Journal: Veterinary Research

Article Title: Protective immunity against Trichinella spiralis in mice elicited by oral vaccination with attenuated Salmonella-delivered TsSP1.2 DNA

doi: 10.1186/s13567-018-0582-2

Figure Lengend Snippet: The in vitro transcription and expression of TsSP1.2 in transfected BHK-21 cells. A TsSP1.2 mRNA transcription in BHK-21 cells was detected by RT-PCR. l. M: DL2000 marker; 1: pcDNA3.1-TsSP1.2 transfected cells; 2: empty pcDNA3.1 transfected cells; 3: non-transfected normal cells; 4: Lipofectamine 2000 control. B TsSP1.2 expression in BHK-21 cells transfected with pcDNA3.1-TsSP1.2 was observed by IFT with anti-rTsSP1.2 serum. C The pcDNA3.1-transfected BHK-21 cells were used as negative control. The scale bar is 50 μm.

Article Snippet: When the BHK 21 cells were grown to 90% confluence, the cells were collected by trypsinization and transfected with pcDNA3.1-TsSP1.2 with a cationic lipid Lipofectamine 2000 (Invitrogen, USA) at the ratio of 0.8 μg DNA: 2 μL lipid per well in serum-free DMEM media at 37 °C for 48 h. Total RNA was extracted from the BHK-21 cells 24 h after transfection, and the transcription levels of TsSP1.2 mRNA in transfected cells were assayed by RT-PCR with TsSP1.2-specific primers as listed above [ ].

Techniques: In Vitro, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Marker, Negative Control

Journal: The Prostate

Article Title: Overexpression of MCM10 promotes cell proliferation and predicts poor prognosis in prostate cancer. Overexpression of MCM10 promotes cell proliferation and predicts poor prognosis in prostate cancer

doi: 10.1002/pros.23703

Figure Lengend Snippet: MCM10 knockdown inhibits its expression levels in DU145 and PC‐3 cells. A‐C, Real‐time reverse transcription PCR analysis showed expression of MCM10 mRNA was reduced after transfection with the indicated shRNAs in DU145 (A) PC‐3 (B) and LNCaP (C) cells. D‐F, Western blot analysis showed protein expression of MCM10 was reduced after transfection with the indicated shRNAs in DU145 (D), PC‐3 (E), and LNCaP (F) cells. Data are represented as the mean ± SD (* P

Article Snippet: Opti‐modified Eagle's medium (Opti‐MEM) which was ideal for use during cationic lipid transfections especially Lipofectamine™ transfection reagents was purchased from Thermo Fisher Scientific, Inc. (Catalog number: 31985062).

Techniques: Expressing, Polymerase Chain Reaction, Transfection, Western Blot

Journal: Journal of Feline Medicine and Surgery

Article Title: FIP: a novel approach to vaccination

doi: 10.1016/j.jfms.2003.08.010

Figure Lengend Snippet: FKCU/LTR reporter cells, transfected with the pHook-3/tas plasmid and stained by the β -galassay. Transfection efficiency approximated as 25–30%, based on the development of blue-coloured cells. (Magnification ×200).

Article Snippet: A cationic lipid-based transfection system (Lipofectamine, Invitrogen) was used to carry plasmid DNA into FKCU/LTR/zeo/β -gal reporter cells (a kind gift from Prof. A. Rethwilm, University of Wurzburg, Germany).

Techniques: Transfection, Plasmid Preparation, Staining

Journal: Nature Communications

Article Title: Neutrophils mediate Salmonella Typhimurium clearance through the GBP4 inflammasome-dependent production of prostaglandins

