HTG+ mice presented reduced polycystin-1 expression, increased apoptosis, and mild fibrosis. Since galectin-3 has been associated with heart dysfunction, we studied it as a potential modifier of the ADPKD cardiac phenotype. Double-mutant Pkd1(cond/cond):Nestin(cre);Lgals3(-/-) (CYG-) and Pkd1(+/-);Lgals3(-/-) (HTG-) mice displayed improved cardiac deformability and systolic parameters compared to single-mutants, not differing from the controls. CYG- and HTG- showed decreased apoptosis and fibrosis. Analysis of a severe cystic model (Pkd1(V/V); VVG+) showed that Pkd1(V/V);Lgals3(-/-) (VVG-) mice have longer survival, decreased cardiac apoptosis and improved heart function compared to VVG+. CYG- and VVG- animals showed no difference in renal cystic burden compared to CYG+ and VVG+ mice. Thus, myocardial dysfunction occurs in different Pkd1-deficient models and suppression of galectin-3 expression rescues this phenotype.

Pulmonary fibrosis (PF) leads to progressive and often irreversible loss of lung functions, posing a health threat with no effective cure. We examined P-Rex1, a PI3K- and G protein βγ-regulated guanine nucleotide exchange factor (GEF) of the Rac small GTPase, for its potential involvement in PF. In a bleomycin-induced PF model, mice deficient in p-rex1 had well-preserved alveolar structure and survived significantly better than their wild type (WT) littermates. The p-rex1(-/-) mice expressed significantly less proinflammatory cytokines and chemokines and had reduced leukocyte infiltration in the lung tissue than their WT littermates. P-Rex1 was detected in lung fibroblasts of WT mice, and its genetic deletion attenuated TGFβ-1-stimulated lung fibroblast migration, Rac1 activation and p38 MAPK phosphorylation. The p-rex1(-/-) mice showed significantly reduced pathological changes including the expression of α-smooth muscle actin, fibronectin and TGFβ-1 compared with their WT controls. Expression of a GEF-deficient P-Rex1 mutant effectively blocked Smads-dependent transcriptional activation, suggesting that P-Rex1 is a downstream mediator of TGFβ-1 signaling. These findings identify P-Rex1 as a novel player of PF, suggesting that targeting P-Rex1 may simultaneously block the inflammatory and fibrogenic processes of PF.

Inflammatory monocyte and tissue macrophages influence the initiation, progression, and resolution of type 2 immune responses, and alveolar macrophages are the most prevalent immune-effector cells in the lung. While we were characterizing the M1- or M2-like macrophages in type 2 allergic inflammation, we discovered that FoxO1 is highly expressed in alternatively activated macrophages. Although several studies have been focused on the fundamental role of FoxOs in hematopoietic and immune cells, the exact role that FoxO1 plays in allergic asthmatic inflammation in activated macrophages has not been investigated. Growing evidences indicate that FoxO1 acts as an upstream regulator of IRF4 and could have a role in a specific inflammatory phenotype of macrophages. Therefore, we hypothesized that IRF4 expression regulated by FoxO1 in alveolar macrophages is required for established type 2 immune mediates allergic lung inflammation. Our data indicate that targeted deletion of FoxO1 using FoxO1-selective inhibitor AS1842856 and genetic ablation of FoxO1 in macrophages significantly decreases IRF4 and various M2 macrophage-associated genes, suggesting a mechanism that involves FoxO1-IRF4 signaling in alveolar macrophages that works to polarize macrophages toward established type 2 immune responses. In response to the challenge of DRA (dust mite, ragweed, and Aspergillus) allergens, macrophage specific FoxO1 overexpression is associated with an accentuation of asthmatic lung inflammation, whereas pharmacologic inhibition of FoxO1 by AS1842856 attenuates the development of asthmatic lung inflammation. Thus, our study identifies a role for FoxO1-IRF4 signaling in the development of alternatively activated alveolar macrophages that contribute to type 2 allergic airway inflammation.

