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  • 99
    Thermo Fisher lipofectamine 2000
    Effect of TRPC1 silencing on Ca 2+  influx and cell migration in stable WT-RhoA transfected cells.  A : representative immunoblots of TRPC1 and RhoA. Cells were transfected with control siRNA (C-siRNA) or siTRPC1 by LipofectAMINE 2000, and whole cell lysates
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 481435 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine rnaimax
    Impact of ANP32A knockdown on viral late gene expression. A. A double-stranded siRNA targeting ANP32A mRNA (ANP) or a universal, non-targeting control siRNA (C) were introduced in A549 cells using <t>lipofectamine</t> <t>RNAiMAX</t> (InVitrogen) as described in Materials and Methods, or were exposed only to lipofectamine (LF) or only to medium (−). Whole cell lysates were prepared 3 days thereafter and ANP32A and β-actin examined by immunoblotting. B. Proliferating A549 cells were treated with ANP32A (ANP) or control (C) siRNAs, or exposed to lipofectamine (LF) or medium (−) only for 72 h. They were then infected with 10 pfu/cell AdEasyE1 or AdeasyE1Δ2347 or mock infected for 24 h. Viral protein V and cellular β-actin in whole cell lysates were examined by immunoblotting.
    Lipofectamine Rnaimax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lipofectamine 3000
    Spred2 overexpression reduced cytokine responses in macrophages. Spred2 was overexpressed by transfecting macrophages with mixtures of <t>Lipofectamine</t> 3000 and Spred2 overexpression plasmid and control plasmid for 24 h, after which the cells were stimulated with PA (500 μM) for 24 h. Cytokine levels in the culture supernatants were measured by ELISA. ** p
    Lipofectamine 3000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 43082 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher taqman microrna reverse transcription kit
    LTP regulates <t>microRNA</t> expression in the dentate gyrus middle molecular layer 5 h post-tetanisation. Regulation of microRNA expression in the middle molecular layer revealed by <t>TaqMan</t> Low Density microRNA Arrays. Eight microRNAs were found to be differentially expressed using dual selection criteria (unadjusted two-tailed Student’s t-test p
    Taqman Microrna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 27939 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher oligofectamine
    Effect of CtBP1 -siRNA on expression of MDR1 mRNA and P-gp a. MDR cell lines, NCI/ADR-RES and A2780DX, were treated with 100 nM of a CtBP1 -targeted siRNA (CtBP1siRNA), or non-targeting RNA, or mock, which only consisted of <t>oligofectamine</t> and OPTI-MEM ® I reduced serum medium. Seventy-two hours later, total RNA was extracted from the cells and the RT reaction was performed. MDR 1 and CtBP1 cDNA were amplified by PCR using the MDR 1 primer (5′-ATATCAGCAGCCCACATCAT-3′; 5′-GAAGCACTGGGATGTCCGGT-3′) and CtBP1 primer (5′-TACCACACCATCACTCTCAC-3′; 5′-CTCTGGACTCGTGTGCCCTC-3′), respectively. GAPDH was used as an internal control. Aliquots of PCR products were electrophoresed on 1.5% agarose gels, and PCR fragments were visualized by ethidium bromide staining. b. NCI/ADR-RES and A2780DX cells were treated with 100 nM of a CtBP1 siRNA (CtBP1siRNA), or non-targeting RNA, or mock, and cell lysates were prepared from the treated cells. Equal amounts (50 μg proteins) of cell lysates were separated by 6% SDS-polyacrylamide gel electrophoresis, and then transferred onto nitrocellulose membrane. The membranes were immunoblotted with monoclonal anti-P-gp, anti-CtBP1, or anti-tubulin antibodies. Detections of proteins were performed using enzyme-linked chemiluminescence. Protein bands were quantified by the Molecular Analyst software. Results are the representative of three similar experiments. c. NCI/ADR-RES and A2780DX cells were treated with 100 nM of CtBP1siRNA-2, and the effect of the siRNA on MDR1 mRNA expression was determined as described above. d. HCT-15 colon carcinoma cells were treated with 100 nM of a CtBP1 siRNA (CtBP1siRNA) or non-targeting RNA, and the effects of the siRNA on MDR1 mRNA and P-gp expression were determined as described above. Results are the representative of two similar experiments.
    Oligofectamine, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18955 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher transfection
    Effect of CtBP1 -siRNA on expression of MDR1 mRNA and P-gp a. MDR cell lines, NCI/ADR-RES and A2780DX, were treated with 100 nM of a CtBP1 -targeted siRNA (CtBP1siRNA), or non-targeting RNA, or mock, which only consisted of <t>oligofectamine</t> and OPTI-MEM ® I reduced serum medium. Seventy-two hours later, total RNA was extracted from the cells and the RT reaction was performed. MDR 1 and CtBP1 cDNA were amplified by PCR using the MDR 1 primer (5′-ATATCAGCAGCCCACATCAT-3′; 5′-GAAGCACTGGGATGTCCGGT-3′) and CtBP1 primer (5′-TACCACACCATCACTCTCAC-3′; 5′-CTCTGGACTCGTGTGCCCTC-3′), respectively. GAPDH was used as an internal control. Aliquots of PCR products were electrophoresed on 1.5% agarose gels, and PCR fragments were visualized by ethidium bromide staining. b. NCI/ADR-RES and A2780DX cells were treated with 100 nM of a CtBP1 siRNA (CtBP1siRNA), or non-targeting RNA, or mock, and cell lysates were prepared from the treated cells. Equal amounts (50 μg proteins) of cell lysates were separated by 6% SDS-polyacrylamide gel electrophoresis, and then transferred onto nitrocellulose membrane. The membranes were immunoblotted with monoclonal anti-P-gp, anti-CtBP1, or anti-tubulin antibodies. Detections of proteins were performed using enzyme-linked chemiluminescence. Protein bands were quantified by the Molecular Analyst software. Results are the representative of three similar experiments. c. NCI/ADR-RES and A2780DX cells were treated with 100 nM of CtBP1siRNA-2, and the effect of the siRNA on MDR1 mRNA expression was determined as described above. d. HCT-15 colon carcinoma cells were treated with 100 nM of a CtBP1 siRNA (CtBP1siRNA) or non-targeting RNA, and the effects of the siRNA on MDR1 mRNA and P-gp expression were determined as described above. Results are the representative of two similar experiments.
    Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1470 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen hiperfect transfection reagent
    Cell viability for each of the three methods: electroporation, <t>Hiperfect,</t> and Nanofectin. Determination of cell viability at 0 h and after 24 h for siRNA <t>transfection</t> experiments using electroporation, Hiperfect, and Nanofectin. The result was plotted as a histogram.
    Hiperfect Transfection Reagent, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 13483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lipofectamine reagent
    Interactions of ATF-2 with JunD in vitro as measured by glutathionine S -transferase (GST)-ATF-2 pull-down assays. A : human ATF-2 amino acid sequence. The basic region and leucine-zipper area were indicated by green and red colors, respectively. B : GST-ATF-2 fusion proteins: schematic diagram depicting various GST-ATF-2 constructs( a ); and GST-ATF-2 fusion proteins as measured by Coomassie blue staining assays( b ). Constructs were transformed into Eschericia coli BL21, and their expression was induced by treatment with isopropyl-b-d-thiogalactopyranoside (IPTG) at the concentration of 0.5 mM. Expressed GST (without ATF-2) or GST-ATF-2 fusion proteins were harvested and purified by equilibrated MagneGST particles. These fusion proteins were monitored by SDS-PAGE analysis and shown by Coomassie blue staining. C : ATF-2 association with JunD in cells overexpressing JunD. Cells were transfected by using the expression vector containing human junD cDNA by <t>LipofectAMINE</t> technique; whole cell lysates were harvested 48 h after the transfection. The magnetic particles bound to GST or GST-ATF-2 fusion proteins were incubated with cell lysate for 30 min, dissolved in 1× SDS loading buffer, and then subjected to SDS-PAGE. Levels of JunD in the complexes pull-down by using GST or GST-ATF-2 fusion proteins were measured by Western blot analysis with the antibody against JunD ( top ), whereas input GST or GST-ATF-2 fusion proteins were examined by using anti-GST antibody ( bottom ). Three experiments were performed that showed similar results. D : levels of JunD protein in the complexes pull-down by GST-ATF-2 fusion proteins GST-505 ( a ) and GST-176 ( b ) from control cells and cells treated with DFMO alone or DFMO plus Put for 6 days.
    Lipofectamine Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 20197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen effectene
    BTK expression is induced in HIV-1 infected cells (A) To assess BTK expression in a series of HIV-1 matched cell targets, 50 µg of whole cell extracts from B cells (BJAB), uninfected T cells (CEM and Jurkat), uninfected monocytic cells (U937 and U937-derived macrophage model cells, mΦ), HIV-1 infected T cells (ACH2 and J1.1), and infected monocytic cells (U1 and U1 mΦ) were run on a 4–20% SDS-PAGE and immunoblotted against BTK and β-actin. (B) Jurkat and U937 cells were transfected with the HIV-1 clone pNL4-3 using <t>Effectene</t> transfection reagent, harvested at 48 hr and lysed in Laemmli sample buffer. Twenty microgram of whole cell extract was assayed by western blot using α-BTK and α-β-actin antibodies. Phosphorylated BTK (p-BTK) is suggestive of BTK activation. (C) Cytosolic and nuclear extracts were prepared from Jurkat and J1.1 cells as described in Materials and Methods. The extracts were analyzed by western blot using anti-BTK antibody. Lamin A was used as a nuclear control. (D) Total cell lysates of both uninfected U937 and infected U1 cells were loaded onto a size-exclusion chromatography column and proteins separated in the presence of high salt buffer (500 mM NaCl) to minimize non-specific binding. No detergent was used during fractionation. A sampling (250 µl) of every 5 th fraction from 10 – 55 was acetone precipitated and resuspended in 30 µl of Laemmli buffer, and then 15 µl were run on a gel for western blotting using BTK and β-actin antibody. More soluble actin was present in the U1 fractions as compared to uninfected U937 cells.
    Effectene, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 19379 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher neon transfection system
