Engineering and characterization of GFP-targeting nanobody: Expression, purification, and post-translational modification analysis.

Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for biological research, diagnosis, and treatment. Given their simple structure, nanobodies are readily produced in multiple systems. However, selecting an appropriate expression system is crucial because different conditions might cause proteins to produce different folds or post-translational modifications (PTMs), and these differences often result in different functions. At present, the strategies of PTMs are rarely reported. The GFP nanobody can specifically target the GFP protein. Here, we engineered a GFP nanobody fused with 6 × His tag and Fc tag, respectively, and expressed in bacteria and mammalian cells. The 6 × His-GFP-nanobody was produced from Escherichia coli at high yields and the pull-down assay indicated that it can precipitate the GFP protein. Meanwhile, the Fc-GFP-nanobody can be expressed in HEK293T cells, and the co-immunoprecipitation experiment can trace and target the GFP-tagged protein in vivo. Furthermore, some different PTMs in antigen-binding regions have been identified after using mass spectrometry (MS) to analyze the GFP nanobodies, which are expressed in prokaryotes and eukaryotes. In this study, a GFP nanobody was designed, and its binding ability was verified by using the eukaryotic and prokaryotic protein expression systems. In addition, this GFP nanobody was transformed into a useful instrument for more in-depth functional investigations of GFP fusion proteins. MS was further used to explore the reason for the difference in binding ability, providing a novel perspective for the study of GFP nanobodies and protein expression purification.

The Nucleolar Protein C1orf131 Is a Novel Gene Involved in the Progression of Lung Adenocarcinoma Cells through the AKT Signalling Pathway.

Lung adenocarcinoma (LUAD) is the most widespread cancer in the world, and its development is associated with complex biological mechanisms that are poorly understood. Here, we revealed a marked upregulation in the mRNA level of C1orf131 in LUAD samples compared to non-tumor tissue samples in The Cancer Genome Atlas (TCGA). Depletion of C1orf131 suppressed cell proliferation and growth, whereas it stimulated apoptosis in LUAD cells. Mechanistic investigations revealed that C1orf131 knockdown induced cell cycle dysregulation via the AKT and p53/p21 signalling pathways. Additionally, C1orf131 knockdown blocked cell migration through the modulation of epithelial-mesenchymal transition (EMT) in lung adenocarcinoma. Notably, we identified the C1orf131 protein nucleolar localization sequence, which included amino acid residues 137-142 (KKRKLT) and 240-245 (KKKRKG). Collectively, C1orf131 has potential as a novel therapeutic marker for patients in the future, as it plays a vital role in the progression of lung adenocarcinoma.

A1CF Binding to the p65 Interaction Site on NKRF Decreased IFN-ß Expression and p65 Phosphorylation (Ser536) in Renal Carcinoma Cells.

Apobec-1 complementation factor (A1CF) functions as an RNA-binding cofactor for APO-BEC1-mediated C-to-U conversion during RNA editing and as a hepatocyte-specific regulator in the alternative pre-mRNA splicing of metabolic enzymes. Its role in RNA editing has not been clearly established. Western blot, co-immunoprecipitation (Co-IP), immunofluorescence (IF), methyl thiazolyl tetrazolium (MTT), and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to examine the role of A1CF beyond RNA editing in renal carcinoma cells. We demonstrated that A1CF interacts with NKRF, independent of RNA and DNA, without affecting its expression or nuclear translocation; however, it modulates p65(Ser536) phosphorylation and IFN-ß levels. Truncation of A1CF or deletion on NKRF revealed that the RRM1 domain of A1CF and the p65 binding motif of NKRF are required for their interaction. Deletion of RRM1 on A1CF abrogates NKRF binding, and the decrease in IFN-ß expression and p65(Ser536) phosphorylation was induced by A1CF. Moreover, full-length A1CF, but not an RRM1 deletion mutant, promoted cell proliferation in renal carcinoma cells. Perturbation of A1CF levels in renal carcinoma cells altered anchorage-independent growth and tumor progression in nude mice. Moreover, p65(Ser536) phosphorylation and IFN-ß expression were lower, but ki67 was higher in A1CF-overexpressing tumor tissues of a xenograft mouse model. Notably, primary and metastatic samples from renal cancer patients exhibited high A1CF expression, low p65(Ser536) phosphorylation, and decreased IFN-ß levels in renal carcinoma tissues compared with the corresponding paracancerous tissues. Our results indicate that A1CF-decreased p65(Ser536) phosphorylation and IFN-ß levels may be caused by A1CF competitive binding to the p65-combined site on NKRF and demonstrate the direct binding of A1CF independent of RNA or DNA in signal pathway regulation and tumor promotion in renal carcinoma cells.