Runt-related transcription factor 2 (RUNX2) is a regulator of embryogenesis and development, but has also been implicated in the progression of certain human cancer. This study aimed to elucidate the role of RUNX2 in the invasive and metastatic potentials of human gastric cancer (GC) and the underlying mechanisms. We found that the levels of RUNX2 expression in gastric cancer tissues were correlated with the differentiation degrees, invasion depth and lymph node metastasis. COX regression analysis indicated that RUNX2 was an independent prognostic indicator for GC patients. RUNX2 significantly increased the migration and invasion ability of GC cells in vitro and enhanced the invasion and metastatic potential of GC cells in an orthotopic GC model of nude mice. Mechanistically, RUNX2 directly bound to the promoter region of the gene coding for the chemokine receptor CXCR4 to enhance its transcription. CXCR4 knockdown or treatment with AMD3100, a CXCR4 inhibitor, attenuated RUNX2-promoted invasion and metastasis. These results demonstrate that RUNX2 promotes the invasion and metastasis of human GC by transcriptionally up-regulating the chemokine receptor CXCR4. Therefore, the RUNX2-CXCR4 axis is a potential therapeutic target for GC.

Autosomal dominant polycystic kidney disease (ADPKD) is estimated to affect 1/600-1/1000 individuals worldwide. The disease is characterized by age dependent renal cyst formation that results in kidney failure during adulthood. Although ultrasound imaging may be an adequate diagnostic tool in at risk individuals older than 30, this modality may not be sufficiently sensitive in younger individuals or for those from PKD2 families who have milder disease. DNA based assays may be indicated in certain clinical situations where imaging cannot provide a definitive clinical diagnosis. The goal of this study was to evaluate the utility of direct DNA analysis in a test sample of 82 individuals who were judged to have polycystic kidney disease by standard clinical criteria. The samples were analyzed using a commercially available assay that employs sequencing of both genes responsible for the disorder. Definite disease causing mutations were identified in 34 (approximately 42%) study participants. An additional 30 (approximately 37%) subjects had either in frame insertions/deletions, non-canonical splice site alterations or a combination of missense changes that were also judged likely to be pathogenic. We noted striking sequence variability in the PKD1 gene, with a mean of 13.1 variants per participant (range 0-60). Our results and analysis highlight the complexity of assessing the pathogenicity of missense variants particularly when individuals have multiple amino acid substitutions. We conclude that a significant fraction of ADPKD mutations are caused by amino acid substitutions that need to be interpreted carefully when utilized in clinical decision-making.

Pancreatic ductal adenocarcinoma (PDAC) is a dominantly (~95%) KRAS-mutant cancer that has extremely poor prognosis, in part this is due to its strong intrinsic resistance towards almost all therapeutic agents. PDAC relies heavily on KRAS-transformed metabolism, including enhanced macropinocytosis and catabolism of extracellular albumin, to maintain its proliferation and progression. However, it has yet to be validated that whether such transformed metabolism could be exploited for the drug delivery to open therapeutic windows of cytotoxic agents in KRAS-mutant PDAC. In this study, we attempt to answer this question by focusing on the impact of two critical regulators of albumin catabolism, KRAS and the neonatal Fc receptor (FcRn), on the sensitivity of PDAC to doxorubicin (DOX, a model cytotoxic agent) and albumin-conjugated doxorubicin (DOX-ALB). Using cell lines and cell-derived xenografts with different KRAS genotypes and FcRn levels, we demonstrated that KRAS-enhanced macropinocytosis and reduced FcRn expression sensitize PDAC to DOX-ALB but not free DOX. In both in vitro and in vivo comparsion, the DOX-ALB demonstrated ~10 times enlarged therapeutic window compared with free DOX, in PDAC with KRAS mutation and reduced FcRn level, two events appear to occur simultaneously in the investigated PDAC. In summary, we conclude that albumin conjugation is an exploitable drug delivery strategy that significantly opens the therapeutic windows of otherwise undevelopable anti-cancer agents for KRAS-mutant PDAC therapy, and creates a new landscape for clinical evaluation and future translation of such compounds.

The aberrant expression of myotubularin-related protein 2 (MTMR2) has been found in some cancers, but little is known about the roles and clinical relevance. The present study aimed to investigate the roles and clinical relevance of MTMR2 as well as the underlying mechanisms in gastric cancer (GC).

Dengue virus (DENV) is a mosquito-borne single-stranded RNA virus causing human disease with variable severity. The production of massive inflammatory cytokines in dengue patients has been associated with dengue disease severity. However, the regulation of these inflammatory responses remains unclear. In this study, we report that SIRT6 is a negative regulator of innate immune responses during DENV infection. Silencing of Sirt6 enhances DENV-induced proinflammatory cytokine and chemokine production. Overexpression of SIRT6 inhibits RIG-I-like receptor (RLR) and Toll-like receptor 3 (TLR3) mediated NF-κB activation. The sirtuin core domain of SIRT6 is required for the inhibition of NF-κB p65 function. SIRT6 interacts with the DNA binding domain of p65 and competes with p65 to occupy the Il6 promoter during DENV infection. Collectively, our study demonstrates that SIRT6 negatively regulates DENV-induced inflammatory response via RLR and TLR3 signaling pathways.

