scholarly journals LEM-domain proteins are lost during human spermiogenesis but BAF and BAF-L persist

Reproduction ◽  
2017 ◽  
Vol 154 (4) ◽  
pp. 387-401 ◽  
Author(s):  
Razan A Elkhatib ◽  
Marine Paci ◽  
Romain Boissier ◽  
Guy Longepied ◽  
Yasmina Auguste ◽  
...  
Keyword(s):  
Western Blotting ◽  
Nuclear Periphery ◽  
Somatic Cells ◽  
Nuclear Lamina ◽  
Full Length ◽  
Rt Pcr ◽  
Male Pronucleus ◽  
Spermatid Nucleus ◽  
Chromatin Proteins

During spermiogenesis the spermatid nucleus is elongated, and dramatically reduced in size with protamines replacing histones to produce a highly compacted chromatin. After fertilisation, this process is reversed in the oocyte to form the male pronucleus. Emerging evidence, including the coordinated loss of the nuclear lamina (NL) and the histones, supports the involvement of the NL in spermatid nuclear remodelling, but how the NL links to the chromatin is not known. In somatic cells, interactions between the NL and the chromatin have been demonstrated: LEM-domain proteins and LBR interact with the NL and respectively, the chromatin proteins BAF and HP1. We therefore sought to characterise the lamina-chromatin interface during spermiogenesis, by investigating the localisation of six LEM-domain proteins, two BAF proteins and LBR, in human spermatids and spermatozoa. Using RT-PCR, IF and western blotting, we show that six of the proteins tested are present in spermatids: LEMD1, LEMD2 (a short isoform), ANKLE2, LAP2β, BAF and BAF-L, and three absent: Emerin, LBR and LEMD3. The full-length LEMD2 isoform, required for nuclear integrity in somatic cells, is absent. In spermatids, no protein localised to the nuclear periphery, but five were nucleoplasmic, receding towards the posterior nuclear pole as spermatids matured. Our study therefore establishes that the lamina-chromatin interface in human spermatids is radically distinct from that defined in somatic cells. In ejaculated spermatozoa, we detected only BAF and BAF-L, suggesting that they might contribute to the shaping of the spermatozoon nucleus and, after fertilisation, its transition to the male pronucleus.

Reduced Paired Box 5 (PAX5) Expression Is Common In Childhood Pro-B Acute Lymphoblastic Leukemic Cells, However, Can Not Be Attributed to PAX5 Genetic Hemizygous Deletion Only

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2505-2505
Author(s):  
Sheng-Kai Chang ◽  
Yen-Ju Chen ◽  
Shiann-Tarng Jou ◽  
Yong-Li Yang ◽  
Shu-Wha Lin ◽  
...  
Keyword(s):  
B Cells ◽  
Protein Expression ◽  
Western Blotting ◽  
Full Length ◽  
Leukemic Cells ◽  
Rt Pcr ◽  
Lineage Differentiation ◽  
Hemizygous Deletion ◽  
Pcr Products ◽  
B Lineage