Transcriptional regulation of MGE progenitor proliferation by PRDM16 controls cortical GABAergic interneuron production.

The mammalian cortex is populated by neurons derived from neural progenitors located throughout the embryonic telencephalon. Excitatory neurons are derived from the dorsal telencephalon, whereas inhibitory interneurons are generated in its ventral portion. The transcriptional regulator PRDM16 is expressed by radial glia, neural progenitors present in both regions; however, its mechanisms of action are still not fully understood. It is unclear whether PRDM16 plays a similar role in neurogenesis in both dorsal and ventral progenitor lineages and, if so, whether it regulates common or unique networks of genes. Here, we show that Prdm16 expression in mouse medial ganglionic eminence (MGE) progenitors is required for maintaining their proliferative capacity and for the production of proper numbers of forebrain GABAergic interneurons. PRDM16 binds to cis-regulatory elements and represses the expression of region-specific neuronal differentiation genes, thereby controlling the timing of neuronal maturation. PRDM16 regulates convergent developmental gene expression programs in the cortex and MGE, which utilize both common and region-specific sets of genes to control the proliferative capacity of neural progenitors, ensuring the generation of correct numbers of cortical neurons.

High-Dose Dexamethasone Manipulates the Tumor Microenvironment and Internal Metabolic Pathways in Anti-Tumor Progression.

High-dose dexamethasone (DEX) is used to treat chemotherapy-induced nausea and vomiting or to control immunotherapy-related autoimmune diseases in clinical practice. However, the underlying mechanisms of high-dose DEX in tumor progression remain unaddressed. Therefore, we explored the effects of high-dose DEX on tumor progression and the potential mechanisms of its anti-tumor function using immunohistochemistry, histological examination, real-time quantitative PCR (qPCR), and Western blotting. Tumor volume, blood vessel invasion, and levels of the cell proliferation markers Ki67 and c-Myc and the anti-apoptotic marker Bcl2 decreased in response to high-dose DEX. However, the cell apoptosis marker cleaved caspase 3 increased significantly in mice treated with 50 mg/kg DEX compared with controls. Some genes associated with immune responses were significantly downregulated following treatment with 50 mg/kg DEX e.g., Cxcl9, Cxcl10, Cd3e, Gzmb, Ifng, Foxp3, S100a9, Arg1, and Mrc1. In contrast, the M1-like tumor-associated macrophages (TAMs) activation marker Nos2 was shown to be increased. Moreover, the expression of peroxisome proliferator-activated receptors α and γ (Pparα and Pparg, respectively) was shown to be significantly upregulated in livers or tumors treated with DEX. However, high-dose DEX treatment decreased the expression of glucose and lipid metabolic pathway-related genes such as glycolysis-associated genes (Glut1, Hk2, Pgk1, Idh3a), triglyceride (TG) synthesis genes (Gpam, Agpat2, Dgat1), exogenous free fatty acid (FFA) uptake-related genes (Fabp1, Slc27a4, and CD36), and fatty acid oxidation (FAO) genes (Acadm, Acaa1, Cpt1a, Pnpla2). In addition, increased serum glucose and decreased serum TG and non-esterified fatty acid (NEFA) were observed in DEX treated-xenografted tumor mice. These findings indicate that high-dose DEX-inhibited tumor progression is a complicated process, not only activated by M1-like TAMs, but also decreased by the uptake and consumption of glucose and lipids that block the raw material and energy supply of cancer cells. Activated M1-like TAMs and inefficient glucose and lipid metabolism delayed tumor cell growth and promoted apoptosis. These findings have important implications for the application of DEX combined with drugs that target key metabolism pathways for tumor therapy in clinical practice.

