scholarly journals Short-term Cultivation of Porcine Cumulus Cells Influences the Cyclin-dependent Kinase 4 (Cdk4) and Connexin 43 (Cx43) Protein Expression—A Real-time Cell Proliferation Approach

2013 ◽  
Vol 59 (4) ◽  
pp. 339-345 ◽  
Author(s):  
Bartosz KEMPISTY ◽  
Agnieszka ZIÓŁKOWSKA ◽  
Hanna PIOTROWSKA ◽  
Sylwia CIESIÓŁKA ◽  
Paweł ANTOSIK ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Real Time ◽  
Connexin 43 ◽  
Cumulus Cells ◽  
Cyclin Dependent Kinase ◽  
Short Term ◽  
Cx43 Protein

scholarly journals Influence of Estradiol-17beta on Progesterone and Estrogen Receptor mRNA Expression in Porcine Follicular Granulosa Cells during Short-Term, In Vitro Real-Time Cell Proliferation

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Sylwia Ciesiółka ◽  
Joanna Budna ◽  
Karol Jopek ◽  
Artur Bryja ◽  
Wiesława Kranc ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Estrogen Receptor ◽  
Progesterone Receptor ◽  
Real Time ◽  
Granulosa Cells ◽  
Housekeeping Genes ◽  
Cumulus Cells ◽  
Short Term ◽  
Estrogen Receptor Mrna

Progesterone (P4) and estradiol (E2) play a significant role in mammalian reproduction. Our study demonstrated that separated porcine cumulus cells (CCs) and/or granulosa cells (GCs) might proliferate in vitro during short-term, real-time primary culture. The GCs were analyzed according to gene expression of the progesterone receptor (nuclear form) (pgr), progesterone receptor membrane component 1 (pgrmc1), and estrogen-related receptor beta 3 (esrrb3) in relation to two housekeeping genes: actb and pbgd. GCs were cultivated in medium with the E2. Both pgr/actb and pgr/pbgd revealed higher expression between 24 and 168 h of IVC of prolonged E2 treatment and at 48 h of IVC after acute E2 administration. The pgrmc1/actb and pgrmc1/pbgd displayed increased expression after prolonged E2 treatment between 24 and 120 h of IVC. The highest level of esrrb3/actb at 120 and 144 h, as well as esrrb3/pbgd at 120 h, in untreated controls as compared to the hormone-stimulated group, was observed. We suggest that E2 significantly influences the upregulation of pgr, pgrmc1, and esrrb3 expression in porcine GCs during real-time cell proliferation. Since esrrb3 expression is stimulated by E2 in both an acute and prolonged manner, estradiol may be recognized as a potential estrogen receptor agonist in GCs.

Effect of SOX9 on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Through WNTβ/Catenin Pathway

2019 ◽  
Vol 9 (10) ◽  
pp. 1429-1434
Author(s):  
Qing Yang ◽  
Cheng Li ◽  
Manli Yan ◽  
Chunhua Fang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Protein Expression ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
Alp Activity ◽  
Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) can be differentiated into different types of cells. SOX9 involves in the development and progression of various diseases. Our study aims to assess SOX9's effect on osteogenic differentiation of BMSCs and its related regulatory mechanisms. Rat BMSCs were isolated and randomly divided into control group, SOX9 group and SOX9 siRNA group, which was transfected with pcDNA-SOX9 plasmid or SOX9 siRNA respectively followed by analysis of SOX9 expression by Real time PCR, cell proliferation by MTT assay, Caspase3 and ALP activity, GSK-3β expression and Wntβ/Catenin Signaling pathway protein expression by Western blot, and expression of osteogenic genes Runx2 and BMP-2 by Real time PCR. Transfection of pcDNA-SOX9 plasmid into BMSCs significantly inhibited cell proliferation, promoted Caspase3 activity, decreased ALP activity and downregulated Runx2 and BMP-2, increased GSK-3β expression and decreased Wntβ/Catenin expression protein expression (P< 0.05). SOX9 siRNA transfection significantly promoted cell proliferation, inhibited Caspase3 activity, increased ALP activity and upregulated Runx2 and BMP-2, downregulated GSK-3β and increased Wntβ/Catenin expression. SOX9 regulates BMSCs proliferation and osteogenic differentiation through Wntβ/Catenin signaling pathway.

