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RNotion Transfection Reagent _ 4 x 1 ml

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[#2201510] RNotion Transfection Reagent _ 4 x 1 ml


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(1) Kruppel-like factor 4 (KLF-4) plays a crucial role in simvastatin (SVT)-induced differentiation of rabbit articular chondrocytes.[TOP]

Pubmed ID :29775609
Publication Date : //
Simvastatin is a cholesterol-lowing reagent that is derived synthetically from the fermentation of Aspergillus terreus. Recently, SVT has been shown to possess a protective effect of chondrocytes. Kruppel-like factor 4 (KLF-4) is a zinc finger transcription factor that plays crucial roles during the development and maintenance of multiple organs. However, the roles of KLF-4 in chondrocytes have not been well unknown. Here, we investigated whether KLF-4 regulates SVT-caused differentiated phenotype of chondrocytes. A KLF-4 cDNA or KLF-4 siRNA was transfected into SVT-treated chondrocytes. Western blot analysis, RT-PCR and immunofluorescence staining analyzed expression of type II collagen and SOX-9, marker proteins of differentiation. The results showed overexpression of KLF-4 accelerates SVT-induced type II collagen expression, as determined by western blot analysis and causes sulfated-proteoglycan synthesis, as detected by Alcian blue staining. RT-PCR revealed that ectopic expression of KLF-4 induces SVT-caused SOX-9, a transcription factor of type II collagen, expression. Transfection of KLF-4 siRNA reversed SVT-caused type II collagen and SOX-9 expression and inhibited SVT-induced sulfated proteoglycan production. This study indicates that KLF-4 plays critical role in SVT-caused chondrocytes differentiation.

Authors : Yu Seon-Mi, Kim Song Ja,

(2) Polyethylene glycol and octa-arginine dual-functionalized nanographene oxide: an optimization for efficient nucleic acid delivery.[TOP]

Pubmed ID :29757340
Publication Date : //
The successful application of nucleic acid-based therapy for the treatment of various cancers is largely dependent on a safe and efficient delivery system. A dual-functionalized graphene oxide (GO)-based nanocarrier with the conjugation of aminated-polyethylene glycol (PEG-diamine) and octa-arginine (R8) for the intracellular delivery of nucleic acids is proposed. The functionalized sites are covalently co-conjugated and the PEG : R8 molar ratio is optimized at 10 : 1 to achieve a hydrocolloidally stable size of 252 ± 2.0 nm with an effective charge of +40.97 ± 1.05 and an amine-rich content of 10.87 ± 0.4 μmol g-1. The uptake of the nanocarrier in breast cancer cell lines, MCF-7 and MDA-MB 231, is investigated. The siRNA and pDNA condensation ability in the presence and absence of enzymes and the endosomal buffering capacity, as well as the intracellular localization of the gene/nanocarrier complex are also evaluated. Furthermore, the delivery of functional genes associated with the nanocarrier is assessed using c-Myc protein knockdown and EGFP gene expression. The effective uptake of the nanocarrier by the cells shows superior cytocompatibility, and protects the siRNA and pDNA against enzyme degradation while inhibiting their migration with N : P ratios of 10 and 5, respectively. The co-conjugation of PEG-diamine and the cationic cell-penetrating peptide (CPP) into the GO nanocarrier also provides a superior internalization efficacy of 85% in comparison with a commercially available transfection reagent. The c-Myc protein knockdown and EGFP expression, which are induced by the nanocarrier, confirm that the optimized PEG-diamine/R8-functionalized GO could effectively deliver pDNA and siRNA into the cells and interfere with gene expression.

Authors : Imani Rana, Prakash Satya, Vali Hojatollah, Faghihi Shahab,

(3) An improved method for increasing the efficiency of gene transfection and transduction.[TOP]

