The influence of serum‐supplemented culture media in a transwell migration assay

Background/Aims: Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s). Methods: Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM) was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay. Results: TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes. Conclusion: This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.

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Hypoxia increases KIAA1199/CEMIP expression and enhances cell migration in pancreatic cancer

Scientific Reports ◽

10.1038/s41598-021-97752-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Takuya Oba ◽

Norihiro Sato ◽

Yasuhiro Adachi ◽

Takao Amaike ◽

Yuzan Kudo ◽

...

Keyword(s):

Cell Migration ◽

Protein Expression ◽

Hypoxia Inducible Factor ◽

Ductal Adenocarcinoma ◽

Rt Pcr ◽

Transwell Migration Assay ◽

Migration Assay ◽

Hypoxic Conditions ◽

Mrna And Protein Expression ◽

Interfering Rna

AbstractPancreatic ductal adenocarcinoma (PDAC) is characterised by dense desmoplasia and hypoxic microenvironment. Our previous reports demonstrated that hyaluronan (HA), especially low-molecular-weight HA, provides a favourable microenvironment for PDAC progression. However, the effect of hypoxia on HA metabolism remains unknown. Using quantitative real-time RT-PCR and western blot analysis, we analysed the changes in the expression of HA-synthesizing enzymes (HAS2 and HAS3) and HA-degrading enzymes (HYAL1, KIAA1199/CEMIP) in PDAC cell lines under hypoxic conditions. Hypoxia increased the mRNA and protein expression of KIAA1199, whereas it decreased HYAL1 expression. The expression of HAS3 was increased and HAS2 remained unchanged in response to hypoxia. The effect of KIAA1199 on hypoxia-induced cell migration was determined using a transwell migration assay and small-interfering RNA (siRNA). Hypoxia enhanced the migratory ability of PDAC cells, which was inhibited by KIAA1199 knockdown. We also used immunohistochemistry to analyse the protein expression of hypoxia inducible factor (HIF) 1α and KIAA1199 in PDAC tissues. There was a significant immunohistochemically positive correlation between KIAA1199 and HIF1α. These findings suggest that hypoxia-induced KIAA1199 expression may contribute to enhanced motility in PDAC.

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IL-1β Pretreatment Improves the Efficacy of Mesenchymal Stem Cells on Acute Liver Failure by Enhancing CXCR4 Expression

Stem Cells International ◽

10.1155/2020/1498315 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11

Author(s):

He Nie ◽

Fangmei An ◽

Jie Mei ◽

Cheng Yang ◽

Qiang Zhan ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Liver Failure ◽

Acute Liver Failure ◽

Inflammatory Diseases ◽

Cxcr4 Expression ◽

Migration Ability ◽

Transwell Migration Assay ◽

Migration Assay ◽

Effective Therapeutic Strategy

Background. Mesenchymal stem cells (MSCs), with the powerful metabolic and functional supporting abilities for inflammatory diseases, may be an effective therapeutic strategy for acute liver failure (ALF). However, the efficacy of MSCs can still be promoted if pretreatment is applied to enhance their poor migration towards the damaged liver. The purpose of this study is to determine the effect of IL-1β pretreatment on the efficacy and homing ability of MSCs in ALF. Methods. MSCs were isolated by the whole bone marrow adherence method and characterized. The efficacy and homing ability of IL-1β-pretreated MSCs (Pre-MSCs) were examined in a rat ALF model and compared with that of MSCs and normal saline. Then, Western blot was performed to detect the c-Met and CXCR4 expression of MSCs and Pre-MSCs and followed by flow cytometry to detect the meaningful indicators. Finally, the migration abilities of different cells and different conditions were tested by the Transwell migration assay. Results. MSCs of ideal purity were successfully isolated and cultured. Comparing with MSCs, Pre-MSCs had significantly better efficacy on improving the survival rate and liver function of ALF rats. Further analyses of damaged liver tissues showed that IL-1β pretreatment significantly enhanced the efficacy of MSCs on suppressing liver necrosis. Besides, Pre-MSCs exhibited better effects in inhibiting apoptosis and activating proliferation. The results of tracing experiments with CM-Dil-labeled cells confirmed that more cells migrated to the damaged liver in the Pre-MSC group. In terms of mechanism, the CXCR4 expression was significantly enhanced by IL-1β pretreatment, and an increased migration ability towards SDF-1 that could be reversed by AMD3100 was found in Pre-MSCs. Conclusion. IL-1β pretreatment could enhance the homing ability of MSCs at least partially by increasing the expression of CXCR4 and further improve the efficacy of MSCs on ALF.