    GBP1 is recruited to the bacterial surface and binds LPS through electrostatic interactions. a Size exclusion chromatograms of recombinant His-tagged GBP1 incubated with E. coli LPS, or with E. coli LPS pre-treated with CaCl 2 (5 mM), Polymyxin B (10 µg/mL) or with alkaline phosphatase. Curves were corrected by subtracting the respective LPS-specific absorbance at 280 nm. Black curves representing control condition were overlayed. b Release of LDH from IFNγ-primed HeLa 5 h after <t>transfection</t> with E. coli LPS, or after transfection with LPS previously treated with alkaline phosphatase. c 3D structure of human GBP1 (PDB 1f5n), highlighting five different negatively charged patches (A to E). Residues comprising patch E are only visible in PDB 6k1z. For each patch, the indicated residues were all mutated to alanines and analyzed for GBP1-LPS interaction by size exclusion chromatography. Purple indicates GTPase domain, green indicates helical domain. d Size exclusion chromatograms of different His-tagged GBP1 mutants incubated with E. coli LPS. Curves were corrected by subtracting LPS-specific absorbance at 280 nm. e Fluorescence confocal microscopy of naive HeLa expressing eGFP-GBP1 wt , eGFP-GBP1 KKK61-63AAA or eGFP-GBP1 KK87-88AA and infected with Salmonella -dsRed for 1 h. DNA was stained with Hoechst (blue). Representative confocal images are shown and scale bar corresponds to 5 µm. f Percentage of eGFP-GBP1 positive Salmonella at 1 h p.i., as quantified by counting between 100–200 bacteria per coverslip. Graphs show the mean ± SD, and data are pooled from three independent experiments performed in duplicate ( f ), four independent experiments performed in triplicate ( b ), or are representative from three ( a , d , e ) independent experiments. *** P
    Neon Transfection System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 15041 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega fugene hd
    Comparison of major commercially available transfection reagents using described flow cytometric method that quantifies transfection efficiency. 293T cells underwent chemical transfection using labeled or standard transfected nucleic acids (pNL4-3 or mCherry plasmids) and different commercially available transfection reagents as described in Methods. Gating on viable cells was performed as shown in Fig 1 . Each experiment was performed in triplicates and six independent times (6 per plasmid and 12 in total) with specific (un)labelled nucleic acids. Data in each experiment were normalized by the average of the experimental control (standard transfected nucleic acids) in each experiment and were then pooled together. This approach increases statistical power while taking into consideration the inherent differences in transfection efficiency among different transfection methods (different chemical transfection reagents and plasmids). The non-parametric statistical Mann-Whitney test was used for comparisons between groups. Median and interquartile range (IQR) are shown. The transfection efficiency was assessed by both amount of labelled DNA (A) and protein (B). The use of TransIT-X2 and Jet Prime gave the best (and comparable) transfection efficiencies, whereas the efficiency was reduced with <t>Lipofectamine</t> 2000 and <t>Fugene</t> HD. There was a major decrease in the amount of detectable labelled DNA with Lipofectamine 2000 and Fugene HD.
    Fugene Hd, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 15825 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega fugene
    Rb phosphorylation at S807 is required for association with Bax. Rb mutant plasmids were generated as described in the Materials and Methods section. Rb-negative <t>C33A</t> cells were transfected using <t>Fugene</t> (Promega). ( A ) Rb plasmids expressing either S807A or S811A alanine mutants were transfected into cells and 48 hours later cell lysates were utilized in GST-Bax fusion protein pull down assays. Proteins associated with GST-Bax were analyzed by immunoblotting with Rb antibodies. ( B ) Rb plasmids expressing either S807A or S807E mutants were transfected into C33A cells and 48 hours later co-immunoprecipitation with Bax antibodies was performed. Immunoprecipitates were analyzed by immunoblotting with Rb and Bax antibodies. Equivalent expression of the Rb mutant proteins in the C33A cells is verified by immunoblotting (lower panel). Data shown is representative of three independent experiments.
    Fugene, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3475 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lipofectin
    Impact of MetAP2 anti-sense oligonucleotide on mesothelioma cell viability and nucleosome formation. A: Mesothelioma cells (HM) were incubated with increasing concentrations of either a specific MetAP2 anti-sense or a scrambled oligonucleotide. After 24 hours total RNA was extracted and blotted with a specific probe for MetAP2. MetAP2 protein levels were determined by Western blotting after 48 hours exposure to the oligonucleotides. β-actin was used as an internal control. B: HM cells were treated for different periods of time with either <t>lipofectin</t> alone (mock) or with 100 nmol/L of the MetAP2 anti-sense or of a scrambled oligonucleotide. At the end of the incubation, cell viability was assessed by trypan blue exclusion. Data points depict mean values ± SD from two experiments with quadruplicate determinations (*, P = 0.02; **, P
    Lipofectin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9571 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of TRPC1 silencing on Ca 2+  influx and cell migration in stable WT-RhoA transfected cells.  A : representative immunoblots of TRPC1 and RhoA. Cells were transfected with control siRNA (C-siRNA) or siTRPC1 by LipofectAMINE 2000, and whole cell lysates