Abstract Abstract 2505 Background: Genetic alterations affecting the B cell transcription factor PAX5 were identified in over 30% of leukemic samples obtained from both pediatric and adult pro-B ALL patients in previous studies using high-resolution single-nucleotide polymorphism (SNP) microarrays. The clinical significance of PAX5 hemizygous deletion has not been fully characterized yet. In order to understand the role of PAX5 alteration in the context of pro-B ALL, we investigated the genetic changes as well as the relevant PAX5 transcript/protein expression in the leukemic cells. Patients and methods: Twenty-four children with pro-B ALL enrolled in Taiwan Pediatric Oncology Group (TPOG) were studied. RNA samples were prepared from the diagnostic bone marrow and PAX5 mRNA expression was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). The PCR products were subjected to cloning and nucleotide sequence analysis to detect mutations/micro-deletion in the PAX5 coding transcripts. PAX5 protein expression in leukemic cells was analyzed by Western blotting in 6 available samples. Mature B cells purified from PBMCs of healthy subjects were served as control in RT-PCR and Western blotting assays. Variation of copy number in PAX5 gene was analyzed in the above 6 patients. To assess the loss of heterozygosity (LOH) at/near PAX5 locus (PAX5/LOH) in the leukemic cells, length polymorphisms in 5 micro-satellite (STR) markers spanning chromosome 9p were compared between the leukemic cells and respective PBMCs obtained at the remission stage. Results: PAX5 transcripts were expressed at a reduced level (less than half of that in the normal mature B cells) in 13 of the 24 leukemic samples. While performing RT-PCR spanning exons 6 to 10 of PAX5, PCR products of different size (corresponding to full length, -E8, -E7/8, -E8/9 etc.) were frequently identified in the leukemic samples, compared to primarily full length- and little -E8-transcripts shown in the normal B cells. We did not identify any mutations or micro-deletion in the amplified PAX5 transcripts. In six leukemic samples available for both PAX5 protein Western blotting and DNA PAX5/LOH analysis, three showed a massive reduction in PAX5 protein while another 3 express PAX5 protein comparable to that of the normal mature B cells. The reduced PAX5 protein production correlated to the reduced level of full-length PAX5 transcripts. Three leukemic samples showed LOH at/near PAX5 locus. However, PAX5/LOH did not correlate with the reduced PAX5 mRNA and protein expression in the leukemic cells: 2 leukemic samples with PAX5/LOH showed PAX5 expression comparable to that of the normal mature B cells. Development of non-B-lineage differentiation was demonstrated in a mouse model with a conditionally knocked-out PAX5 in the committed B-cells. Although statistically non-significant, presence of non-B-lineage differentiation markers was more frequently present in the leukemic cells reduced PAX5 expression. Conclusion: LOH at or near PAX5 locus is common, while point mutation or micro-deletion in PAX5 is rarely found in childhood pro-B ALL. Reduced PAX5 expression (in mRNA and protein) is common in leukemic cells. However, it does not correlate to the PAX5 hemizygous deletion status in the corresponding cells. Mechanisms other than haplo-insufficiency might determine PAX5 expression in the pro-B ALL cells. In leukemic samples, different variant forms at the C-terminal part of PAX5 transcript were frequently identified. The significance of the reduced PAX5 expression and the presence of the variant PAX5 transcripts, in leukemogenesis and the clinical settings in pro-B ALL remained to be studied. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair

2021 ◽  
Author(s):  
Laura A Murray-Nerger ◽  
Joshua L Justice ◽  
Pranav Rekapalli ◽  
Josiah E Hutton ◽  
Ileana M Cristea
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Posttranslational Modifications ◽  
Cell Cycle Progression ◽  
Nuclear Periphery ◽  
Virus Production ◽  
Nuclear Lamina ◽  
Regulatory Function ◽  
Herpesvirus Type ◽  
Lamin B1

Abstract The integrity and regulation of the nuclear lamina is essential for nuclear organization and chromatin stability, with its dysregulation being linked to laminopathy diseases and cancer. Although numerous posttranslational modifications have been identified on lamins, few have been ascribed a regulatory function. Here, we establish that lamin B1 (LMNB1) acetylation at K134 is a molecular toggle that controls nuclear periphery stability, cell cycle progression, and DNA repair. LMNB1 acetylation prevents lamina disruption during herpesvirus type 1 (HSV-1) infection, thereby inhibiting virus production. We also demonstrate the broad impact of this site on laminar processes in uninfected cells. LMNB1 acetylation negatively regulates canonical nonhomologous end joining by impairing the recruitment of 53BP1 to damaged DNA. This defect causes a delay in DNA damage resolution and a persistent activation of the G1/S checkpoint. Altogether, we reveal LMNB1 acetylation as a mechanism for controlling DNA repair pathway choice and stabilizing the nuclear periphery.

scholarly journals The Progesterone Receptor in Human Term Amniochorion and Placenta Is Isoform C

Endocrinology ◽  
2006 ◽  
Vol 147 (2) ◽  
pp. 687-693 ◽  
Author(s):  
Anthony H. Taylor ◽  
Penny C. McParland ◽  
David J. Taylor ◽  
Stephen C. Bell
Keyword(s):  
Progesterone Receptor ◽  
Western Blotting ◽  
Cell Types ◽  
Specific Role ◽  
Fetal Membranes ◽  
Rt Pcr ◽  
Reproductive Tissues ◽  
Pcr Techniques ◽  
Human Parturition ◽  
Extraembryonic Tissues