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UPLC-PDA-QTOFMS-guided isolation of prenylated xanthones and benzoylphloroglucinols from the leaves of Garcinia oblongifolia and their migration-inhibitory activity

Scientific Reports ◽

10.1038/srep35789 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 5

Author(s):

Hong Zhang ◽

Dan Zheng ◽

Zhi-Jie Ding ◽

Yuan-Zhi Lao ◽

Hong-Sheng Tan ◽

...

Keyword(s):

Spectroscopic Analysis ◽

Soluble Fraction ◽

2D Nmr ◽

Acetone Extract ◽

Cancer Cell Migration ◽

Transwell Migration Assay ◽

Migration Assay ◽

Protein Levels ◽

Snail Protein ◽

New Compounds

Abstract A UPLC-PDA-QTOFMS-guided isolation strategy was employed to screen and track potentially new compounds from Garcinia oblongifolia. As a result, two new prenylated xanthones, oblongixanthones D and E (1–2), six new prenylated benzoylphloroglucinol derivatives, oblongifolins V–Z (3–7) and oblongifolin AA (8), as well as a known compound oblongifolin L (9), were isolated from the EtOAc-soluble fraction of an acetone extract of the leaves of Garcinia oblongifolia guided by UPLC-PDA-QTOFMS analysis. The structures of the new compounds were elucidated by 1D- and 2D-NMR spectroscopic analysis and mass spectrometry. Experimental and calculated ECD spectra were used to determine the absolute configurations. The results of wound healing and transwell migration assay showed that oblongixanthones D (1), E (2), and oblongifolin L (9) have the ability to inhibit cancer cell migration in lower cytotoxic concentrations. Western blotting results showed that these compounds exhibited an anti-metastasis effect mainly through downregulating RAF protein levels. In addition, 2 and 9 could inhibit phospho-MEK and phospho-ERK at downstream. Moreover, 1, 2, and 9 could inhibit snail protein level, suggesting that they could regulate the EMT pathway.

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The high level of RANTES in the ectopic milieu recruits macrophages and induces their tolerance in progression of endometriosis

Journal of Molecular Endocrinology ◽

10.1677/jme-09-0177 ◽

2010 ◽

Vol 45 (5) ◽

pp. 291-299 ◽

Cited By ~ 37

Author(s):

Xiao-Qiu Wang ◽

Jing Yu ◽

Xue-Zhen Luo ◽

Ying-Li Shi ◽

Yun Wang ◽

...

Keyword(s):

U937 Cells ◽

Rt Pcr ◽

Normal Endometrium ◽

Endometrial Stromal Cells ◽

Eutopic Endometrium ◽

Transwell Migration Assay ◽

Migration Assay ◽

Macrophage Recruitment ◽

17Β Estradiol ◽

High Level

RANTES (C–C chemokine, regulated on activation, normal T cell expressed and secreted) is involved in progression of endometriosis, but the precise mechanism is understood inadequately. This study is to elucidate the roles of RANTES in macrophage recruitment and tolerance in the endometriotic milieu. The expression of RANTES was analyzed by immunohistochemistry. The cell co-cultures were applied to simulate the endometriotic milieu to investigate the regulation of RANTES secretion and its receptor CCR1 expression. Transwell migration assay was used for chemotaxis of U937 cells (macrophage line) to endometrial stromal cells (ESCs) and/or human pelvic mesothelial cells. The expression of CCR1 was analyzed by RT-PCR and qPCR in transcription and by western blot in translation respectively. Concentrations of RANTES, IL10, and IL12p70 were determined by ELISA. The phenotype of U937 cells and apoptosis of ESCs were analyzed by flow cytometry. We have found that the expression of RANTES is significantly higher in the endometriotic tissue and eutopic endometrium than that of the normal endometrium without endometriosis. The combination of 17β-estradiol and dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin increases significantly RANTES secretion in the endometriosis-associated cell co-culture which can recruit more macrophages, upregulate CCR1 expression, and induce tolerant phenotype, which inhibits the apoptosis of ESC in the milieu. In conclusion, the higher levels of RANTES in the ectopic milieu facilitate the onset and progression of endometriosis by macrophage recruitment and tolerance that in turn inhibits apoptosis and enhances growth of ESC.