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: RhoA enhances store-operated Ca2+ entry and intestinal epithelial restitution by interacting with TRPC1 after wounding

    doi: 10.1152/ajpgi.00185.2015

    Figure Lengend Snippet: Effect of TRPC1 silencing on Ca 2+ influx and cell migration in stable WT-RhoA transfected cells. A : representative immunoblots of TRPC1 and RhoA. Cells were transfected with control siRNA (C-siRNA) or siTRPC1 by LipofectAMINE 2000, and whole cell lysates

    Article Snippet: Tissue culture media, LipofectAMINE 2000, and dialyzed fetal bovine serum (dFBS) were obtained from Invitrogen (Carlsbad, CA), and biochemicals were obtained from Sigma (St. Louis, MO).

    Techniques: Migration, Transfection, Western Blot

    CBX2 is a negative regulator of neuronal differentiation. (A) M17 cells were co-transfected with each construct as indicated in the image and the MCS-EF1-copGFP-T2A-puro vector (System Biosciences) by using Lipofectamine 2000 reagent. Puromycin was added to the culture medium 24 h later at a concentration of 1 μg/ml to eliminate non-transfected cells. Immunoblotting analysis was performed 72 h later. Immunoblotting for CBX2 expression showed that shCBX2-1 and shCBX2-2 reduced the amount of endogenous CBX2 in M17 cells. (B) The morphology of the transfected M17 cells was monitored on the 3rd day after differentiation by observing the GFP fluorescence. (C) Knocked-down of CBX2 increased the neurite length in differentiated M17 cells. Scale bar: 100 μm. The data are presented as mean ± SD, n = 30 cells, ∗∗∗ P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: CBX2 Inhibits Neurite Development by Regulating Neuron-Specific Genes Expression

    doi: 10.3389/fnmol.2018.00046

    Figure Lengend Snippet: CBX2 is a negative regulator of neuronal differentiation. (A) M17 cells were co-transfected with each construct as indicated in the image and the MCS-EF1-copGFP-T2A-puro vector (System Biosciences) by using Lipofectamine 2000 reagent. Puromycin was added to the culture medium 24 h later at a concentration of 1 μg/ml to eliminate non-transfected cells. Immunoblotting analysis was performed 72 h later. Immunoblotting for CBX2 expression showed that shCBX2-1 and shCBX2-2 reduced the amount of endogenous CBX2 in M17 cells. (B) The morphology of the transfected M17 cells was monitored on the 3rd day after differentiation by observing the GFP fluorescence. (C) Knocked-down of CBX2 increased the neurite length in differentiated M17 cells. Scale bar: 100 μm. The data are presented as mean ± SD, n = 30 cells, ∗∗∗ P

    Article Snippet: Twenty-four hours after plating, the neurons were transfected with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Transfection, Construct, Plasmid Preparation, Concentration Assay, Expressing, Fluorescence

    Tetracycline inducibility of BAC E11-IGR-β-catenin-ERα. As a gene of interest, the coding sequence of β-catenin-ERα fusion protein is engineered into BAC. (A) The activity of firefly luciferase, a surrogate marker is assayed to reflect the activity of P tet bi. HTB56 cells cultured in 6-well plates (300,000 per well) were cotransfected with 1 µg supercoiled BAC E11-IGR-β-catenin-ERα and 0.1 µg of renilla luciferase-encoding pRLSV40 DNA by lipofectamine reagent. Cells were left untreated or exposed to indicated concentrations of Tet or Dox 24 hours after transfection and luciferase activity was measured in cell extracts 24 hours later. Data from 3 independent experiments are represented as means plus standard deviation (SD). The SD values are too small to be visible in the first five bars. The relative luciferase activity of Tet and Dox untreated cells were set as 1 arbitrarily (* P

    Journal: PLoS ONE

    Article Title: Construction and Application of an Inducible System for Homogenous Expression Levels in Bulk Cell Lines

    doi: 10.1371/journal.pone.0006445

    Figure Lengend Snippet: Tetracycline inducibility of BAC E11-IGR-β-catenin-ERα. As a gene of interest, the coding sequence of β-catenin-ERα fusion protein is engineered into BAC. (A) The activity of firefly luciferase, a surrogate marker is assayed to reflect the activity of P tet bi. HTB56 cells cultured in 6-well plates (300,000 per well) were cotransfected with 1 µg supercoiled BAC E11-IGR-β-catenin-ERα and 0.1 µg of renilla luciferase-encoding pRLSV40 DNA by lipofectamine reagent. Cells were left untreated or exposed to indicated concentrations of Tet or Dox 24 hours after transfection and luciferase activity was measured in cell extracts 24 hours later. Data from 3 independent experiments are represented as means plus standard deviation (SD). The SD values are too small to be visible in the first five bars. The relative luciferase activity of Tet and Dox untreated cells were set as 1 arbitrarily (* P

    Article Snippet: Transfection was performed with Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA, USA) by using 1 µg of super-coiled BAC DNA per well.

    Techniques: BAC Assay, Sequencing, Activity Assay, Luciferase, Marker, Cell Culture, Transfection, Standard Deviation

    The EGFP is an effective selection marker for FACS. (A) 32D cells were mock transfected or transfected with I- Sce I-linearized BAC E11-IGR-β-catenin-ERα DNA by electroporation. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. The EGFP fluorescence was compared by flow cytometry. In the range of the indicated marker, there are 0.84, 7.97 and 67.76% of EGFP positive cells in mock, unsorted and sorted cell populations respectively. (B) HTB56 cells were mock transfected or transfected with supercoiled BAC E11-IGR-β-catenin-ERα DNA by lipofectamine reagent. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. Compared by flow cytometry, there are 0.16, 9.80 and 79.05% of EGFP positive cells in mock, unsorted and sorted cell populations respectively in the range of the indicated marker.