The mechanism that initiates human parturition has been proposed to be functional progesterone withdrawal whereby the 116-kDa B isoform of the progesterone receptor (PR-B) switches in favor of the 94-kDa A isoform (PR-A) in reproductive tissues. Recently other PR isoforms, PR-S, PR-C, and PR-M generated from the same gene have been identified and partially characterized. Using immunohistochemical, Western blotting, and RT-PCR techniques, evidence is provided that the major PR isoform present in human term fetal membranes (amnion and chorion) and syncytiotrophoblast of the placenta is neither of the classical nuclear PR-B or PR-A isoforms but is the N terminally truncated 60-kDa PR-C isoform. Evidence is also provided that the PR-C isoform resides in the cytoplasm of the expressing cell types. Data are also presented to show that PR-B, PR-A, and PR-S isoforms are essentially absent from the amnion and chorion, whereas PR isoforms A, B, C, and S are all present in the decidua, with PR-A being the major isoform. The syncytiotrophoblast of the placenta contains the cytoplasmic PR-C isoform but not PR-A, PR-B, or PR-S. The major PR isoform in the amnion, chorion, and placenta is PR-C, suggesting that the cytoplasmic PR-C isoform has a specific role in extraembryonic tissues and may be involved in the regulation of human parturition.

scholarly journals Expression of β-catenin in human trophoblast and its role in placenta accreta and placenta previa

2018 ◽  
Vol 47 (1) ◽  
pp. 206-214
Author(s):  
Qing Han ◽  
Lianghui Zheng ◽  
Zhaodong Liu ◽  
Jinying Luo ◽  
Rongxin Chen ◽  
...  
Keyword(s):  
Western Blotting ◽  
Cell Invasion ◽  
Placenta Accreta ◽  
Placenta Previa ◽  
Control Group ◽  
Rt Pcr ◽  
Lesion Area ◽  
Human Trophoblast ◽  
Chorionic Villi ◽  
Pcr Techniques

Objectives To investigate the expression of β-catenin in chorionic villi, and to explore its roles in placenta accreta and placenta previa. Methods We compared β-catenin expression in the control group, placenta accreta group (lesion area and normal zones), and placenta previa group (placental central and placental edge zones) by immunohistochemistry, Western blotting, and RT-PCR techniques. Results Compared with the normal group, the placenta accreta group had a longer length of stay, greater bleeding volume, and lower newborn birth weight. Further, the expression of β-catenin was lower in both placenta previa and placenta accreta groups than in the control group, as measured by immunohistochemistry. Compared with the control group, expression of β-catenin was significantly lower in the placenta previa and placenta accreta groups by Western blotting and RT-PCR. Importantly, the level of placental β-catenin was significantly different when compared between the lesion and normal zones of placenta. Conclusion The expression of β-catenin in placenta accreta might play an important role in the regulation of placental cell invasion; low expression of β-catenin in placenta accreta might be responsible for excessive trophoblastic invasion.

scholarly journals Nuclear Cytoskeleton in Virus Infection

2022 ◽  
Vol 23 (1) ◽  
pp. 578
Author(s):  
Lenka Horníková ◽  
Kateřina Bruštíková ◽  
Sandra Huérfano ◽  
Jitka Forstová
Keyword(s):  
Virus Infection ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
Nuclear Actin ◽  
Nuclear Egress ◽  
Viral Particles ◽  
Replication Sites ◽  
Main Component ◽  
Virus Egress ◽  
Natural Barrier

The nuclear lamina is the main component of the nuclear cytoskeleton that maintains the integrity of the nucleus. However, it represents a natural barrier for viruses replicating in the cell nucleus. The lamina blocks viruses from being trafficked to the nucleus for replication, but it also impedes the nuclear egress of the progeny of viral particles. Thus, viruses have evolved mechanisms to overcome this obstacle. Large viruses induce the assembly of multiprotein complexes that are anchored to the inner nuclear membrane. Important components of these complexes are the viral and cellular kinases phosphorylating the lamina and promoting its disaggregation, therefore allowing virus egress. Small viruses also use cellular kinases to induce lamina phosphorylation and the subsequent disruption in order to facilitate the import of viral particles during the early stages of infection or during their nuclear egress. Another component of the nuclear cytoskeleton, nuclear actin, is exploited by viruses for the intranuclear movement of their particles from the replication sites to the nuclear periphery. This study focuses on exploitation of the nuclear cytoskeleton by viruses, although this is just the beginning for many viruses, and promises to reveal the mechanisms and dynamic of physiological and pathological processes in the nucleus.

scholarly journals Μελέτη γλυκοζαμινογλυκανών και πρωτεογλυκανών σε λευχαιμικά κύτταρα

1998 ◽  
Author(s):  
Ευδοκία Μακατσώρη
Keyword(s):  
Western Blotting ◽  
Rt Pcr