Celecoxib Ameliorates Seizure Susceptibility in Autosomal Dominant Lateral Temporal Epilepsy.

Autosomal dominant lateral temporal epilepsy (ADLTE) is an inherited syndrome caused by mutations in the leucine-rich glioma inactivated 1 (LGI1) gene. It is known that glutamatergic transmission is altered in LGI1 mutant mice, and seizures can be reduced by restoring LGI1 function. Yet, the mechanism underlying ADLTE is unclear. Here, we propose that seizures in male LGI1-/- mice are due to nonsynaptic epileptiform activity in cortical neurons. We examined the intrinsic excitability of pyramidal neurons in the temporal cortex of male LGI1-/- mice and found that the voltage-gated K+ channel Kv1.2 was significantly downregulated. We also found that cytosolic phospholipase A2 (cPLA2)-cyclooxygenase 2 (Cox2) signaling was enhanced in LGI1-/- mice. Interestingly, Cox2 inhibition effectively restored the dysregulated Kv1.2 and reduced the intrinsic excitability of pyramidal neurons. Moreover, in vivo injection of celecoxib, an FDA-approved nonsteroidal anti-inflammatory drug, rescued the defective Kv1.2 (an ∼1.9-fold increase), thereby alleviating the seizure susceptibility and extending the life of LGI1-/- mice by 5 d. In summary, we conclude that LGI1 deficiency dysregulates cPLA2-Cox2 signaling to cause hyperexcitability of cortical pyramidal neurons, and celecoxib is a potential agent to manage human ADLTE.SIGNIFICANCE STATEMENT Haploinsufficiency of the leucine-rich glioma inactivated 1 (LGI1) gene is the major pathogenic basis for ADLTE, an inherited syndrome with no cure to date. Existing studies suggest that altered glutamatergic transmission in the hippocampus causes this disease, but the data are paradoxical. We demonstrate that the loss of LGI1 decreases Kv1.2 expression, enhances intrinsic excitability, and thereby causes epilepsy. Interestingly, for the first time, we show that an FDA-approved drug, celecoxib, rescues the Kv1.2 defect and alleviates seizure susceptibility in LGI1-/- mice, as well as improving their survival. Thus, we suggest that celecoxib is a promising drug for the treatment of ADLTE patients.

Heparinized silk fibroin hydrogels loading FGF1 promote the wound healing in rats with full-thickness skin excision.

BACKGROUND: Silk fibroin hydrogel, derived from Bombyx mori cocoons, has been shown to have potential effects on wound healing due to its excellent biocompatibility and less immunogenic and biodegradable properties. Many studies suggest silk fibroin as a promising material of wound dressing and it can support the adhesion and proliferation of a variety of human cells in vitro. However, lack of translational evidence has hampered its clinical applications for skin repair. Herein, a heparin-immobilized fibroin hydrogel was fabricated to deliver FGF1 (human acidic fibroblast growth factor 1) on top of wound in rats with full-thickness skin excision by performing comprehensive preclinical studies to fully evaluate its safety and effectiveness. The wound-healing efficiency of developed fibroin hydrogels was evaluated in full-thickness wound model of rats, compared with the chitosan used clinically. RESULTS: The water absorption, swelling ratio, accumulative FGF1 releasing rate and biodegradation ratio of fabricated hydrogels were measured. The regenerated fibroin hydrogels with good water uptake properties rapidly swelled to a 17.3-fold maximum swelling behavior over 12 h and a total amount of 40.48 ± 1.28% hydrogels was lost within 15 days. Furthermore, accumulative releasing data suggested that heparinized hydrogels possessed effective release behavior of FGF1. Then full-thickness skin excision was created in rats and left untreated or covered with heparinized fibroin hydrogels-immobilized recombinant human FGF1. The histological evaluation using hematoxylin and eosin (HE) and Masson's trichrome (MT) staining was performed to observe the dermic formation and collagen deposition on the wound-healing site. To evaluate the wound-healing mechanisms induced by fibroin hydrogel treatment, wound-healing scratch and cell proliferation assay were performed. it was found that both fibroin hydrogels and FGF1 can facilitate the migration of fibroblast L929 cells proliferation and migration. CONCLUSION: This study provides systematic preclinical evidence that the silk fibroin promotes wound healing as a wound-healing dressing, thereby establishing a foundation toward its further application for new treatment options of wound repair and regeneration.