    Journal: PLoS ONE

    Article Title: Construction and Application of an Inducible System for Homogenous Expression Levels in Bulk Cell Lines

    doi: 10.1371/journal.pone.0006445

    Figure Lengend Snippet: The EGFP is an effective selection marker for FACS. (A) 32D cells were mock transfected or transfected with I- Sce I-linearized BAC E11-IGR-β-catenin-ERα DNA by electroporation. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. The EGFP fluorescence was compared by flow cytometry. In the range of the indicated marker, there are 0.84, 7.97 and 67.76% of EGFP positive cells in mock, unsorted and sorted cell populations respectively. (B) HTB56 cells were mock transfected or transfected with supercoiled BAC E11-IGR-β-catenin-ERα DNA by lipofectamine reagent. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. Compared by flow cytometry, there are 0.16, 9.80 and 79.05% of EGFP positive cells in mock, unsorted and sorted cell populations respectively in the range of the indicated marker.

    Article Snippet: Transfection was performed with Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA, USA) by using 1 µg of super-coiled BAC DNA per well.

    Techniques: Selection, Marker, FACS, Transfection, BAC Assay, Electroporation, Incubation, Fluorescence, Flow Cytometry, Cytometry

    Axin1 expression in the intestinal epithelial cells changes inflammatory responses. Transient transfections were performed with Lipofectamine2000 (LF2000). (A) IL-8 mRNA in Axin1-overexpressing HCT116 cells after Salmonella colonization. (B) IL-8 protein secretion in cell culture media in Axin1-overexpressing HCT116 cells after Salmonella colonization. (C) IL-8 mRNA in cells with Axin1 blockage by siRNA of Axin1 overexpression and IL-8 protein. (D) Salmonella -induced IL-8 protein levels in cells with Axin1 blockage by using siRNA against Axin1. (E) Salmonella -induced IL-8 protein levels in cells with Axin1 mutations. Axin1 ΔDIX lost the effect to inhibit IL-8 secretion. Data are expressed as mean ± SD. * P

    Journal: PLoS ONE

    Article Title: Axin1 Prevents Salmonella Invasiveness and Inflammatory Response in Intestinal Epithelial Cells

    doi: 10.1371/journal.pone.0034942

    Figure Lengend Snippet: Axin1 expression in the intestinal epithelial cells changes inflammatory responses. Transient transfections were performed with Lipofectamine2000 (LF2000). (A) IL-8 mRNA in Axin1-overexpressing HCT116 cells after Salmonella colonization. (B) IL-8 protein secretion in cell culture media in Axin1-overexpressing HCT116 cells after Salmonella colonization. (C) IL-8 mRNA in cells with Axin1 blockage by siRNA of Axin1 overexpression and IL-8 protein. (D) Salmonella -induced IL-8 protein levels in cells with Axin1 blockage by using siRNA against Axin1. (E) Salmonella -induced IL-8 protein levels in cells with Axin1 mutations. Axin1 ΔDIX lost the effect to inhibit IL-8 secretion. Data are expressed as mean ± SD. * P

    Article Snippet: Transient transfections were performed with Lipofectamine2000 (Invitrogen, San Diego, CA) in accordance with the manufacturer's instructions.

    Techniques: Expressing, Transfection, Cell Culture, Over Expression

    Impact of ANP32A knockdown on viral late gene expression. A. A double-stranded siRNA targeting ANP32A mRNA (ANP) or a universal, non-targeting control siRNA (C) were introduced in A549 cells using lipofectamine RNAiMAX (InVitrogen) as described in Materials and Methods, or were exposed only to lipofectamine (LF) or only to medium (−). Whole cell lysates were prepared 3 days thereafter and ANP32A and β-actin examined by immunoblotting. B. Proliferating A549 cells were treated with ANP32A (ANP) or control (C) siRNAs, or exposed to lipofectamine (LF) or medium (−) only for 72 h. They were then infected with 10 pfu/cell AdEasyE1 or AdeasyE1Δ2347 or mock infected for 24 h. Viral protein V and cellular β-actin in whole cell lysates were examined by immunoblotting.

    Journal: Virology

    Article Title: Normal human cell proteins that interact with the adenovirus type 5 E1B 55 kDa protein

    doi: 10.1016/j.virol.2017.01.013

    Figure Lengend Snippet: Impact of ANP32A knockdown on viral late gene expression. A. A double-stranded siRNA targeting ANP32A mRNA (ANP) or a universal, non-targeting control siRNA (C) were introduced in A549 cells using lipofectamine RNAiMAX (InVitrogen) as described in Materials and Methods, or were exposed only to lipofectamine (LF) or only to medium (−). Whole cell lysates were prepared 3 days thereafter and ANP32A and β-actin examined by immunoblotting. B. Proliferating A549 cells were treated with ANP32A (ANP) or control (C) siRNAs, or exposed to lipofectamine (LF) or medium (−) only for 72 h. They were then infected with 10 pfu/cell AdEasyE1 or AdeasyE1Δ2347 or mock infected for 24 h. Viral protein V and cellular β-actin in whole cell lysates were examined by immunoblotting.