doi: 10.1038/ncomms12077

Figure Lengend Snippet: Gbp4 and Gbp4 KS/AA localize to the speck in the presence of Asc. HEK293T cells were transfected with zebrafish Gbp4-GFP, Gbp4 KS/AA -GFP or Gbp4 ▵CARD -GFP in the presence or absence of zebrafish Asc-Myc, fixed at 48 h post-transfection and labelled in red with specific antibodies to the Myc epitope. ( a ) Representative frontal (xy) and lateral (xz and yz) views of maximum-intensity projection images of HEK293T cells stained with anti-Myc antibodies (Asc, Red). Gbp4, Gbp4 KS/AA and Gbp4 ▵CARD are visualized in green thanks to GFP, while nuclei are labelled with DAPI (blue). ( b ) Quantitation of the percentage of Gbp4 specks in relation to the total number of Gbp4 transfected cells. ( c ) Cells were lysed and anti-GFP and anti-c-Myc antibodies were used to validate the transfection assays by detecting Gbp4 and Asc, respectively, by western blot. The mass weights for all the proteins are indicated. Scale bars, 10 μm. The sample size for each treatment is Gbp4: 1300 cells; Gbp4+Asc: 1270 cells; Gbp4 KS/AA : 369 cells; Gbp4 KS/AA +Asc: 768 cells; Gbp4 ▵CARD : 773 cells; Gbp4 ▵CARD +Asc: 961cells. NS, not significant; *** P

Article Snippet: Transfections were performed with a cationic lipid-based transfection reagent (LyoVec, Invivogen) according to the manufacturer's instructions.

Techniques: Transfection, Staining, Quantitation Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: Ca2+ enrichment in culture medium potentiates effect of oligonucleotides

doi: 10.1093/nar/gkv626

Figure Lengend Snippet: CEM effect on subcellular localization of 2′, 4′-BNA ASO. ( A ) Huh-7 cells were lipofected with Cy3-labeled ApoB -ASO at 100 nM using Lipofectamine 2000 (Life Technologies). After 24 h of incubation, nuclei were stained with 0.5 μM Hoechst 33342 (Life Technologies) for 30 min, following by staining of lysosomes with LysoTraker ® Green DND-26 (Life Technologies) at 75 nM for 30 min at 37°C. After staining, the medium was replaced with HBSS and the cells were observed using a confocal laser scanning microscope (Leica, TCS SP5). ( B and C ) Huh-7 cells were treated with Cy3-labeled ApoB -ASO at 500 nM with or without 9 mM CaCl 2 . After 24 h of incubation, the subcellular localization of Cy3-labeled ApoB -ASO was visualized by confocal microscopy. ( D ) Huh-7 cells were transfected with the CalPhos™ Mammalian Transfection kit according to manufacturer's procedure. After 24 h of incubation, the subcellular localization of Cy3-labeled ApoB -ASO was visualized by confocal microscopy.

Article Snippet: Specifically, ZsG-N1–2R/HLE cells were transfected with each of fourteen ZsGreen1-ASOs using the cationic lipid transfection reagent Lipofectamine RNAiMAX (Invitrogen) with or without the addition of 9 mM CaCl2 .

Techniques: Allele-specific Oligonucleotide, Labeling, Incubation, Staining, Laser-Scanning Microscopy, Confocal Microscopy, Transfection

Journal: Nucleic Acids Research

Article Title: Ca2+ enrichment in culture medium potentiates effect of oligonucleotides

doi: 10.1093/nar/gkv626

Figure Lengend Snippet: CEM method reflects the activity of antisense oligonucleotides delivered by free-uptake and in vivo silencing activity more strictly than does lipofection. ( A ) ZsG-N1–2R/HLE cells were treated with each of 14 different ZsGreen1-ASOs at 2.5 μM in the presence of CaCl 2 in the medium. After 4 days, the fluorescence of ZsGreen1 and DsRed were measured. To compare the knockdown activity of each ASO with free-uptake, ZsG-N1–2R/HLE cells were treated with each respective ZsGreen1-ASO at 5 μM in the absence of CaCl 2 in the medium. After 6 days, the fluorescence of ZsGreen1 and DsRed were measured. Each data point represents the mean of three independent experiments. The mean value of knockdown activity was plotted in the two-dimensional space to analyze the correlation between transfection methods. ( B ) To compare the knockdown activities to those obtained by lipofection, ZsG-N1–2R/HLE cells were lipofected using RNAiMAX (Invitrogen). After 48 h of transfection, the fluorescence of ZsGreen1 and DsRed were measured and knockdown activity was calculated. Each data point represents the mean of three independent experiments. The mean value of knockdown activity was plotted in the two-dimensional space to analyze the correlation between transfection methods. ( C and D ) To compare the in vitro activity of CEM and lipofection with in vivo activity, Huh-7 cells were treated with each of ten different ApoB -ASOs at 10 nM in the presence of 9 mM CaCl 2 in the medium or transfected using RNAiMAX. After 24 h, total RNA was extracted and ApoB mRNA was quantitated by qRT-PCR. Mice (C57BL/6, n = 3/group) were injected subcutaneously (at 10 mg/kg) with a single dose of each of 10 ApoB -ASOs. Mice were sacrificed 72 h later and livers were analyzed for reductions in ApoB mRNA levels. The relative quantification of ApoB mRNA was normalized against the expression of the GAPDH gene. Each data point represents the mean of three independent experiments. The mean value of knockdown activity was plotted in the two-dimensional space to analyze the correlation between transfection methods.