Myocardial ischemia/reperfusion (MIR) injury, which occurs following acute myocardial infarction, can cause secondary damage to the heart. Tripartite interaction motif (TRIM) proteins, a class of E3 ubiquitin ligases, have been recognized as critical regulators in MIR injury. Zenglv Fumai Granule (ZFG) is a clinical prescription for the treatment of sick sinus syndrome, a disease that is associated with MIR injury. The present study aimed to investigate the effect of ZFG on MIR injury and to determine whether ZFG exerts its effects via regulation of TRIM proteins. In order to establish an in vitro MIR model, human cardiomyocyte cell line AC16 was cultured under hypoxia for 5 h and then under normal conditions for 1 h. Following hypoxia/reoxygenation (H/R) treatment, these cells were cultured with different ZFG concentrations. ZFG notably inhibited H/R-induced cardiomyocyte apoptosis. The expression levels of four TRIM proteins, TRIM7, TRIM14, TRIM22 and TRIM28, were also detected. These four proteins were significantly upregulated in H/R-injured cardiomyocytes, whereas their expression was inhibited following ZFG treatment. Moreover, TRIM28 knockdown inhibited H/R-induced cardiomyocyte apoptosis, whereas TRIM28 overexpression promoted apoptosis and generation of reactive oxygen species (ROS) in cardiomyocytes. However, the effects of TRIM28 overexpression were limited by the action of ROS inhibitor N-acetyl-L-cysteine. In addition, the mRNA and protein levels of antioxidant enzyme glutathione peroxidase (GPX)1 were significantly downregulated in H/R-injured cardiomyocytes. TRIM28 knockdown restored GPX1 protein levels but had no effect on mRNA expression levels. Co-immunoprecipitation and ubiquitination assays demonstrated that TRIM28 negatively regulated GPX1 via ubiquitination. In sum, the present study revealed that ZFG attenuated H/R-induced cardiomyocyte apoptosis by regulating the TRIM28/GPX1/ROS pathway. ZFG and TRIM28 offer potential therapeutic options for the treatment of MIR injury.

The radioprotective effect of herb epimedium (or yin yang huo) extract (5 g/kg, oral administration daily for 4 weeks) on neurogenesis and cognition after acute radiation exposure with 5.5 Gy was evaluated in Balb/c mice by behavioral tests and immunohistochemical study. The results indicated that epimedium extract could improve animal weight loss, locomotor activity and spatial learning and memory which are similar to pre-irradiation intraperitoneal injection (100 mg/kg) of amifostine phosphate, a well- known radioprotective drug. Immunohistochemical study showed that epimedium extract prevented the loss of proliferation cells, newly generated neurons, and interneurons in the hilus, in particular, the subgranular zone of the dentate gyrus. It suggests that herb epimedium may be a promising radio-neuro-protective drug to prevent radiation-induced neuropsychological disorders.

Autosomal recessive polycystic kidney disease (OMIM 263200) is a serious condition of the kidney and liver caused by mutations in a single gene, PKHD1. This gene encodes fibrocystin/polyductin (FPC, PD1), a large protein shown by in vitro studies to undergo Notch-like processing. Its cytoplasmic tail, reported to include a ciliary targeting sequence, a nuclear localization signal, and a polycystin-2 binding domain, is thought to traffic to the nucleus after cleavage. We now report a novel mouse line with a triple HA-epitope "knocked-in" to the C-terminus along with lox P sites flanking exon 67, which encodes most of the C-terminus (Pkhd1Flox67HA). The triple HA-epitope has no functional effect as assayed by phenotype and allows in vivo tracking of Fibrocystin. We used the HA tag to identify previously predicted Fibrocystin cleavage products in tissue. In addition, we found that Polycystin-2 fails to co-precipitate with Fibrocystin in kidney samples. Immunofluorescence studies with anti-HA antibodies demonstrate that Fibrocystin is primarily present in a sub-apical location the in kidney, biliary duct, and pancreatic ducts, partially overlapping with the Golgi. In contrast to previous studies, the endogenous protein in the primary cilia was not detectable in mouse tissues. After Cre-mediated deletion, homozygous Pkhd1Δ67 mice are completely normal. Thus, Pkhd1Flox67HA is a valid model to track Pkhd1-derived products containing the C-terminus. Significantly, exon 67 containing the nuclear localization signal and the polycystin-2 binding domain is not essential for Fibrocystin function in our model.