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Leishmania major Promastigotes Inhibit Dendritic Cell Motility In Vitro

Infection and Immunity ◽

10.1128/iai.70.2.1023-1026.2002 ◽

2002 ◽

Vol 70 (2) ◽

pp. 1023-1026 ◽

Cited By ~ 24

Author(s):

Heather Jebbari ◽

Andrew J. Stagg ◽

Robert N. Davidson ◽

Stella C. Knight

Keyword(s):

Dendritic Cells ◽

Dendritic Cell ◽

Cell Motility ◽

Leishmania Major ◽

Protective Role ◽

Transwell Migration Assay ◽

Migration Assay ◽

Dose Dependent

ABSTRACT Using an in vitro transwell migration assay, we have demonstrated that products secreted by Leishmania major promastigotes inhibit the motility of dendritic cells (DC) by up to 93%. Inhibition was dose dependent and reversible. By inhibiting DC migration in vivo, L. major may therefore subvert DC from their potentially protective role during leishmaniasis.

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TSLC1 inhibits bladder cancer cells proliferation and promotes apoptosis by targeting miR-125b

10.21203/rs.2.22221/v1 ◽

2020 ◽

Author(s):

Jun Zhu ◽

Rui Hu ◽

NingJing Ou ◽

Zhen Liang ◽

Wei Zhang ◽

...

Keyword(s):

Bladder Cancer ◽

Cell Line ◽

Migration And Invasion ◽

P53 Expression ◽

Transwell Migration Assay ◽

Migration Assay ◽

Bladder Cancer Cells ◽

Cancer Tissues ◽

The Relationship

Abstract Backgroud: The aim of this study was to investigate the relationship between the expression of tumor suppressor in lung cancer-1 (TSLC1) and miRNA-125b in bladder cancer (BC) pathogenesis. Methods: The expression of miRNA-125b,TSLC1 and p53 in BC cell line was detected by real-time quantitative RT-PCR (RT-qPCR) or western blot. Transwell migration assay was used in the in vitro migration and invison anssay. TSLC1 and p53 expression was evaluated by immunohistochemistric staining in bladder cancer tissues. Results: We showed that the expression of miRNA-125b was significantly decreased in BC cell line(T24) transfection of miR-125b inhibitor.Knockdown of miRNA-125b promoted the growth and metastasis of T24 cells,while overexpression of miRNA-125b had the opposite effects. Furthermore,TSLC1 was significantly positive correlated with miRNA-125b expression and negative correlated with p53 expression in T24 cells.TSLC1 transfection increased the expression of miRNA-125b,and inhibited BC cell migration and invasion in vitro,and promoted apoptosis. The expression of TSLC1 and p53 was opposite in bladder cancer tissues. Conclusions: Our data provided strong evidence that TSLC1 inhibited tumorigenesis and development of BC through up-regulating tumor-suppressive miRNA-125b.

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Hypoxia Increases KIAA1199/CEMIP Expression and Enhances Cell Migration in Pancreatic Cancer

10.21203/rs.3.rs-219524/v1 ◽

2021 ◽

Author(s):

Takuya Oba ◽

Norihiro Sato ◽

Yasuhiro Adachi ◽

Takao Amaike ◽

Yuzan Kudo ◽

...

Keyword(s):

Cell Migration ◽

Protein Expression ◽

Hypoxia Inducible Factor ◽

Hypoxic Condition ◽

Ductal Adenocarcinoma ◽

Rt Pcr ◽

Transwell Migration Assay ◽

Migration Assay ◽

Mrna And Protein Expression ◽

Interfering Rna

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense desmoplasia and hypoxic microenvironment. We previously demonstrated that hyaluronan (HA), especially low-molecular weight HA, provides a favorable microenvironment for progression of PDAC. However, the effect of hypoxia on HA metabolism is unknown. Using quantitative real-time RT-PCR and Western blot analysis, we analyzed changes in expression of HA-synthesizing enzymes (HAS2, HAS3) and HA-degrading enzymes (HYAL1, KIAA1199/CEMIP) in PDAC cell lines under hypoxic condition. Hypoxia increased mRNA and protein expression of KIAA1199, whereas it decreased expression of HYAL1. Expression of HAS2 and HAS3 remained unchanged in response to hypoxia. The effect of KIAA1199 on hypoxia-induced cell migration was determined by transwell migration assay and small-interfering RNA (siRNA). Hypoxia enhanced migratory ability of PDAC cells, which was inhibited by knockdown of KIAA1199 expression. We also used immunohistochemistry to analyze Hypoxia Inducible Factor (HIF) 1α and KIAA1199 protein expression in PDAC tissues. There was a significant immunohistochemically positive correlation between KIAA1199 and HIF1α. These findings suggest that hypoxia-induced KIAA1199 expression may contribute to enhanced motility in PDAC.