    Article Snippet: All three siRNAs were introduced into HFFs using lipofectamine RNAiMAX (InVitrogen), raising the possibility that this reagent is deleterious to HFFs and/or viral genome replication.

    Techniques: Expressing, Aqueous Normal-phase Chromatography, Infection

    Functional characteristics of miR-548aq-3p as anti-angiogenic miRNAs. ( A ) Healthy ECFCs were transduced with mock lentivirus (M) or lentivirus overexpressing miR-548aq-3p (OE). 48 hours after transduction, total RNA was extracted and the expression levels of miR-548aq-3p were quantified by RT-qPCR. ( B ) Representative images from the tube formation assays of non-transduced controls treated with or without 10 nM vinblastine (VB), mock infected (M) and miR-548aq-3p overexpressed (OE) healthy ECFCs (original magnification 10×, scale bar 300 μm). ( C ) Quantitative data of total tube length (left panel), number of tubes ( > 30 μm) (middle panel) and number of branched cells (right panel) in ( B ). ( D ) CAD ECFCs were transfected with mock (M) or miR-548aq-3p inhibitor (KD) using Lipofectamine RNAiMAX. 48 hours after transfection, total RNA was extracted and the expression levels of miR-548aq-3p were quantified by RT-qPCR. ( E ) Representative images from the tube formation assay of CAD ECFCs treated with or without 10 nM vinblastine (VB), mock transfected (M) and miR-548aq-3p knockdown (KD) (original magnification 10×, scale bar 300 μm). ( F ) Quantitative data of total tube length (left panel), number of tubes ( > 30 μm) (middle panel) and number of branched cells (right panel) in ( E ).

    Journal: Scientific Reports

    Article Title: miR-548aq-3p is a novel target of Far infrared radiation which predicts coronary artery disease endothelial colony forming cell responsiveness

    doi: 10.1038/s41598-020-63311-1

    Figure Lengend Snippet: Functional characteristics of miR-548aq-3p as anti-angiogenic miRNAs. ( A ) Healthy ECFCs were transduced with mock lentivirus (M) or lentivirus overexpressing miR-548aq-3p (OE). 48 hours after transduction, total RNA was extracted and the expression levels of miR-548aq-3p were quantified by RT-qPCR. ( B ) Representative images from the tube formation assays of non-transduced controls treated with or without 10 nM vinblastine (VB), mock infected (M) and miR-548aq-3p overexpressed (OE) healthy ECFCs (original magnification 10×, scale bar 300 μm). ( C ) Quantitative data of total tube length (left panel), number of tubes ( > 30 μm) (middle panel) and number of branched cells (right panel) in ( B ). ( D ) CAD ECFCs were transfected with mock (M) or miR-548aq-3p inhibitor (KD) using Lipofectamine RNAiMAX. 48 hours after transfection, total RNA was extracted and the expression levels of miR-548aq-3p were quantified by RT-qPCR. ( E ) Representative images from the tube formation assay of CAD ECFCs treated with or without 10 nM vinblastine (VB), mock transfected (M) and miR-548aq-3p knockdown (KD) (original magnification 10×, scale bar 300 μm). ( F ) Quantitative data of total tube length (left panel), number of tubes ( > 30 μm) (middle panel) and number of branched cells (right panel) in ( E ).

    Article Snippet: To knockdown miR-548aq-3p in ECFCs, a commercial synthetic miRIDIAN microRNA Hairpin Inhibitor (hsa-miR-548aq-3p, IH-302531-01-0005) (Dharmacon, Lafayette, CO, USA) was added to the culture medium at a final concentration of 20 nM at 70~80% cell confluence using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA).

    Techniques: Functional Assay, Transduction, Expressing, Quantitative RT-PCR, Infection, Transfection, Tube Formation Assay

    Intracellular amount of emodin in K562/ADM and K562 cells. Both cells were incubated with various concentrations of emodin (0.1–10 μM) at 37°C or 4°C for 2 h (A); K562/ADM cells were co-treated with 0, 1, 3, 10 μM of verapamil or 0, 2, 4, 10 μM of cyclosporine A and emodin (5 μM) for 2 h (B). RNAi of P-gp (C). A-: K562/ADM cells transfected with negative control siRNA; AL: K562/ADM cells transfected with Lipofectamine RNAiMAX Reagent; AP: K562/ADM cells transfected with P-gp siRNA; A: K562/ADM cells; S: K562 cells. After transfection with P-gp siRNA, intracellular amount of emodin (5 μM) was enhanced compared with other control groups (D). Data were represented as the mean ± S.D. of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Emodin reverses leukemia multidrug resistance by competitive inhibition and downregulation of P-glycoprotein

    doi: 10.1371/journal.pone.0187971

    Figure Lengend Snippet: Intracellular amount of emodin in K562/ADM and K562 cells. Both cells were incubated with various concentrations of emodin (0.1–10 μM) at 37°C or 4°C for 2 h (A); K562/ADM cells were co-treated with 0, 1, 3, 10 μM of verapamil or 0, 2, 4, 10 μM of cyclosporine A and emodin (5 μM) for 2 h (B). RNAi of P-gp (C). A-: K562/ADM cells transfected with negative control siRNA; AL: K562/ADM cells transfected with Lipofectamine RNAiMAX Reagent; AP: K562/ADM cells transfected with P-gp siRNA; A: K562/ADM cells; S: K562 cells. After transfection with P-gp siRNA, intracellular amount of emodin (5 μM) was enhanced compared with other control groups (D). Data were represented as the mean ± S.D. of three independent experiments. * P

    Article Snippet: The Lipofectamine RNAiMAX Reagent was purchased from Invitrogen Trading Co., Ltd (Shanghai, China).

