Vol. 22 (2017)



DOI 10.1186/s11658-016-0030-0 Volume 22 (2017)
Title Avoiding the side effects of electric current pulse application to electroporated cells in disposable small volume cuvettes assures good cell survival
Authors Maciej Grys, Zbigniew Madeja* and Włodzimierz Korohoda*
Abstract Background: The harmful side effects of electroporation to cells due to local changes in pH, the appearance of toxic electrode products, temperature increase, and the heterogeneity of the electric field acting on cells in the cuvettes used for electroporation were observed and discussed in several laboratories. If cells are subjected to weak electric fields for prolonged periods, for example in experiments on cell electrophoresis or galvanotaxis the same effects are seen. In these experiments investigators managed to reduce or eliminate the harmful side effects of electric current application.
Methods: For the experiments, disposable 20 μl cuvettes with two walls made of dialysis membranes were constructed and placed in a locally focused electric field at a considerable distance from the electrodes. Cuvettes were mounted into an apparatus for horizontal electrophoresis and the cells were subjected to direct current electric field (dcEF) pulses from a commercial pulse generator of exponentially declining pulses and from a custom-made generator of double and single rectangular pulses.
Results: More than 80% of the electroporated cells survived the dcEF pulses in both systems. Side effects related to electrodes were eliminated in both the flow through the dcEF and in the disposable cuvettes placed in the focused dcEFs. With a disposable cuvette system, we also confirmed the sensitization of cells to a dcEF using procaine by observing the loading of AT2 cells with calceine and using a square pulse generator, applying 50 ms single rectangular pulses.
Conclusions: We suggest that the same methods of avoiding the side effects of electric current pulse application as in cell electrophoresis and galvanotaxis should also be used for electroporation. This conclusion was confirmed in our electroporation experiments performed in conditions assuring survival of over 80% of the electroporated cells. If the amplitude, duration, and shape of the dcEF pulse are known, then electroporation does not depend on the type of pulse generator. This knowledge of the characteristics of the pulse assures reproducibility of electroporation experiments using different equipment.
Keywords Avoiding side effects of electric current pulses, Disposable cuvettes, Reversible electroporation, Fluorescent dyes, Cell viability, Flow through electric field, Direct current electric field, Focused electric field
Address and Contact Information Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Cracow, Poland
* Correspondence: z.madeja@uj.edu.pl; w.korohoda@uj.edu.pl
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DOI 10.1186/s11658-016-0032-y Volume 22 (2017)
Title MITF and PU.1 inhibit adipogenesis of ovine primary preadipocytes by restraining C/EBPβ
Authors ChongMei Ruan1†, Xiu Li2†, JunJie Hu1, Yong Zhang1 and XingXu Zhao1*
Abstract Background: PU box-binding protein (PU.1) is a master gene of hematopoietic lineage and an important specific transcription factor in osteoclast lineage. There is proof of its expression in adipose tissue, and it is known to significantly and negatively affect adipogenesis. However, it is unclear whether there are any other molecules involved in this process.
Methods0: We wished to explore the effect of PU.1’s co-activator microphthalmia-associated transcription factor (MITF) on the adipogenic differentiation of ovine primary preadipocytes. The expression vectors pcDNA-MITF and pcDNA-PU.1, and MITF siRNA and PU.1 siRNA were transfected or co-transfected into ovine tail primary preadipocytes. Real-time PCR and western blot analysis were applied to investigate the expression levels of PU.1 and MITF. The morphologic changes in the cells were observed under a microscope at a magnification of × 200 after staining with Oil Red O. The triglyceride (TG) content in cells was also determined after transfection.
Results: MITF and its co-activator PU.1 synergistically exhibited an opposite expression pattern to that of CCAAT-enhancer-binding protein-β (C/EBPβ) during adipogenic differentiation of ovine primary preadipocytes. Before induction of differentiation, overexpression of MITF or PU.1 inhibited the expression of C/EBPβ and adipogenesis in the cells; and knockdown of MITF or PU.1 promoted the expression of C/EBPβ and adipogenesis in the cells. The inhibitory or promotive effect was enhanced when MITF and PU.1 were co-overexpressed or co-silenced. However, when MITF and/or PU.1 were overexpressed after day 2 of differentiation, no changes in adipogenesis of the cells were observed. Conclusions: MITF and its co-activator PU.1 inhibited adipogenesis of ovine primary preadipocytes by restraining C/EBPβ.
