Application of in Utero Electroporation of G-Protein Coupled Receptor (GPCR) Genes, for Subcellular Localization of Hardly Identifiable GPCR in Mouse Cerebral Cortex

Kim, Nam-Ho; Kim, Seunghyuk; Hong, Jun; Jeon, Sung Ho; Huh, Sung‐Oh

doi:10.14348/molcells.2014.0159

Cited by 5 publications

(5 citation statements)

References 47 publications

(49 reference statements)

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“…The Ab labeling for each of the tested LPARs showed the expected plasma and nuclear membrane staining patterns; the EDG family of LPARs (LPA1-3) has been reported to be present on both of these membranes in other systems. 17,18 We have seen the same distribution for LPA1 and LPA3 in both human GF and PDLF grown in vitro on plastic cell culture plates or glass slides. 19 For our laboratory, the IHC assay of whole tooth was described here, and further, it has been a valuable tool to complement our pharmacologic studies that have characterized the LPARs expressed by human PDLF and the role they play in homeostasis and in periodontal disease.…”

Section: Discussionsupporting

confidence: 57%

A High-resolution Immunohistochemical Method for studying Receptor Expression on the Periodontal Ligament of Whole-mount Human Tooth Roots

Cerutis¹,

Headen²,

Ogunleye³

et al. 2016

International Journal of Experimental Dental Science

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Aims Our laboratory has found that lysophosphatidic acid (LPA) and its cognate receptors [LPARs, (LPA1–6)] expressed by human gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) play key roles in oral fibroblast homeostasis and are implicated in the inflammation seen in periodontal disease. We have reported that PDLF express LPA1 and LPA3; however, information on the gross topographic distribution of LPARs in the periodontal ligament (PDL) was lacking, and therefore, we developed a simple method for in situ labeling of LPARs in the PDL of extracted teeth. Materials and methods Sectioning or grinding thin sections of demineralized or native teeth and periodontium have long been the standard methodologies used to assess biomarker distribution in the PDL; however, we modified traditional immunohistochemical labeling and used whole teeth with fixed, solvent permeabilized PDLs. Results LPA1 and LPA3 were specifically labeled in the PDL and could be visualized at both the macroand micro-levels. Conclusion This technique effectively labeled LPARs, and it can serve as a basis for the in situ visualization of other biomolecules expressed in the PDL. Clinical Significance The ability to observe PDL LPAR distribution at the macro-level complements the microscopic data, and it is useful for detecting and documenting molecular changes in the PDL/PDLF that were brought about by age, experimental treatments, or pathologies like periodontal disease. How to cite this article Cerutis DR, Headen KV, Ogunleye AO, Williams DE. A High-resolution Immunohistochemical Method for studying Receptor Expression on the Periodontal Ligament of Whole-mount Human Tooth Roots. Int J Experiment Dent Sci 2016;5(2):99-103.

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Section: Discussionsupporting

confidence: 57%

A High-resolution Immunohistochemical Method for studying Receptor Expression on the Periodontal Ligament of Whole-mount Human Tooth Roots

Cerutis¹,

Headen²,

Ogunleye³

et al. 2016

International Journal of Experimental Dental Science

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“…In utero electroporation (IUE) was conducted following the previously described method [ 78 ]. After anesthetizing by isoflurane, the embryos in the uterus of pregnant mice were microinjected with plasmids by using pulled glass capillaries.…”

Section: Methodsmentioning

confidence: 99%

N-Acetyl-d-Glucosamine Kinase Interacts with NudC and Lis1 in Dynein Motor Complex and Promotes Cell Migration