Pubmed ID :29755642
Publication Date : //
Transfection and transduction using lentivirus has gained attention in biomedical research. To date, how to reach the maximum transfection and viral transduction efficiency is still challenging. Here we compared the transfection and viral transduction efficiency using commercially available transfection reagents including FuGENE 6, Lipofectamine 2000 and Lipofectamine 3000 in different cell lines and primary cultured cells. Enhanced green fluorescent protein (EGFP) was clearly seen in Eppendorf tubes from harvested cells using Lipofectamine 3000 without using a microscope and UV activation. Strong expression of EGFP was observed in HEK293 cells, mouse primary cortical neurons and human umbilical vein endothelial cells (HUVECs) using confocal microscopy. Western blot showed the strongest EGFP expression using cell lysates from Lipofectamine 3000 transfected HEK293 cells and transduced HUVECs compared with Lipofectamine 2000 or FuGENE 6 reagents. Using Cx43 shRNA lentivirus combined with Lipofectamine 3000 transfection reagent, we can achieve about 90% Cx43 knockdown efficacy in HUVECs. Therefore, our results suggest that a much higher transfection and viral transduction efficiency can be attained by using Lipofectamine 3000 transfection reagent.

Authors : Shi Baomin, Xue Mengzhou, Wang Yi, Wang Yufeng, Li Davey, Zhao Xiaomin, Li Xinbo,

(4) Inhibiting EZH2 rescued bupivacaine-induced neuronal apoptosis in spinal cord dorsal root ganglia in mice.[TOP]

Pubmed ID :29752567
Publication Date : //
In the present work, we intended to explore the function of enhancer of zeste homolog 2 (EZH2) in modulated anesthetic reagent bupivacaine-induced neuronal apoptosis in spinal cord dorsal root ganglia (DRG).

Authors : Zheng Jinwei, Chen Junping, Wu Guorong, Wu Chaoshuang, Wang Ruichun, Wang Wei,

(5) Filaggrin and tight junction proteins are crucial for IL-13-mediated esophageal barrier dysfunction.[TOP]

Pubmed ID :29746170
Publication Date : //
Eosinophilic esophagitis (EoE) is an allergy-mediated disease that is accompanied by IL-13 overexpression and an impaired esophageal barrier. Filaggrin (FLG) and tight junction (TJ) proteins are considered to contribute to epithelial barrier function. However, their functional involvement in EoE has not been elucidated. Here, we aimed to determine the IL-13-mediated barrier dysfunction and expression of TJ-related proteins in EoE, and to characterize interactions among TJ-related proteins involved in the barrier function of the esophageal epithelium. Biopsy specimens from EoE patients were analyzed. Primary human esophageal epithelial cells (HEECs) were cultured using an air-liquid interface (ALI) system. The permeability of TJs was assayed by biotinylation. Transepithelial electrical resistance (TEER) was measured after stimulation with IL-13 and after siRNA silencing of FLG expression. FLG and TJ genes and proteins were assessed by qRT-PCR, western blotting, and immunofluorescent staining. The biotinylation reagent diffused through the paracellular spaces of whole stratified epithelial layers in EoE biopsy samples. The TEER decreased in ALI-cultured HEECs after IL-13 stimulation. Although the protein level of FLG decreased, that of the TJ proteins increased in the mucosa of EoE biopsy samples and in ALI-cultured HEECs after IL-13 stimulation. IL-13 altered the staining patterns of TJ proteins and the epithelial morphology. FLG siRNA transfection significantly decreased TEER.The IL-13-mediated reduced esophageal barrier is associated with the altered expression pattern, but not with the levels of TJ-associated proteins. A deficiency of FLG altered the stratified epithelial barrier.

Authors : Wu Liping, Oshima Tadayuki, Li Min, Tomita Toshihiko, Fukui Hirokazu, Watari Jiro, Miwa Hiroto,

(6) Photoluminescent Cationic Carbon Dots as efficient Non-Viral Delivery of Plasmid SOX9 and Chondrogenesis of Fibroblasts.[TOP]

Pubmed ID :29728593
Publication Date : //
With the increasing demand for higher gene carrier performance, a multifunctional vector could immensely simplify gene delivery for disease treatment; nevertheless, the current non- viral vectors lack self-tracking ability. Here, a type of novel, dual-functional cationic carbon dots (CDs), produced through one-step, microwave-assisted pyrolysis of arginine and glucose, have been utilized as both a self-imaging agent and a non-viral gene vector for chondrogenesis from fibroblasts. The cationic CDs could condense the model gene plasmid SOX9 (pSOX9) to form ultra-small (10-30 nm) nanoparticles which possessed several favorable properties, including high solubility, tunable fluorescence, high yield, low cytotoxicity and outstanding biocompatibility. The MTT assay indicated that CDs/pSOX9 nanoparticles had little cytotoxicity against mouse embryonic fibroblasts (MEFs) compared to Lipofectamine2000 and PEI (25 kDa). Importantly, the CDs/pSOX9 nanoparticles with tunable fluorescence not only enabled the intracellular tracking of the nanoparticles, but also could successfully deliver the pSOX9 into MEFs with significantly high efficiency. Furthermore, the CDs/pSOX9 nanoparticles-mediated transfection of MEFs showed obvious chondrogenic differentiation. Altogether, these findings demonstrated that the CDs prepared in this study could serve as a paradigmatic example of the dual-functional reagent for both self-imaging and effective non-viral gene delivery.