ER-localized protein-Herpud1 is a new mediator of IL-4-induced macrophage polarization and migration.

ER-localized proteins have been reported function in endoplasmic reticulum, unfolded protein degradation and destruction of misfolded proteins by the ER-associated protein degradation (ERAD) system, but their function in the chemotaxis of macrophage cells remained un-addressed. Here, we showed that ER protein with ubiquitin like domain 1(Herpud1) was upregulated in IL-4-treated M2 macrophage cells and its expression pattern was similar with macrophage polarization markers, such as Arg1, Mrc1 and Fizz1. Inhibition of Herpud1 by using specific target shRNA decreased these marker's expression at mRNA and protein level in IL-4-treated or -untreated M2 macrophage cells. IL-4 treatment promoted M2 macrophage cell migration and polarization, but this promotion was weakened by Herpud1 depletion and we got similar results by inhibition of ER stress response with chemical molecule 4-phenylbutyric acid (4-PBA) in IL-4-treated or untreated-M2 macrophage cells with Herpud1 overexpression. These results indicated that depending on ER-associated protein degradation (ERAD) to help unfolded protein degradation or destruction is not the only function of Herpud1 and acting as a mediator of IL-4 induced macrophage activation and polarization maybe another unrevealed function, elucidating the role of Herpud1-associated M2 macrophage cell polarization and activation are helpful for exploration the function of macrophage cells in immune response.

Tumor-suppressive activity of Hnf1ß in Wilms' tumor.

Hepatocyte nuclear factor 1ß (HNF1ß) is a transcription factor belonging to the HNF-1 family and has been implicated in a number of cancers, but its role in Wilms' tumor (nephroblastoma) has not been addressed. Here, we compared its expression between Wilms' tumor patient kidney tissue and adjacent tissue based on the Oncomine database ( www.oncomine.com ). Cell proliferation, apoptosis, migration, and HNF1ß expression level were analyzed in Wilms' tumor-derived G401 cells. Using a variety of mouse tissues (lung, heart, kidney, etc.), we found that HNF1ß is the highest expression in the kidneys. Oncomine analysis further demonstrated that HNF1ß has a lower expression in Wilms' tumor tissue than in paracancerous tissues. Overexpression of HNF1ß decreased cell proliferation and migration, but promoted cell apoptosis. Knockdown of HNF1ß produced the opposite results. These results indicated that HNF1ß may play important roles in kidney development and function, and its activation may negatively regulate Wilms' tumor progression.

PPP3CB Inhibits Migration of G401 Cells via Regulating Epithelial-to-Mesenchymal Transition and Promotes G401 Cells Growth.

PPP3CB belongs to the phosphoprotein phosphatases (PPPs) group. Although the majority of the PPP family play important roles in the epithelial-to-mesenchymal transition (EMT) of tumor cells, little is known about the function of PPP3CB in the EMT process. Here, we found PPP3CB had high expression in kidney mesenchymal-like cells compared with kidney epithelial-like cells. Knock-down of PPP3CB downregulated epithelial marker E-cadherin and upregulated mesenchymal marker Vimentin, promoting the transition of cell states from epithelial to mesenchymal and reorganizing the actin cytoskeleton which contributed to cell migration. Conversely, overexpression of PPP3CB reversed EMT and inhibited migration of tumor cells. Besides, in vitro and in vivo experiments indicated that the loss of PPP3CB suppressed the tumor growth. However, the deletion of the phosphatase domain of PPP3CB showed no effect on the expression of E-cadherin, migration, and G401 cell proliferation. Together, we demonstrate that PPP3CB inhibits G401 cell migration through regulating EMT and promotes cell proliferation, which are both associated with the phosphatase activity of PPP3CB.

Gulo regulates the proliferation, apoptosis and mesenchymal-to-epithelial transformation of metanephric mesenchyme cells via inhibiting Six2.