Keywords Adipogenesis, Lineage-specific transcription factor, Microphthalmia-associated transcription factor, PU box-binding protein, CCAAT-enhancer-binding protein-β
Address and Contact Information 1College of Veterinary Medicine, Gansu Agriculture University, Lanzhou 730070, China.
2College of Animal Science and Technology, Anhui Agriculture University, Hefei 230036, China.
* Correspondence: zhaoxx@gsau.edu.cn
Equal contributors
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DOI 10.1186/s11658-017-0033-5 Volume 22 (2017)
Title MicroRNA-210 induces endothelial cell apoptosis by directly targeting PDK1 in the setting of atherosclerosis
Authors Ying Li1,2, Chunyan Yang1, Lili Zhang3 and Ping Yang1*
Abstract Background: Atherosclerosis is a chronically inflammatory disease and one of the leading causes of deaths worldwide. Endothelial cell apoptosis plays a crucial role in its development. Several microRNAs (miRNAs) are reportedly involved in atherosclerotic plaque formation, including miRNA-210 (miR-210). However, the underlying mechanism of its role in endothelial cell apoptosis during atherosclerosis is still largely unknown.
Methods: A mouse model with atherosclerosis induced by a high-fat diet (HFD) was built in ApoE (-/-) mice. The levels of endothelial cell apoptosis were determined via flow cytometry. The expressions of miR-210 and PDK1 in purified CD31+ endothelial cells from mouse aorta were measured via RT-qPCR and western blot. Binding between miR-210 and the 3′-untranslated region (UTR) of PDK1 mRNA was predicted using bioinformatics analyses and confirmed with a dual luciferase reporter assay. The effects of miR-210 were further analyzed in an in vitro model using human aortic endothelial cells (HAECs) treated with oxidized low-density lipoprotein (ox-LDL).
Results: We found that the HFD mice developed atherosclerosis in 12 weeks and had a significantly higher percentage of endothelial cell apoptosis. The upregulated level of miR-210 in the HFD mice and HAECs inversely correlated with the level of PDK1. Inhibiting miR-210 expression significantly reduced HAEC apoptosis, as evidenced by the results of the MTT and flow cytometry experiments. Further analysis identified PDK1 as the target of miR-210 and showed that PDK1 overexpression reversed the pro-apoptotic effect of miR-210 through mediation of the P13K/Akt/mTOR pathways.
Conclusion: Our study suggests a novel role for miR-210 in the progression of atherosclerosis through the regulation of endothelial apoptosis. This indicates that miR-210 might have potential in treatment of atherosclerosis.
Keywords Atherosclerosis, miR-210, PDK1, Endothelial cell apoptosis, ApoE (-/-)
Address and Contact Information 1Department of Cardiology, China-Japan Union Hospital of Jilin University, 130033 Changchun, China.
2Department of Neonatology, The First Hospital of Jilin University, 130021 Changchun, China.
3Department of Ultrasonography, Eastern Division of First Hospital of Jilin University, 130021 Changchun, China.
* Correspondence: pingyangjida@163.com
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DOI: 10.1186/s11658-017-0035-3 Volume 22 (2017)
Title The regulatory mechanisms of NG2/CSPG4 expression
Authors Emmanuel Ampofo*, Beate M. Schmitt, Michael D. Menger and Matthias W. Laschke
Abstract Neuron-glial antigen 2 (NG2), also known as chondroitin sulphate proteoglycan 4 (CSPG4), is a surface type I transmembrane core proteoglycan that is crucially involved in cell survival, migration and angiogenesis. NG2 is frequently used as a marker for the identification and characterization of certain cell types, but little is known about the mechanisms regulating its expression. In this review, we provide evidence that the regulation of NG2 expression underlies inflammation and hypoxia and is mediated by methyltransferases, transcription factors, including Sp1, paired box (Pax) 3 and Egr-1, and the microRNA miR129-2. These regulatory factors crucially determine NG2-mediated cellular processes such as glial scar formation in the central nervous system (CNS) or tumor growth and metastasis. Therefore, they are potential targets for the establishment of novel NG2-based therapeutic strategies in the treatment of CNS injuries, cancer and other conditions of these types.