    Techniques: Incubation, Transfection, Negative Control

    Spred2 overexpression reduced cytokine responses in macrophages. Spred2 was overexpressed by transfecting macrophages with mixtures of Lipofectamine 3000 and Spred2 overexpression plasmid and control plasmid for 24 h, after which the cells were stimulated with PA (500 μM) for 24 h. Cytokine levels in the culture supernatants were measured by ELISA. ** p

    Journal: Frontiers in Immunology

    Article Title: Spred2 Regulates High Fat Diet-Induced Adipose Tissue Inflammation, and Metabolic Abnormalities in Mice

    doi: 10.3389/fimmu.2019.00017

    Figure Lengend Snippet: Spred2 overexpression reduced cytokine responses in macrophages. Spred2 was overexpressed by transfecting macrophages with mixtures of Lipofectamine 3000 and Spred2 overexpression plasmid and control plasmid for 24 h, after which the cells were stimulated with PA (500 μM) for 24 h. Cytokine levels in the culture supernatants were measured by ELISA. ** p

    Article Snippet: For each transfection, 0.75 μL Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) and 0.5 μg Spred2 expression plasmid (kindly provided by Dr. Masakiyo Sakaguchi, Okayama university, Japan) were added to 50 μL Opti-MEM (Life Technologies) and incubated for 10 min at room temperature before the mixtures were added to the cells.

    Techniques: Over Expression, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    MiR-181d-5p targets KLF6 to ameliorate H/R injury. HK-2 cells were co-transfected with the KLF6 plasmid and miR-181d-5p mimic with Lipofectamine 3000 and, 72 h later, were treated with hypoxia (l% oxygen) for 24 h/reoxygenation for 3 h. (A,B) Quantitative analysis of HIF1-α, KIM-1, and caspase-3 expression in HK-2 cells treated with or without miR-181d-5p and KLF6 ( n = 4 or 5 per group). (C) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 4 per group). The data are presented as the means ± SDs. ∗ P

    Journal: Frontiers in Physiology

    Article Title: MiR-181d-5p Targets KLF6 to Improve Ischemia/Reperfusion-Induced AKI Through Effects on Renal Function, Apoptosis, and Inflammation

    doi: 10.3389/fphys.2020.00510

    Figure Lengend Snippet: MiR-181d-5p targets KLF6 to ameliorate H/R injury. HK-2 cells were co-transfected with the KLF6 plasmid and miR-181d-5p mimic with Lipofectamine 3000 and, 72 h later, were treated with hypoxia (l% oxygen) for 24 h/reoxygenation for 3 h. (A,B) Quantitative analysis of HIF1-α, KIM-1, and caspase-3 expression in HK-2 cells treated with or without miR-181d-5p and KLF6 ( n = 4 or 5 per group). (C) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 4 per group). The data are presented as the means ± SDs. ∗ P

    Article Snippet: After 24 h, 293T cells were co-transfected with the miR-181d mimic or a scrambled miRNA sequence and PGL3-KLF6-wt or PFL3-KLF6-mut using Lipofectamine 3000 transfection reagent (L3000015, Thermo Fisher Scientific, Waltham, MA, United States).

    Techniques: Transfection, Plasmid Preparation, Expressing

    KLF6 overexpression exacerbated the hypoxia-induced decline in renal function, renal tubular cell apoptosis, and inflammatory response. HK-2 cells were transfected with KLF6 plasmid and KLF6 shRNA plasmid or scrambled plasmid with Lipofectamine 3000 and, 72 h later, were incubated in normoxia (control) or treated with hypoxia (1% oxygen) for 24 h/reoxygenation for 3 h. (A) KLF6 protein expression in HK-2 cells treated with or without KLF6 ( n = 3 per group). (B,C) qRT-PCR was used to measure miR-181d-5p, KIM-1 and HIF1-α levels after KLF6 transfection ( n = 5 per group). (D) Annexin V-FITC/PI double staining was utilized to evaluate apoptosis after KLF6 transfection. This experiment was repeated three times. (E) KLF6 increased NF-KB expression. HK-2 cells were transfected with or without the KLF6 plasmid. The results shown are from Western blot analysis of NF-KB and I-KB. β-Actin and Lamin-A were used as internal controls for I-KB and NF-KB, respectively ( n = 3 per group). (F) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 3 per group). The data are presented as the means ± SDs. * P