During kidney development, the balance between self-renewal and differentiation of metanephric mesenchyme (MM) cells, mainly regulated by Sine oculis-related homeobox 2 (Six2), is critical for forming mature kidney. L-gulono-γ-lactone oxidase (Gulo), a crucial enzyme for vitamin C synthesis, reveals a different expression at various stages during kidney development, but its function in the early renal development remains unknown. In this work, we aim to study the role of Gulo in MM cells at two differentiation stages. We found that Gulo expression in undifferentiated MM (mK3) cells was lower than in differentiated MM (mK4) cells. Over-expression of Gulo can promote mesenchymal-to-epithelial transformation (MET) and apoptosis and inhibit the proliferation in mK3 cells. Knock-down of Gulo in mK4 cells made its epithelial character cells unstabilized, facilitated the proliferation and restrained the apoptosis. Furthermore, we found that Six2 was negatively regulated by Gulo, and over-expression or knock-down of Six2 was able to rescue partially the MET, proliferation and apoptosis of MM cells caused by Gulo. In conclusion, these findings reveal that Gulo promotes the MET and apoptosis, and inhibits proliferation in MM cells by down-regulating Six2.

The small GTPase ROP10 of Medicago truncatula is required for both tip growth of root hairs and nod factor-induced root hair deformation.

Rhizobia preferentially enter legume root hairs via infection threads, after which root hairs undergo tip swelling, branching, and curling. However, the mechanisms underlying such root hair deformation are poorly understood. Here, we showed that a type II small GTPase, ROP10, of Medicago truncatula is localized at the plasma membrane (PM) of root hair tips to regulate root hair tip growth. Overexpression of ROP10 and a constitutively active mutant (ROP10CA) generated depolarized growth of root hairs, whereas a dominant negative mutant (ROP10DN) inhibited root hair elongation. Inoculated with Sinorhizobium meliloti, the depolarized swollen and ballooning root hairs exhibited extensive root hair deformation and aberrant infection symptoms. Upon treatment with rhizobia-secreted nodulation factors (NFs), ROP10 was transiently upregulated in root hairs, and ROP10 fused to green fluorescent protein was ectopically localized at the PM of NF-induced outgrowths and curls around rhizobia. ROP10 interacted with the kinase domain of the NF receptor NFP in a GTP-dependent manner. Moreover, NF-induced expression of the early nodulin gene ENOD11 was enhanced by the overexpression of ROP10 and ROP10CA. These data suggest that NFs spatiotemporally regulate ROP10 localization and activity at the PM of root hair tips and that interactions between ROP10 and NF receptors are required for root hair deformation and continuous curling during rhizobial infection.

Numb deficiency in cerebellar Purkinje cells impairs synaptic expression of metabotropic glutamate receptor and motor coordination.

Protein Numb, first identified as a cell-fate determinant in Drosophila, has been shown to promote the development of neurites in mammals and to be cotransported with endocytic receptors in clathrin-coated vesicles in vitro. Nevertheless, its function in mature neurons has not yet been elucidated. Here we show that cerebellar Purkinje cells (PCs) express high levels of Numb during adulthood and that conditional deletion of Numb in PCs is sufficient to impair motor coordination despite maintenance of a normal cerebellar cyto-architecture. Numb proved to be critical for internalization and recycling of metabotropic glutamate 1 receptor (mGlu1) in PCs. A significant decrease of mGlu1 and an inhibition of long-term depression at the parallel fiber-PC synapse were observed in conditional Numb knockout mice. Indeed, the trafficking of mGlu1 induced by agonists was inhibited significantly in these mutants, but the expression of ionotropic glutamate receptor subunits and of mGlu1-associated proteins was not affected by the loss of Numb. Moreover, transient and persistent forms of mGlu1 plasticity were robustly induced in mutant PCs, suggesting that they do not require mGlu1 trafficking. Together, our data demonstrate that Numb is a regulator for constitutive expression and dynamic transport of mGlu1.

Apobec-1 complementation factor regulates cell migration and apoptosis through Dickkopf1 by acting on its 3' untranslated region in MCF7 cells.