Keywords NG2, CSPG4, Inflammation, Hypoxia, Methylation, Transcription MiRNA
Address and Contact Information Institute for Clinical & Experimental Surgery, Saarland University
* Correspondence: emmanuel.ampofo@uks.eu
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DOI: 10.1186/s11658-017-0034-4 Volume 22 (2017)
Title A simple and effective protocol for fast isolation of human Tenon’s fibroblasts from a single trabeculectomy biopsy – a comparison of cell behaviour in different culture media
Authors Agata Przekora1*, Tomasz Zarnowski2 and Grazyna Ginalska1
Abstract Background: Human Tenon’s fibroblasts (HTFs) play a crucial role in wound healing. They cause postoperative scarring of the filtering bleb and are thus responsible for trabeculectomy failure. This study aimed to find an effective and fast protocol for HTF isolation from trabeculectomy biopsies. The protocol was compared with the commonly recommended HTF isolation procedure, which uses Dulbecco’s modified Eagle’s medium (DMEM). We used Eagle’s minimum essential medium (EMEM) enriched with fibroblast growth factor (FGF), which selectively promoted the proliferation of HTF cells. A secondary goal was to compare HTF morphology, metabolism and growth during parallel cultivation of the isolated cells in FGF-enriched EMEM and DMEM.
Results: Standard procedures for HTF isolation from tissue biopsies require a 20- to 30-day culture of the explants to obtain the first monolayer. Our protocol yielded the first monolayer after approx. 15 days. More importantly, the majority of the cells were fibroblasts with only individual epithelium-derived cells present. Using FGF-enriched EMEM allowed 1.3 × 106 vimentin-positive fibroblasts to be obtained from a single biopsy within approx. 25 days. Using DMEM resulted in isolation failure and required exchange to FGF-enriched medium to recover the fibroblast culture. HTFs maintained in FGF-enriched EMEM also showed faster proliferation and a different type I collagen production ability compared to HTFs cultured in DMEM. Thus, FGF-enriched EMEM is recommended for fast propagation of HTFs unless the aim of the study is to assess the effect of a tested agent on proliferation ability or type I collagen production.
Conclusions: Our fast protocol for HTF isolation allows easy setup of cell banks by researchers under laboratory conditions and could be very useful during testing of novel ophthalmologic anti-fibrotic agents in vitro. Molecular analysis of HTFs isolated from patients with known treatment histories may provide valuable information on the effects of some medications taken before glaucoma surgery on the subsequent wound-healing process and potential for trabeculectomy failure.
Keywords Glaucoma Ocular disorders, Cell banking, Isolation protocol, Primary culture
Address and Contact Information 1 Department of Biochemistry and Biotechnology, Medical University of Lublin, Lublin, Poland
2 Department of Ophthalmology, Medical University of Lublin, Lublin, Poland
* Correspondence: agata.przekora@umlub.pl
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DOI: 10.1186/s11658-017-0037-1 Volume 22 (2017)
Title PPARγ agonist through the terminal differentiation phase is essential for adipogenic differentiation of fetal ovine preadipocytes
Authors Yong Pu and Almudena Veiga-Lopez*
Abstract Background: Although the 3T3-L1 preadipocyte cell line represents an informative model for in vitro adipogenesis research, primary cultured cells are often needed to understand particular human or animal metabolic phenotypes. As demonstrated by in vitro cultured preadipocytes from large mammalian species, primary cultured cells require specific adipogenic differentiation conditions different to that of the 3T3-L1 cell line. These conditions are also species-specific and require optimization steps. However, efficient protocols to differentiate primary preadipocytes using alternative species to rodents are scarce. Sheep represent an amenable animal model for fetal biology and developmental origins of health and disease studies. In this work, we present with the first detailed procedure to efficiently differentiate primary fetal and adult ovine preadipocytes.
Methods: Fetal and adult ovine adipose and skin tissue harvest, preadipocyte and fibroblast isolation, proliferation, and standardization and optimization of a new adipogenic differentiation protocol. Use of commercial cell lines (3T3-L1 and NIH-3T3) for validation purposes. Oil red O stain and gene expression were used to validate adipogenic differentiation. ANOVA and Fisher’s exact test were used to determine statistical significance.
Results: Our optimized adipogenic differentiation method included a prolonged adipogenic cocktail exposure time from 2 to 8 days, higher insulin concentration, and supplementation with the peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone. This protocol was optimized for both, fetal and adult preadipocytes.
Conclusions: Our protocol enables successful adipogenic differentiation of fetal and adult ovine preadipocytes. This work demonstrates that compared to the 3T3-L1 cell line, fetal ovine preadipocytes require a longer exposure to the differentiation cocktail, and the need for IMBX, dexamethasone, and/or the PPARγ agonist rosiglitazone through the terminal differentiation phase. They also require higher insulin concentration during differentiation to enhance lipid accumulation and similar to human primary preadipocytes, PPARγ agonist supplementation is also required for ovine adipogenic differentiation. This work highlights species-specific differences requirements for adipogenic differentiation and the need to develop standardized methods to investigate comparative adipocyte biology.