Η ΠΑΡΟΥΣΑ ΔΙΑΤΡΙΒΗ ΕΙΧΕ ΣΤΟΧΟ ΤΗ ΜΕΛΕΤΗ ΠΡΩΤΕΟΓΛΥΚΑΝΩΝ (PGS)ΚΑΙ ΓΛΥΚΟΖΑΜΙΝΟΓΛΥΚΑΝΩΝ (GAGS)ΛΕΥΧΑΙΜΙΚΩΝ ΛΕΜΦΟΚΥΤΤΑΡΩΝ ΚΑΙ ΜΟΝΟΚΥΤΤΑΡΩΝ ΚΑΙ ΤΗ ΜΕΛΕΤΗ ΤΗΣ ΔΡΑΣΗΣ ΜΙΤΟΓΟΝΩΝ ΣΤΗ ΣΥΝΘΕΣΗ ΚΑΙ ΚΑΤΑΝΟΜΗ ΤΩΝ GAGS ΟΠΩΣ ΕΠΙΣΗΣ ΤΗ ΜΕΛΕΤΗ ΔΡΑΣΗΣ ΕΞΩΓΕΝΩΝ GAGS ΣΤΟΝ ΚΥΤΤΑΡΙΚΟ ΠΟΛΛΑΠΛΑΣΙΑΣΜΟ,ΜΟΝΩΝ ΤΟΥΣ,Η ΣΕ ΣΥΝΔΥΑΣΜΟ ΜΕ ΜΙΤΟΓΟΝΑ.ΕΠΙΛΕΧΘΗΚΑΝ ΤΡΕΙΣ ΛΕΥΧΑΙΜΙΚΕΣ ΚΥΤΤΑΡΙΚΕΣ ΣΕΙΡΕΣ :JURKAT (ΟΞΕΙΑ Τ-ΛΕΜΦΟΚΥΤΤΑΡΙΚΗ ΛΕΥΧΑΙΜΙΑ),DAUDI (Β-ΛΕΜΦΟΚΥΤΤΑΡΙΚΗ ΛΕΥΧΑΙΜΙΑ)ΚΑΙ THP-1 (ΟΞΕΙΑ ΜΟΝΟΚΥΤΤΑΡΙΚΗ ΛΕΥΧΑΙΜΙΑ).ΑΡΧΙΚΑ ΜΕ RT-PCR ΜΕΛΕΤΗΘΗΚΕ Η ΕΜΦΑΣΗ ΠΡΩΤΕΟΓΛΥΚΑΝΩΝ ΣΕ ΕΠΙΠΕΔΟ MRNA,ΚΑΙ ΒΡΕΘΗΚΕ ΟΤΙ ΚΑΙ ΟΙ ΤΡΕΙΣ ΚΥΤΑΡΙΚΕΣ ΣΕΙΡΕΣ ΕΧΟΥΝ ΤΗ ΔΥΝΑΤΟΤΗΤΑ ΣΥΝΘΕΣΗΣ ΣΥΝΔΕΙΝΗΣ-1,-2,-4,ΓΛΥΠΙΚΑΝΗΣ,CD44,ΒΕΡΣΙΚΑΝΗΣ-0 ΚΑΙ ΒΕΡΣΙΚΑΝΗΣ-1(ΤΥΠΟΣΕΝΑΛΛΑΚΤΙΚΟΥ ΜΑΤΙΣΜΑΤΟΣ ΤΗΣ ΒΕΡΣΙΚΑΝΗΣ). ΤΑ DAUDI ΕΧΟΥΝ ΕΠΙΠΛΕΟΝ ΤΗ ΔΥΝΑΤΟΤΗΤΑ ΣΥΝΘΕΣΗΣ ΙΝΟΜΟΝΤΟΥΛΙΝΗΣ,ΕΝΩ ΤΑ THP-1 ΜΠΟΡΕΙ ΝΑ ΣΥΝΘΕΤΟΥΝ ΠΕΡΛΕΚΑΝΗ ΚΑΙ ΘΡΟΜΒΟΜΟΝΤΟΥΛΙΝΗ. ΠΙΣΤΟΠΟΙΗΘΗΚΕ ΜΕ WESTERN BLOTTING Η ΠΑΡΟΥΣΙΑ ΣΥΝΔΕΙΝΗΣ -1,ΔΥΟΤΥΠΩΝ ΣΥΝΔΕΙΝΗΣ -4 ΔΙΑΦΟΡΕΤΙΚΗΣ ΓΛΥΚΟΖΙΛΙΩΣΗΣ,Η ΜΕΓΑΛΥΤΕΡΗΣ ΘΕΙΩΣΗΣ,ΘΡΟΜΒΟΜΟΝΤΟΥΛΙΝΗΣ, ΒΕΡΣΙΚΑΝΗΣ-0,ΒΕΡΣΙΚΑΝΗΣ-1 ΜΙΑΣ PG ΘΕΙΙΚΗΣ ΚΕΡΑΤΑΝΗΣ ΚΑΙ ΠΕΡΛΕΚΑΝΗΣΑΝΑΛΟΓΑ ΜΕ ΤΗΝ ΚΑΘΕ ΚΥΤΤΑΡΙΚΗ ΣΕΙΡΑ.ΜΕ ΧΡΗΣΗ ΕΝΖΥΜΙΚΩΝ ΚΑΤΕΡΓΑΣΙΩΝ ΚΑΙ ΑΝΑΛΥΣΗΣ HPLC ΒΡΕΘΗΚΕ ΠΩΣ ΚΑΜΙΑ ΚΥΤΤΑΡΙΚΗ ΣΕΙΡΑ ΔΕ ΣΥΝΘΕΤΕΙ HA.ΣΥΝΘΕΤΟΥΝ ΚΥΡΙΩΣ CSΚΑΙ ΣΕ ΜΙΚΡΟΤΕΡΟ ΠΟΣΟΣΤΟ HS.ΑΚΟΛΟΥΘΩΣ,ΕΛΕΓΘΗΚΕ Η ΕΠΙΔΡΑΣΗ ΕΞΩΓΕΝΩΝ GAGS ΣΤΟΝ ΠΟΛΛΑΠΛΑΣΙΑΣΜΟ ΤΩΝ ΚΥΤΤΑΡΩΝ,ΟΠΩΣ ΕΠΙΣΗΣ ΚΑΙ Η ΔΡΑΣΗ ΓΛΥΚΟΖΑΜΙΝΟΓΛΥΚΑΝΩΝ ΠΑΡΟΥΣΙΑ ΜΙΤΟΓΟΤΩΝ ΣΤΟΝ ΠΟΛΛΑΠΛΑΣΙΑΣΜΟ ΤΩΝ ΚΥΤΤΑΡΩΝ ,ΜΕ ΣΚΟΠΟ ΝΑ ΔΙΑΠΙΣΤΩΘΕΙ ΤΟ ΚΑΤΑ ΠΟΣΟ ΣΥΜΜΕΤΕΧΟΥΝ ΟΙ GAGS ΣΤΟΥΣ ΜΗΧΑΝΙΣΜΟΥΣ ΔΙΕΓΕΡΣΗΣ.