Islam

Choi

Dash

et al. 2020

IJMS

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Recently, we showed that N-acetylglucosamine kinase (NAGK), an enzyme of amino sugar metabolism, interacts with dynein light chain roadblock type 1 (DYNLRB1) and promotes the functions of dynein motor. Here, we report that NAGK interacts with nuclear distribution protein C (NudC) and lissencephaly 1 (Lis1) in the dynein complex. Yeast two-hybrid assays, pull-down assays, immunocytochemistry, and proximity ligation assays revealed NAGK–NudC–Lis1–dynein complexes around nuclei, at the leading poles of migrating HEK293T cells, and at the tips of migratory processes of cultured rat neuroblast cells. The exogenous expression of red fluorescent protein (RFP)-tagged NAGK accelerated HEK293T cell migration during in vitro wound-healing assays and of neurons during in vitro neurosphere migration and in utero electroporation assays, whereas NAGK knockdown by short hairpin RNA (shRNA) delayed migration. Finally, a small NAGK peptide derived from the NudC interacting domain in in silico molecular docking analysis retarded the migrations of HEK293T and SH-SY5Y cells. These data indicate a functional interaction between NAGK and dynein–NudC–Lis1 complex at the nuclear envelope is required for the regulation of cell migration.

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“…Pregnant mice were anesthetized with isoflurane. IUE was performed essentially as described previously ( Kim et al, 2014 ). The uterine horns were exposed by way of a laparotomy, and plasmids (1 μl) containing 0.01% fast green (Sigma-Aldrich, USA) were microinjected through the uterus into the lateral ventricles of embryos by pulled glass capillaries.…”

Section: Methodsmentioning

confidence: 99%

“…Western blot was performed essentially as described previously ( Kim et al, 2014 ). HEK cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM Na 3 VO 4 , 5 mM NaF, and protease inhibitor cocktail).…”

Section: Methodsmentioning

confidence: 99%

Depletion of Inositol Polyphosphate 4-Phosphatase II Suppresses Callosal Axon Formation in the Developing Mice

Ji¹,

Kim²,

Huh³

et al. 2016

Molecules and Cells

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The corpus callosum is a bundle of nerve fibers that connects the two cerebral hemispheres and is essential for coordinated transmission of information between them. Disruption of early stages of callosal development can cause agenesis of the corpus callosum (AgCC), including both complete and partial callosal absence, causing mild to severe cognitive impairment. Despite extensive studies, the etiology of AgCC remains to be clarified due to the complicated mechanism involved in generating AgCC. The biological function of PI3K signaling including phosphatidylinositol-3,4,5-trisphosphate is well established in diverse biochemical processes including axon and dendrite morphogenesis, but the function of the closely related phosphatidylinositol-3,4,-bisphosphate (PI(3,4)P2) signaling, particularly in the nervous system, is largely unknown. Here, we provide the first report on the role of inositol polyphosphate 4-phosphatase II (INPP4B), a PI(3,4)P2 metabolizing 4-phosphatase in the regulation of callosal axon formation. Depleting INPP4B by in utero electroporation suppressed medially directed callosal axon formation. Moreover, depletion of INPP4B significantly attenuated formation of Satb2-positive pyramidal neurons and axon polarization in cortical neurons during cortical development. Taken together, these data suggest that INPP4B plays a role in the regulating callosal axon formation by controlling axon polarization and the Satb2-positive pyramidal neuron population. Dysregulation of INPP4B during cortical development may be implicated in the generation of partial AgCC.

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Application of in Utero Electroporation of G-Protein Coupled Receptor (GPCR) Genes, for Subcellular Localization of Hardly Identifiable GPCR in Mouse Cerebral Cortex

Cited by 5 publications

References 47 publications

A High-resolution Immunohistochemical Method for studying Receptor Expression on the Periodontal Ligament of Whole-mount Human Tooth Roots

A High-resolution Immunohistochemical Method for studying Receptor Expression on the Periodontal Ligament of Whole-mount Human Tooth Roots

N-Acetyl-d-Glucosamine Kinase Interacts with NudC and Lis1 in Dynein Motor Complex and Promotes Cell Migration

Depletion of Inositol Polyphosphate 4-Phosphatase II Suppresses Callosal Axon Formation in the Developing Mice

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