Keywords Sheep, Fetal, Preadipocyte, Adipogenic differentiation
Address and Contact Information Department of Animal Science, Michigan State University
* Correspondence: veiga@msu.edu
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DOI: 10.1186/s11658-017-0036-2 Volume 22 (2017)
Title ABCC6 knockdown in HepG2 cells induces a senescent-like cell phenotype
Authors Rocchina Miglionico1, Angela Ostuni1, Maria Francesca Armentano1, Luigi Milella1, Elvira Crescenzi2, Monica Carmosino1* and Faustino Bisaccia1*
Abstract Background
Pseudoxanthoma elasticum (PXE) is characterized by progressive ectopic mineralization of elastic fibers in dermal, ocular and vascular tissues. No effective treatment exists. It is caused by inactivating mutations in the gene encoding for the ATP-binding cassette, sub-family C member 6 transporter (ABCC6), which is mainly expressed in the liver. The ABCC6 substrate (s) and the PXE pathomechanism remain unknown. Recent studies have shown that overexpression of ABCC6 in HEK293 cells results in efflux of ATP, which is rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since the latter inhibits mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the characteristic ectopic mineralization. These studies also demonstrated that the presence of ABCC6 modifies cell secretory activity and suggested that ABCC6 can change the cell phenotype.
Methods
Stable ABCC6 knockdown HepG2 clones were generated using small hairpin RNA (shRNA) technology. The intracellular glutathione and ROS levels were determined. Experiments using cell cycle analysis, real-time PCR and western blot were performed on genes involved in the senescence phenotype.
Results
To shed light on the physiological role of ABCC6, we focused on the phenotype of HepG2 cells that lack ABCC6 activity. Interestingly, we found that ABCC6 knockdown HepG2 cells show: 1) intracellular reductive stress; 2) cell cycle arrest in G1 phase; 3) upregulation of p21Cip p53 independent; and 4) downregulation of lamin A/C.
Conclusions
These findings show that the absence of ABCC6 profoundly changes the HepG2 phenotype, suggesting that the PXE syndrome is a complex metabolic disease that is not exclusively related to the absence of pyrophosphate in the bloodstream.
Keywords ABCC6, Cell cycle, Senescence, Reductive stress
Address and Contact Information 1Department of Sciences, University of Basilicata
2Institute of Experimental Endocrinology and Oncology (IEOS), National Research Council (CNR)
* Correspondence: monica.carmosino@unibas.it or faustino.bisaccia@unibas.it
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DOI: 10.1186/s11658-017-0038-0 Volume 22 (2017)
Title Survivin, a molecular target for therapeutic interventions in squamous cell carcinoma
Authors Zakir Khan†1,2*, Abdul Arif Khan†3, Hariom Yadav4, Godavarthi B. K. S. Prasad1 and Prakash Singh Bisen1*
Abstract Squamous cell carcinoma (SCC) is the most common cancer worldwide. The treatment of locally advanced disease generally requires various combinations of radiotherapy, surgery, and systemic therapy. Despite aggressive multimodal treatment, most of the patients relapse. Identification of molecules that sustain cancer cell growth and survival has made molecular targeting a feasible therapeutic strategy. Survivin is a member of the Inhibitor of Apoptosis Protein (IAP) family, which is overexpressed in most of the malignancies including SCC and totally absent in most of the normal tissues. This feature makes survivin an ideal target for cancer therapy. It orchestrates several important mechanisms to support cancer cell survival including inhibition of apoptosis and regulation of cell division. Overexpression of survivin in tumors is also associated with poor prognosis, aggressive tumor behavior, resistance to therapy, and high tumor recurrence. Various strategies have been developed to target survivin expression in cancer cells, and their effects on apoptosis induction and tumor growth attenuation have been demonstrated. In this review, we discuss recent advances in therapeutic potential of survivin in cancer treatment.
Keywords Squamous cell carcinoma (SCC), Survivin, Apoptosis, Cancer immunotherapy, YM155, Hsp90 inhibitors, CDK inhibitors
Address and Contact Information 1School of Studies in Biotechnology, Jiwaji University
2Department of Biomedical Sciences, Department of Pathology, Cedars-Sinai Medical Center
3Department of Pharmaceutics, College of Pharmacy, King Saud University
4National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health
†Contributed equally
* Correspondence: zakirq.khan@gmail.com, zakir.khan@cshs.org or psbisen@gmail.com
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