Expression and cellular distribution of estrogen and progesterone receptors and the real-time proliferation of porcine cumulus cells

Zygote ◽  
2014 ◽  
Vol 23 (6) ◽  
pp. 836-845 ◽  
Author(s):  
Bartosz Kempisty ◽  
Agnieszka Ziółkowska ◽  
Sylwia Ciesiółka ◽  
Hanna Piotrowska ◽  
Paweł Antosik ◽  
...  
Keyword(s):  
Protein Expression ◽  
Real Time ◽  
Progesterone Receptors ◽  
Cumulus Cells ◽  
Proliferation Index ◽  
Estrogen And Progesterone Receptors ◽  
Real Time Quantitative Pcr ◽  
Qrt Pcr ◽  
Cell Proliferation Index

SummaryAlthough the expression of estrogen and progesterone receptors within porcine ovary and cumulus–oocyte complexes (COCs) is well recognized, still little information is known regarding expression of the progesterone receptor (PGR), PGR membrane component 1 (PGRMC1) and of estrogen-related receptors (ERRγ and ERRβ/γ) in separated cumulus cells in relation to real-time proliferation. In this study, a model of oocytes-separated cumulus cells was used to analyze the cell proliferation index and the expression PGR, PGRMC1 and of ERRγ and ERRβ/γ during 96-h cultivation in vitro using real-time quantitative PCR (qRT-PCR) and confocal microscopic observation. We found that PGR protein expression was increased at 0 h, compared with PGR protein expression after 96 h of culture (P < 0.001). The expression of PGRMC1, ERRγ and ERRβ/γ was unchanged. After using qRT-PCR we did not found statistical differences in expression of PGR, PGRMC1, ERRγ and ERRβ/γ during 96 h of cumulus cells in vitro culture (IVC). We supposed that the differential expression of the PGR protein at 0 h and after 96 h is related to a time-dependent down-regulation, which may activate a negative feedback. The distribution of PGR, PGRMC1 proteins may be linked with the translocation of receptors to the cytoplasm after the membrane binding of respective agonists and intra-cytoplasmic signal transduction. Furthermore, cumulus cells analyzed at 0 h were characterized by decreased proliferation index, whereas those after 96 h of culture revealed a significant increase of proliferation index, which may be associated with differentiation/luteinization of these cells during real-time proliferation.

Negative Transcriptional Regulation of Connexin 43 by Tbx2 in Rat Immature Coronal Sutures and ROS 17/2.8 Cells in Culture

2003 ◽  
Vol 40 (3) ◽  
pp. 284-290 ◽  
Author(s):  
James L. Borke ◽  
Jung-Ren Chen ◽  
Jack C. Yu ◽  
Roni J. Bollag ◽  
Maria F. Orellana ◽  
...  
Keyword(s):  
Protein Expression ◽  
Connexin 43 ◽  
Protein Localization ◽  
Cell Communication ◽  
Negative Regulator ◽  
Cranial Sutures ◽  
Transcriptional Level ◽  
Cx43 Expression ◽  
Cx43 Protein