A1CF (apobec-1 complementation factor) acts as a component of the apolipoprotein-B messenger RNA editing complex. Previous researches mainly focused on its post-transcriptional cytidine to uridine RNA editing. However, few study reported its role in progression of breast carcinoma cells. Wound healing assay and flow cytometry were applied to detect the migration and apoptosis; western blot, real-time polymerase chain reaction, and dual-luciferase assays were applied to investigate the potential regulation mechanism of A1CF-mediated cell migration and apoptosis. Knockdown of A1CF decreased cell migration and enhanced cell apoptosis in MCF7 cells in vitro. Western blot analysis showed that knockdown of A1CF decreased Dickkopf1 but increased c-Myc and ß-catenin expression, and overexpression of A1CF can get opposite results. Knockdown of Dickkopf1 in A1CF-overexpressed cells decreased cell migration and enhanced cell apoptosis compared with A1CF-overexpressed cells. Luciferase-fused 3' untranslated region of human Dickkopf1 activity was highly upregulated in A1CF-overexpressed MCF7 cells, but this upregulation can be inhibited by mutating conserved binding motifs of Dickkopf1 3' untranslated region. A1CF played a crucial role in cell migration and survival through affecting 3' untranslated region of Dickkopf1 to upregulate its expression in MCF7 cells.

Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells.

Excitatory amino acid transporter 4 (EAAT4) is believed to be critical to the synaptic activity of cerebellar Purkinje cells by limiting extracellular glutamate concentrations and facilitating the induction of long-term depression. However, the modulation of EAAT4 expression has not been elucidated. It has been shown that Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) signaling plays essential roles in the regulation of protein translation, cell size, and cell growth. In addition, we previously found that a cascade including mTOR suppression and Akt activation induces increased expression of EAAT2 in astrocytes. In the present work, we explored whether Rheb/mTOR signaling is involved in the regulation of EAAT4 expression using conditional Rheb1 knockout mice. Our results demonstrated that Rheb1 deficiency resulted in the downregulation of EAAT4 expression, as well as decreased activity of mTOR and increased activity of Akt. The downregulation of EAAT4 was also confirmed by reduced EAAT4 currents and slowed kinetics of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor-mediated currents. On the other hand, conditional knockout of Rheb1 did not alter the morphology of Purkinje cell layer and the number of Purkinje cells. Overall, our findings suggest that small GTPase Rheb1 is a modulator in the expression of EAAT4 in Purkinje cells.

Gulo Acts as a de novo Marker for Pronephric Tubules in Xenopus laevis.

BACKGROUNDS/AIMS: Vitamin C is an antioxidant and acts as a cofactor for several key enzymatic catalytic reactions in animals. Amphibians produce vitamin C in their kidneys, as opposed to mammals that produce vitamin C in their liver. Gulo serves as a crucial enzyme for vitamin C synthesis in mammals, but the characteristics and localization of its homologous genes during kidney development in Xenopus laevis, an amphibian, remains unknown. METHODS: We aligned amino acid sequences of Gulo across different species by using bioinformatics methods and detected patterns of expression for Gulo during kidney development by using RT-PCR and in situ hybridization. RESULTS: We identified a new site on the X. laevis genome, LOC495407. Sequence alignment analysis indicated this fragment is highly conserved and homologous to gulo genes in mammals. RT-PCR and in situ hybridization results reveal that X. laevis gulo is maternally expressed during the early stages of embryonic development, particularly, in the tubules of the pronephros from the middle tail-bud stage and onward in embryos. CONCLUSION: Gulo is a novel specific marker for pronephros tubules in X. laevis, and may be used as a potential marker for kidney development studies and disease diagnosis in mammals.

Zeb1 Is a Potential Regulator of Six2 in the Proliferation, Apoptosis and Migration of Metanephric Mesenchyme Cells.