scholarly journals H3K9me2 orchestrates inheritance of spatial positioning of peripheral heterochromatin through mitosis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Andrey Poleshko ◽  
Cheryl L Smith ◽  
Son C Nguyen ◽  
Priya Sivaramakrishnan ◽  
Karen G Wong ◽  
...  
Keyword(s):  
Cell Division ◽  
Spatial Organization ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
Positional Information ◽  
Specific Gene ◽  
Cell Type ◽  
Histone 3 ◽  
Spatial Positioning ◽  
Peripheral Heterochromatin

Cell-type-specific 3D organization of the genome is unrecognizable during mitosis. It remains unclear how essential positional information is transmitted through cell division such that a daughter cell recapitulates the spatial genome organization of the parent. Lamina-associated domains (LADs) are regions of repressive heterochromatin positioned at the nuclear periphery that vary by cell type and contribute to cell-specific gene expression and identity. Here we show that histone 3 lysine 9 dimethylation (H3K9me2) is an evolutionarily conserved, specific mark of nuclear peripheral heterochromatin and that it is retained through mitosis. During mitosis, phosphorylation of histone 3 serine 10 temporarily shields the H3K9me2 mark allowing for dissociation of chromatin from the nuclear lamina. Using high-resolution 3D immuno-oligoFISH, we demonstrate that H3K9me2-enriched genomic regions, which are positioned at the nuclear lamina in interphase cells prior to mitosis, re-associate with the forming nuclear lamina before mitotic exit. The H3K9me2 modification of peripheral heterochromatin ensures that positional information is safeguarded through cell division such that individual LADs are re-established at the nuclear periphery in daughter nuclei. Thus, H3K9me2 acts as a 3D architectural mitotic guidepost. Our data establish a mechanism for epigenetic memory and inheritance of spatial organization of the genome.