Background Tbx2 is a member of the T-box family of transcriptional regulatory genes with an extensive but not yet fully understood role in embryonic development. This study explores the potential role of Tbx2 in calvarial morphogenesis. Objectives To explore the hypothesis that Tbx2 has a negative regulatory effect on the expression of connexin 43 (Cx43), a protein necessary for cell-to-cell communication; document the presence of Tbx2 protein in the developing cranial sutures; and determine the spatial pattern of expression of this developmentally regulated transcription factor in calvariae. Design The osteoblast-like cell line ROS 17/2.8 was stably transfected with sense or antisense Tbx2. Immunohistochemistry and Western blotting was used to study Tbx2 and Cx43 expression in these cells and sections of embedded developing coronal sutures. Results The ROS 17/2.8 cells transfected with antisense Tbx2 showed a decrease in expression of Tbx2 protein and an increase in expression of endogenous Cx43. The reverse is seen with sense-transfected cells. Both of these proteins are expressed in rat developing coronal sutures. The pattern of Tbx2 expression in the developing was also reciprocal to the pattern of Cx43 expression. Tbx2 protein is concentrated in the center of the sutural blastema, an area devoid of Cx43 protein localization. Conversely, Tbx2 protein expression is low in the periphery of the sutures, in which there is high Cx43 protein expression. Conclusions Taken together, these studies suggest that Tbx2 protein is a negative regulator of Cx43 expression at the transcriptional level in cranial sutures in vivo.

scholarly journals Connexin 43 Communication Channels in Follicular Dendritic Cell Development and in Follicular Lymphomas

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Hajnalka Rajnai ◽  
Ivett Teleki ◽  
Gergo Kiszner ◽  
Nora Meggyesházi ◽  
Peter Balla ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cell ◽  
Connexin 43 ◽  
Ex Vivo ◽  
Bone Marrow Involvement ◽  
Germinal Centers ◽  
Cx43 Expression ◽  
Cx43 Protein ◽  
Follicular Dendritic ◽  
Follicular Lymphomas

Follicular dendritic cells (FDC) show homo- and heterocellular metabolic coupling through connexin 43 (Cx43) gap junctions and support B cell selection and maturation in germinal centers. In follicular lymphomas B cells escape apoptosis while FDC develop abnormally. Here we tested Cx43 channels in reactive FDC development and follicular lymphomas. In culture, the treatment of FDC-B cell clusters (resembling to “ex vivo” germinal centers) with Gap27 peptide, mimicking the 2nd extracellular loop of Cx43 protein, significantly impaired FDC-B cell cluster formation and cell survival. In untreated cultures of intact clusters, cell proliferation showed a moderate reduction. In tissues, Cx43 protein levels run parallel with the density of FDC both in reactive germinal centers and in malformed follicles of follicular lymphomas and showed strong upregulation in newly generated and/or degrading bi-/multinuclear FDC of rudimentary processes. However, the inverse correlation between Cx43 expression and B cell proliferation seen in reactive germinal centers was not detected in follicular lymphomas. Furthermore, Cx43 levels were not associated with either lymphoma grade or bone marrow involvement. Our results suggest that Cx43 channels are critical in FDC and “ex vivo” germinal center development and in the persistence of FDC in follicular lymphomas but do not affect tumor progression.

scholarly journals Connexin 43 plays a role in proliferation and migration of pulmonary arterial fibroblasts in response to hypoxia

2020 ◽  
Vol 10 (3) ◽  
pp. 204589402093713 ◽  
Author(s):  
Andrew J. McNair ◽  
Kathryn S. Wilson ◽  
Patricia E. Martin ◽  
David J. Welsh ◽  
Yvonne Dempsie
Keyword(s):  
Pulmonary Artery ◽  
Protein Expression ◽  
Connexin 43 ◽  
Pulmonary Vasculature ◽  
Hypoxic Exposure ◽  
In Vivo Models ◽  
Cx43 Protein ◽  
And Migration ◽  
Proliferation And Migration