Authors : Cao Xia, Wang Jianping, Deng Wenwen, Chen Jingjing, Wang Yan, Zhou Jie, Du Pan, Xu Wenqian, Wang Qiang, Wang Qilong, Yu Qingtong, Spector Myron, Yu Jiangnan, Xu Ximing,

(7) [Downregulation of PTTG1 expression inhibits the proliferation and invasiveness and promotes the apoptosis of human prostate cancer LNCaP-AI cells].[TOP]

Pubmed ID :29723450
Publication Date : //
To investigate the effects of down-regulation of PTTG1 expression on the proliferation, invasiveness and apoptosis of androgen-independent human prostate cancer LNCaP-AI cells and their sensitivity to androgen antagonists.

Authors : Cao Xi-Liang, Wei Yang-Yang, Song Xiao-Ming, Lu Ke-Quan, Yu Wen-Chao, Chen Yong-Qiang, Liu Yong-Liang, Gao Jiang-Ping,

(8) Metformin induces autophagy and G0/G1 phase cell cycle arrest in myeloma by targeting the AMPK/mTORC1 and mTORC2 pathways.[TOP]

Pubmed ID :29554968
Publication Date : //
Metformin is a commonly used drug for the treatment of diabetes. Accumulating evidence suggests that it exerts anti-tumor effects in many cancers, including multiple myeloma (MM); however, the underlying molecular mechanisms have not been clearly elucidated.

Authors : Wang Yan, Xu Wenbin, Yan Zixun, Zhao Weili, Mi Jianqing, Li Junmin, Yan Hua,

(9) shRNA targeting of ferritin heavy chain activates H19/miR-675 axis in K562 cells.[TOP]

Pubmed ID :29544765
Publication Date : //
The heavy subunit of the iron storage protein ferritin (FHC) is essential for the intracellular iron metabolism and, at the same time, it represents a central hub of iron-independent pathways, such as cell proliferation, angiogenesis, p53 regulation, chemokine signalling, stem cell expansion, miRNAs expression. In this work we have explored the ability of FHC to modulate gene expression in K562 cells, through the up-regulation of the lncRNA H19 and its cognate miR-675.

Authors : Di Sanzo M, Chirillo R, Aversa I, Biamonte F, Santamaria G, Giovannone E D, Faniello M C, Cuda G, Costanzo F,

(10) Non-viral Methodology for Efficient Co-transfection.[TOP]

Pubmed ID :29524139
Publication Date : //
The potential impact of CRISPR/Cas9, TALE, and zinc finger technology is immense, both with respect to their use as tools for understanding the roles and functions of the genomic elements and epigenome modifications in an endogenous context and as new methods for treatment of diseases. Application of such technologies has drawn attention, however, to the prevailing lack of effective delivery methods. Promising viral and non-viral methods both currently fall short when the efficient delivery of large plasmids or multiple plasmids is required. Therefore, the use of TALE and CRISPR platforms has been severely limited in applications where selection methods to increase the relative proportion of treated cells are not applicable, and it represents a significant bottleneck in the further application of these tools as therapeutics.The protocol presented here describes the synthesis of a dendronized polymer as a highly efficient and nontoxic transfection agent. Furthermore, the optimization of the polymer as a co-transfection reagent for large and multiple plasmids in cell lines is described, in addition to general considerations for co-transfection experiments. Usage of this method has allowed for significantly improved large plasmid co-transfection efficiency over Lipofectamine 2000 in multiple cell lines, allowing an improved delivery of CRISPR/dCas9 and TALE systems.

Authors : Kretzmann Jessica A, Evans Cameron W, Norret Marck, Blancafort Pilar, Swaminathan Iyer K,