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Methylation regulation of MUC6 correlates with metastasis of gastric cancer

10.21203/rs.2.23506/v1 ◽

2020 ◽

Author(s):

Ding Shi ◽

Xiaoxia Xi

Keyword(s):

Cell Lines ◽

Promoter Region ◽

Quantitative Polymerase Chain Reaction ◽

Migration And Invasion ◽

Sgc7901 Cell ◽

Transwell Migration Assay ◽

Migration Assay ◽

Normal Tissues ◽

Polymerase Chain ◽

Cancer Tissues

Abstract Background: The aim of this study was to investigate the mechanism of the downregulation of MUC6 and its influence on GC metastasis.Methods: The expression of MUC6 was examined in cancer tissues and their corresponding adjacent normal tissues in 40 gastric adenocarcinoma patients. The investigation of methylation level of MUC6 promoter region in gastric cell lines and gastric specimen tissues was performed through immunohistochemistry and/or quantitative polymerase chain reaction (qPCR)s. MUC6 was knocked down in GES-1 cell lines and overexpressed in SGC7901 cell lines; the effects of MUC6 knockdown and overexpression on cell migration and invasion were examined using Transwell migration assay. The effects of demethylation and methylation on MUC6 expression were examined using Western blot, qPCR, or double luciferase report experiment.Results: The expression of MUC6 in GC tissues was significantly lower than that in normal paracancerous tissues. While the cells migration and invasion abilities were decreased significantly after overexpression of MUC6, these abilities increased significantly after the knocking down of MUC6. The methylation levels of MUC6 in GC tissues and GC cell lines (MGC803, MKN45, AGS, SGC7901, and BGC823) were significantly higher than those in paracancerous tissues and gastric epithelial cells. The promoter methylation could significantly reduce the binding of MUC6 promoter region to the related transcription factors. The expression of MUC6 increased with the concentration of demethylated drugs and the time of action.Conclusion: The expression of MUC6 was regulated by methylation of its promoter, and this methylation of MUC6 promoter may lead to significant downregulation of MUC6 in GC and promote the metastasis of GC.

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Enhanced engraftment of hematopoietic stem/progenitor cells by the transient inhibition of an adaptor protein, Lnk

Blood ◽

10.1182/blood-2005-05-2138 ◽

2006 ◽

Vol 107 (7) ◽

pp. 2968-2975 ◽

Cited By ~ 36

Author(s):

Hitoshi Takizawa ◽

Chiyomi Kubo-Akashi ◽

Ikuo Nobuhisa ◽

Sang-Mo Kwon ◽

Masanori Iseki ◽

...

Keyword(s):

Progenitor Cells ◽

Adaptor Protein ◽

Dominant Negative ◽

Cellular Interaction ◽

Vascular Cell Adhesion ◽

Hematopoietic Stem ◽

Transwell Migration Assay ◽

Migration Assay ◽

Long Time ◽

Cell Adhesion Molecule 1

AbstractHematopoietic stem cells (HSCs) are the key elements responsible for maintaining blood-cell production throughout life and for lymphohematopoietic reconstitution following bone marrow (BM) transplantation. Enhancement of the engrafting potential and expansion capabilities of HSCs as well as hematopoietic progenitor cells (HPCs) has been a long-time desire as a means of reducing the risks and difficulties that accompany BM transplantation. The ability of HSCs/HPCs to reconstitute the hematopoietic system of irradiated hosts is negatively regulated by an intracellular adaptor protein, Lnk. Here we have identified the functional domains of Lnk and developed a dominant-negative (DN) Lnk mutant that inhibits the functions of Lnk endogenously expressed in the HSCs/HPCs and thereby potentiates the HSCs/HPCs for engraftment. Importantly, even transient expression of DN-Lnk in HSCs/HPCs facilitated their engraftment under nonmyeloablative conditions and fully reconstituted the lymphoid compartments of immunodeficient host animals. HPCs expressing DN-Lnk were efficiently trapped by immobilized vascular cell adhesion molecule-1 (VCAM-1) in a transwell migration assay, suggesting involvement of Lnk in the regulation of cell mobility or cellular interaction in microenvironments. Transient inhibition of Lnk or Lnk-mediated pathways could be a potent approach to augment engraftment of HSCs/HPCs without obvious side effects.

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