Article Snippet: Specifically, ZsG-N1–2R/HLE cells were transfected with each of fourteen ZsGreen1-ASOs using the cationic lipid transfection reagent Lipofectamine RNAiMAX (Invitrogen) with or without the addition of 9 mM CaCl2 .

Techniques: Activity Assay, In Vivo, Fluorescence, Allele-specific Oligonucleotide, Transfection, In Vitro, Quantitative RT-PCR, Mouse Assay, Injection, Expressing

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Neurokinin1 receptors regulate morphine-induced endocytosis and desensitization of mu opioid receptors in CNS neurons

doi: 10.1523/JNEUROSCI.4315-08.2009

Figure Lengend Snippet: Mutational disruption of NK1R-mediated sequestration of β-arrestin prevents inhibition of F-MOR endocytosis in N2A cells (A) Confocal micrographs of N2A cells co-expressing either full-length HA-NK1R (left panels, red) or truncated HA-NK1R (right panels, red) with β-arrestin2-EGFP (green). Incubation with 10μM SP in cells expressing the truncated HA-NK1R (355x) resulted in less co-localization with receptors in endosomal clusters and higher cytoplasmic β-arrestin2-EGFP distribution (left column), compared to β-arrestin2-EGFP distribution in the wild type HA-NK1R under the same conditions (right column). Scale bar, 10μm. (B) In N2A cells co-expressing F-MOR (red) and HA-NK1 355x receptors (green), both morphine and DAMGO were able to drive internalization of F-MORs even in the presence of substance P. Scale bar, 10μm. (C) Quantification using ratiometric staining of F-MORs in N2A cells co-expressing HA-NK1 355x revealed that co-incubation with SP produced comparable levels of F-MOR internalization compared to treatment with either MS or DG alone. Bar graphs represent mean internalization determined from ~50 cell bodies selected at random in the N2A cultures and averaged over four independent transfections. (D) Biochemical assay of F-MOR internalization by surface biotinylation confirmed that F-MOR internalization induced by either MS or DG (lanes 3 and 5) was not significantly inhibited by co-application of SP (lanes 4 and 6; n= 3 independent experiments).

Article Snippet: Transfections were performed using a cationic lipid transfection reagent (Effectene; Qiagen, Hilden, Germany).

Techniques: Inhibition, Expressing, Incubation, Staining, Produced, Mass Spectrometry, Transfection

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Neurokinin1 receptors regulate morphine-induced endocytosis and desensitization of mu opioid receptors in CNS neurons

doi: 10.1523/JNEUROSCI.4315-08.2009

Figure Lengend Snippet: Heterologous regulation of F-MOR endocytosis in mouse neuroblastoma (N2A) cells (A) Dual-labeled confocal fluorescence micrographs show F-MOR (red) and HA-NK1R (green) transiently expressed in N2A cells. In untreated cells, both F-MOR and HA-NK1R showed a plasma membrane distribution, with minimal labeled receptors present internally in the cell (top row). Incubation with either 10μM MS or 10μM SP for 30 minutes resulted in a selective increase in either labeled F-MORs (second row, first panel) or HA-NK1Rs (third row, middle panel), respectively, present internally in the cell. Simultaneous incubation with MS and SP produced visibly less F-MOR internalization compared to incubation with MS alone (bottom row, first panel). Scale bar, 10μm. (B) Quantification of ratiometric staining in N2A cells show inhibition of F-MOR internalization in response to both MS and DG when SP is also present. Bar graphs represent mean internalization determined from ~50 cell bodies selected at random in N2A cultures and averaged over five independent transfections (**p

Article Snippet: Transfections were performed using a cationic lipid transfection reagent (Effectene; Qiagen, Hilden, Germany).

Techniques: Labeling, Fluorescence, Incubation, Mass Spectrometry, Produced, Staining, Inhibition, Transfection