Pulmonary hypertension (PH) is a disease associated with vasoconstriction and remodelling of the pulmonary vasculature. Pulmonary artery fibroblasts (PAFs) play an important role in hypoxic-induced remodelling. Connexin 43 (Cx43) is involved in cellular communication and regulation of the pulmonary vasculature. Using both in vitro and in vivo models of PH, the aims of this study were to (i) investigate the role of Cx43 in hypoxic-induced proliferation and migration of rat PAFs (rPAFs) and rat pulmonary artery smooth muscle cells (rPASMCs) and (ii) determine whether Cx43 expression is dysregulated in the rat sugen5416/hypoxic model of PH. The role of Cx43 in hypoxic-induced proliferation and migration was investigated using Gap27 (a pharmacological inhibitor of Cx43) or genetic knockdown of Cx43 using siRNA. Cx43 protein expression was increased by hypoxia in rPAFs but not rPASMCs. Hypoxic exposure, in the presence of serum, resulted in an increase in proliferation of rPAFs but not rPASMCs. Hypoxic exposure caused migration of rPAFs but not rPASMCs. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) and ERK1/2 were increased by hypoxia in rPAFs. The effects of hypoxia on proliferation, migration and MAPK phosphorylation in rPAFs were attenuated in the presence of Gap27 or Cx43 siRNA. Cx43 protein expression was increased in sugen5416/hypoxic rat lung; this increased expression was not observed in sugen5416/hypoxic rats treated with the MAPK pathway inhibitor GS-444217. In conclusion, Cx43 is involved in the proliferation and migration of rPAFs in response to hypoxia via the MAPK signalling pathway.

scholarly journals Expression and cellular distribution of cyclin-dependent kinase 4 (Cdk4) and connexin 43 (Cx43) in porcine oocytes before and after in vitro maturation

2014 ◽  
Vol 62 (1) ◽  
pp. 84-95 ◽  
Author(s):  
Bartosz Kempisty ◽  
Agnieszka Ziółkowska ◽  
Hanna Piotrowska ◽  
Paweł Antosik ◽  
Dorota Bukowska ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Oocyte Maturation ◽  
Zona Pellucida ◽  
Connexin 43 ◽  
Cumulus Cell ◽  
Cyclin Dependent Kinase ◽  
Before And After ◽  
Cx43 Mrna

It is recognised that connexin 43 (Cx43) and cyclin-dependent kinase 4 (Cdk4) are involved in the cumulus cell-oocyte communication via gap junctions and the control of cell cycle progress. However, little is known about their mRNA expression pattern and encoded proteins distribution in porcine oocytes during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were collected from 31 puberal crossbred Landrace gilts and analysed for their Cdk4 and Cx43 mRNA expression using RQ-PCR and for the respective protein expression by confocal microscopic observations. An increased Cdk4 and Cx43 mRNA expression was found in oocytes after IVM (P < 0.001 and P < 0.05, respectively). Confocal microscopic observations revealed a significant increase of Cdk4 protein expression in the cytoplasm of oocytes during the maturation process. The localisation of Cx43 changed from zona pellucida before to cytoplasm of oocytes after IVM. It is supposed that the increased expression of Cdk4 and Cx43 mRNA in oocytes after IVM is linked with the accumulation of a large amount of templates during the process of oocyte maturation. The translocation especially of Cx43 from the zona pellucida into the cytoplasm may be associated with a decrease in gap junction activity in fully grown porcine oocytes. Both Cdk4 and Cx43 can be used as ‘checkpoints’ of oocyte maturation.

scholarly journals T3 Up-Regulates PDZK1 to Promote the Proliferation of Papillary Thyroid Cancer Cells

2021 ◽  
Author(s):  
Yan Wang ◽  
Yanan Cheng ◽  
Rongxin Sun ◽  
Jianan Lang ◽  
Longyan Yang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
Thyroid Cancer ◽  
Protein Expression ◽  
Real Time ◽  
Papillary Thyroid Cancer ◽  
Immunofluorescence Staining ◽  
Cell Analysis ◽  
Papillary Thyroid ◽  
Rna Seq