The nuclear lamina and heterochromatin: a complex relationship

2011 ◽  
Vol 39 (6) ◽  
pp. 1705-1709 ◽  
Author(s):  
Erin M. Bank ◽  
Yosef Gruenbaum
Keyword(s):  
Nuclear Periphery ◽  
Gene Activation ◽  
Nuclear Lamina ◽  
Current Evidence ◽  
Specific Gene ◽  
Nuclear Interior ◽  
Gene Retention ◽  
Gene Positioning ◽  
Review Current ◽  
Active Genes

In metazoan cells, the heterochromatin is generally localized at the nuclear periphery, whereas active genes are preferentially found in the nuclear interior. In the present paper, we review current evidence showing that components of the nuclear lamina interact directly with heterochromatin, which implicates the nuclear lamina in a mechanism of specific gene retention at the nuclear periphery and release to the nuclear interior upon gene activation. We also discuss recent data showing that mutations in lamin proteins affect gene positioning and expression, providing a potential mechanism for how these mutations lead to tissue-specific diseases.

scholarly journals Large-scale RT-PCR recovery of full-length cDNA clones

BioTechniques ◽  
2004 ◽  
Vol 36 (4) ◽  
pp. 690-700 ◽  
Author(s):  
Jia Qian Wu ◽  
Angela M. Garcia ◽  
Steven Hulyk ◽  
Anna Sneed ◽  
Carla Kowis ◽  
...  
Keyword(s):  
Large Scale ◽  
Full Length ◽  
Cdna Clones ◽  
Rt Pcr ◽  
Full Length Cdna

scholarly journals Visfatin Induces Inflammation and Insulin Resistance via the NF-κB and STAT3 Signaling Pathways in Hepatocytes

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Yu Jung Heo ◽  
Sung-E Choi ◽  
Ja Young Jeon ◽  
Seung Jin Han ◽  
Dae Jung Kim ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Signaling Pathways ◽  
Western Blotting ◽  
Insulin Signaling ◽  
Proinflammatory Cytokine ◽  
Hepg2 Cells ◽  
Molecular Mechanisms ◽  
Immunocytochemical Staining ◽  
Mrna Levels ◽  
Rt Pcr

Background. It has been suggested that visfatin, which is an adipocytokine, exhibits proinflammatory properties and is associated with insulin resistance. Insulin resistance and inflammation are the principal pathogeneses of nonalcoholic fatty liver disease (NAFLD), but the relationship, if any, between visfatin and NAFLD remains unclear. Here, we evaluated the effects of visfatin on hepatic inflammation and insulin resistance in HepG2 cells and examined the molecular mechanisms involved. Methods. After treatment with visfatin, the inflammatory cytokines IL-6, TNF-α, and IL-1β were assessed by real-time polymerase chain reaction (RT-PCR) and immunocytochemical staining in HepG2 cells. To investigate the effects of visfatin on insulin resistance, we evaluated insulin-signaling pathways, such as IR, IRS-1, GSK, and AKT using immunoblotting. We assessed the intracellular signaling molecules including STAT3, NF-κB, IKK, p38, JNK, and ERK by western blotting. We treated HepG2 cells with both visfatin and either AG490 (a JAK2 inhibitor) or Bay 7082 (an NF-κB inhibitor); we examined proinflammatory cytokine mRNA levels using RT-PCR and insulin signaling using western blotting. Results. In HepG2 cells, visfatin significantly increased the levels of proinflammatory cytokines, reduced the levels of proteins (e.g., phospho-IR, phospho-IRS-1 (Tyr612), phospho-AKT, and phospho-GSK-3α/β) involved in insulin signaling, and increased IRS-1 S307 phosphorylation compared to controls. Interestingly, visfatin increased the activities of the JAK2/STAT3 and IKK/NF-κB signaling pathways but not those of the JNK, p38, and ERK pathways. Visfatin-induced inflammation and insulin resistance were regulated by JAK2/STAT3 and IKK/NF-κB signaling; together with AG490 or Bay 7082, visfatin significantly reduced mRNA levels of IL-6, TNF-α and IL-1β and rescued insulin signaling. Conclusion. Visfatin induced proinflammatory cytokine production and inhibited insulin signaling via the STAT3 and NF-κB pathways in HepG2 cells.

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