Nephron progenitor cells surround around the ureteric bud tips (UB) and inductively interact with the UB to originate nephrons, the basic units of renal function. This process is determined by the internal balance between self-renewal and consumption of the nephron progenitor cells, which is depending on the complicated regulation networks. It has been reported that Zeb1 regulates the proliferation of mesenchymal cells in mouse embryos. However, the role of Zeb1 in nephrons generation is not clear, especially in metanephric mesenchyme (MM). Here, we detected cell proliferation, apoptosis and migration in MM cells by EdU assay, flow cytometry assay and wound healing assay, respectively. Meanwhile, Western and RT-PCR were used to measure the expression level of Zeb1 and Six2 in MM cells and developing kidney. Besides, the dual-luciferase assay was conducted to study the molecular relationship between Zeb1 and Six2. We found that knock-down of Zeb1 decreased cell proliferation, migration and promoted cell apoptosis in MM cells and Zeb1 overexpression leaded to the opposite data. Western-blot and RT-PCR results showed that knock-down of Zeb1 decreased the expression of Six2 in MM cells and Zeb1 overexpression contributed to the opposite results. Similarly, Zeb1 promoted Six2 promoter reporter activity in luciferase assays. However, double knock-down of Zeb1 and Six2 did not enhance the apoptosis of MM cells compared with control cells. Nevertheless, double silence of Zeb1 and Six2 repressed cell proliferation. In addition, we also found that Zeb1 and Six2 had an identical pattern in distinct developing phases of embryonic kidney. These results indicated that there may exist a complicated regulation network between Six2 and Zeb1. Together, we demonstrate Zeb1 promotes proliferation and apoptosis and inhibits the migration of MM cells, in association with Six2.

Six2 Is a Coordinator of LiCl-Induced Cell Proliferation and Apoptosis.

The metanephric mesenchyme (MM) cells are a subset of kidney progenitor cells and play an essential role in mesenchymal-epithelial transition (MET), the key step of nephron generation. Six2, a biological marker related to Wnt signaling pathway, promotes the proliferation, inhibits the apoptosis and maintains the un-differentiation of MM cells. Besides, LiCl is an activator of Wnt signaling pathway. However, the role of LiCl in cellular regulation of MM cells remains unclear, and the relationship between LiCl and Six2 in this process is also little known. Here, we performed EdU assay and flow cytometry assay to, respectively, detect the proliferation and apoptosis of MM cells treated with LiCl of increasing dosages. In addition, reverse transcription-PCR (RT-PCR) and Western-blot were conducted to measure the expression of Six2 and some maker genes of Wnt and bone-morphogenetic-protein (BMP) signaling pathway. Furthermore, luciferase assay was also carried out to detect the transcriptional regulation of Six2. Then we found LiCl promoted MM cell proliferation at low-concentration (10, 20, 30, and 40 mM). The expression of Six2 was dose-dependently increased in low-concentration (10, 20, 30, and 40 mM) at both mRNA and protein level. In addition, both of cell proliferation and Six2 expression in MM cells declined when dosage reached high-concentration (50 mM). However, Six2 knock-down converted the proliferation reduction at 50 mM. Furthermore, Six2 deficiency increased the apoptosis of MM cells, compared with negative control cells at relative LiCl concentration. However, the abnormal rise of apoptosis at 30 mM of LiCl concentration implies that it might be the reduction of GSK3ß that increased cell apoptosis. Together, these demonstrate that LiCl can induce the proliferation and apoptosis of MM cells coordinating with Six2.

Distinct functions of nuclear distribution proteins LIS1, Ndel1 and NudCL in regulating axonal mitochondrial transport.

Neurons critically depend on the long-distance transport of mitochondria. Motor proteins kinesin and dynein control anterograde and retrograde mitochondrial transport, respectively in axons. The regulatory molecules that link them to mitochondria need to be better characterized. Nuclear distribution (Nud) family proteins LIS1, Ndel1 and NudCL are critical components of cytoplasmic dynein complex. Roles of these Nud proteins in neuronal mitochondrial transport are unknown. Here we report distinct functions of LIS1, Ndel1 and NudCL on axonal mitochondrial transport in cultured hippocampal neurons. We found that LIS1 interacted with kinsein family protein KIF5b. Depletion of LIS1 enormously suppressed mitochondrial motility in both anterograde and retrograde directions. Inhibition of either Ndel1 or NudCL only partially reduced retrograde mitochondrial motility. However, knocking down both Ndel1 and NudCL almost blocked retrograde mitochondrial transport, suggesting these proteins may work together to regulate retrograde mitochondrial transport through linking dynein-LIS1 complex. Taken together, our results uncover novel roles of LIS1, Ndel1 and NudCL in the transport of mitochondria in axons.