Abstract Aim: Thyroid cancer (TC) is the most common malignant tumor of the endocrine system. Studies have showed that Triiodothyronine (T3) promotes the proliferation of papillary thyroid cancer (PTC) cells, but the specific mechanism remains unclear. Several studies have showed that PDZK1 played important roles in the occurrence and development of cancer. However, the biological function of PDZK1 in PTC remains unclear. Therefore, the aims of this study were to investigate the effect of PDZK1 in PTC and the underlying mechanism.Materials and Methods: The effect of T3 on proliferation of PTC cell (TPC-1) was analyzed by colony formation assay and real time cell analysis. Immunofluorescence staining aimed to analyze protein expression. RNA-seq was used to analyze the expression of PDZK1. Meanwhile, Western blot was used to verify the protein expression. The effect of PDZK1 on PTC cell proliferation was investigated by Cell Counting Kit-8(CCK8), real time cell analysis, flow cytometry and transwell assay, respectively. Results: We found that T3 increased the expression of PDZK1 in TPC-1 cells and promote the proliferation of TPC-1 cells which can be weakened after PDZK1 was knocked down. Immunofluorescence staining showed that the expression of PDZK1 was higher in PTC than paracancerous tissues. And the analysis of 6 benign thyroid nodules and 4 thyroid cancer tissues by RNA-Seq showed that the expression of PDZK1 was increased in PTC tissues, the expression of PDZK1 was also increased in PTC cells compared with the normal thyroid epithelium. In addition, PDZK1 promoted TPC-1 cell proliferation was detected by real-time cell analysis (RTCA) and CCK8. Flow cytometry analysis showed that PDZK1 increased cell cycle at S phase and decreased at G1 phase of TPC-1. PDZK1 promoted the invasion of TPC-1 cell was tested by transwell. Conclusion: These results suggest that T3 can promote the proliferation of PTC cells, which may be mediated by PDZK1. This study illustrates a possible mechanism by which T3 promotes the proliferation of PTC and provides a theoretical basis for the prevention and treatment of PTC.

Vitamin C supplementation had no side effect in non-cancer, but had anticancer properties in ovarian cancer cells

2020 ◽  
pp. 1-11 ◽  
Author(s):  
Ewa Lucja Gregoraszczuk ◽  
Karolina Zajda ◽  
Joanna Tekla ◽  
Natalia Respekta ◽  
Paweł Zdybał ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Proliferation ◽  
Vitamin C ◽  
Protein Expression ◽  
Cancer Cells ◽  
Basal Medium ◽  
Ovarian Cancer Cells ◽  
Cyclin Dependent Kinase ◽  
Apoptosis Pathway ◽  
Vit C

Abstract. Vitamin C (Vit C) has been widely used in the treatment and prevention of cancer. Nevertheless, the clinical results are still inconclusive. Using non-cancer (HOSEpiC) and cancer OVCAR-3 cells cultured in basal medium or in ovarian cancer-associated fibroblast (CAF)-supplemented medium, we estimated the dose-dependent effect of Vit C on sodium–ascorbate co -transporters (SVCT1, SVCT2) and g lucose transporter (GLUT1) protein expression. Additionally, the action of Vit C on cell proliferation (alamarBlue), membrane permeability (LDH assay), caspase3 activity, the selected cell cycle and apoptosis pathway, poly(ADP-ribose) polymerase-1 (PARP) protein expression, and reactive oxygen species (ROS) activity was determined. We showed different effects of Vit C on the expression of the co-transporter in non-cancer and cancer cells. In non-cancer cells, Vit C, at a pharmacological concentration, increased SVCT2 and decreased GLUT1, while the opposite effect was noted in cancer cells. In cancer cells, Vit C, in a pharmacological dose, decreased cell proliferation through an inhibitory effect on cyclin-dependent kinase 2 (CDK2) (4.4-fold; p < 0.01), mainly due to the stimulatory effect on the expression of cyclin-dependent kinase (CDK) inhibitors, such as p21 and p53 (3.2- and 2.8-fold, respectively; p < 0.001), but not caspase pathway. The tumour microenvironment caused inefficiency of the lower doses of Vit C in ovarian cancer cells. At a pharmacological dose of 1 mM, Vit C decreased PARP expression (1.5-fold; p < 0.05). We suggest that it’s nontoxic effects on non-cancer cells may be an indicator of its prophylactic use, while in a pharmacological dose Vit C should be considered a possible adjunctive drug in ovarian cancer. However, it is necessary to consider the effect of the CAF.

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