    Journal: Frontiers in Physiology

    Article Title: MiR-181d-5p Targets KLF6 to Improve Ischemia/Reperfusion-Induced AKI Through Effects on Renal Function, Apoptosis, and Inflammation

    doi: 10.3389/fphys.2020.00510

    Figure Lengend Snippet: KLF6 overexpression exacerbated the hypoxia-induced decline in renal function, renal tubular cell apoptosis, and inflammatory response. HK-2 cells were transfected with KLF6 plasmid and KLF6 shRNA plasmid or scrambled plasmid with Lipofectamine 3000 and, 72 h later, were incubated in normoxia (control) or treated with hypoxia (1% oxygen) for 24 h/reoxygenation for 3 h. (A) KLF6 protein expression in HK-2 cells treated with or without KLF6 ( n = 3 per group). (B,C) qRT-PCR was used to measure miR-181d-5p, KIM-1 and HIF1-α levels after KLF6 transfection ( n = 5 per group). (D) Annexin V-FITC/PI double staining was utilized to evaluate apoptosis after KLF6 transfection. This experiment was repeated three times. (E) KLF6 increased NF-KB expression. HK-2 cells were transfected with or without the KLF6 plasmid. The results shown are from Western blot analysis of NF-KB and I-KB. β-Actin and Lamin-A were used as internal controls for I-KB and NF-KB, respectively ( n = 3 per group). (F) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 3 per group). The data are presented as the means ± SDs. * P

    Article Snippet: After 24 h, 293T cells were co-transfected with the miR-181d mimic or a scrambled miRNA sequence and PGL3-KLF6-wt or PFL3-KLF6-mut using Lipofectamine 3000 transfection reagent (L3000015, Thermo Fisher Scientific, Waltham, MA, United States).

    Techniques: Over Expression, Transfection, Plasmid Preparation, shRNA, Incubation, Expressing, Quantitative RT-PCR, Double Staining, Western Blot

    siRNA-mediated silencing of PCGF isoforms increases  E. chaffeensis  infection. THP-1 cells were transfected with isoform-specific siRNA and then infected with  E. chaffeensis  at 24 h posttransfection. (A) Alexa Fluor 488-conjugated siRNA-transfected cell, showing high efficiency of RNA transfection using Lipofectamine 3000. (B) Western blot analysis of the total cell lysate from control and siRNA-transfected THP-1 cells confirmed the decrease in PCGF2, PCGF3, PCGF4, and PCGF5 48 h posttransfection. GAPDH was used as the loading control. The relative abundance of PCGF isoforms in siRNA-transfected cells was determined after normalization to the loading control and then represented as the percentage remaining after the knockdown. (C) Table representing the percentage increase in ehrlichial morulae and the average number of morulae/cell for each PCGF isoform-specific knockdown. The average morula counts were determined by counting the number of morula present in each field of view and then dividing that by the number of cells counted. The experiment was repeated three times in duplicate, and the values shown are means ± standard deviations (Stdev). (D) The fold change in ehrlichial infection was determined by comparing the ehrlichial  dsb  to the host cell  gapdh  in individual PCGF knockdown using real-time qPCR at 48 hpi ( n  = 3; *,  P  ≤ 0.05).

    Journal: Infection and Immunity

    Article Title: Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection

    doi: 10.1128/IAI.00845-17

    Figure Lengend Snippet: siRNA-mediated silencing of PCGF isoforms increases E. chaffeensis infection. THP-1 cells were transfected with isoform-specific siRNA and then infected with E. chaffeensis at 24 h posttransfection. (A) Alexa Fluor 488-conjugated siRNA-transfected cell, showing high efficiency of RNA transfection using Lipofectamine 3000. (B) Western blot analysis of the total cell lysate from control and siRNA-transfected THP-1 cells confirmed the decrease in PCGF2, PCGF3, PCGF4, and PCGF5 48 h posttransfection. GAPDH was used as the loading control. The relative abundance of PCGF isoforms in siRNA-transfected cells was determined after normalization to the loading control and then represented as the percentage remaining after the knockdown. (C) Table representing the percentage increase in ehrlichial morulae and the average number of morulae/cell for each PCGF isoform-specific knockdown. The average morula counts were determined by counting the number of morula present in each field of view and then dividing that by the number of cells counted. The experiment was repeated three times in duplicate, and the values shown are means ± standard deviations (Stdev). (D) The fold change in ehrlichial infection was determined by comparing the ehrlichial dsb to the host cell gapdh in individual PCGF knockdown using real-time qPCR at 48 hpi ( n = 3; *, P ≤ 0.05).

    Article Snippet: Briefly, specific siRNA (3 μl) and Lipofectamine 3000 reagent (7.5 μl) were added to Opti-MEM medium (250 μl) (Invitrogen), incubated for 5 min at room temperature, and then added to the cell suspension in a 6-well plate.

    Techniques: Infection, Transfection, Western Blot, Real-time Polymerase Chain Reaction

    LTP regulates microRNA expression in the dentate gyrus middle molecular layer 5 h post-tetanisation. Regulation of microRNA expression in the middle molecular layer revealed by TaqMan Low Density microRNA Arrays. Eight microRNAs were found to be differentially expressed using dual selection criteria (unadjusted two-tailed Student’s t-test p

    Journal: PLoS ONE

    Article Title: MicroRNAs, miR-23a-3p and miR-151-3p, Are Regulated in Dentate Gyrus Neuropil following Induction of Long-Term Potentiation In Vivo

    doi: 10.1371/journal.pone.0170407

    Figure Lengend Snippet: LTP regulates microRNA expression in the dentate gyrus middle molecular layer 5 h post-tetanisation. Regulation of microRNA expression in the middle molecular layer revealed by TaqMan Low Density microRNA Arrays. Eight microRNAs were found to be differentially expressed using dual selection criteria (unadjusted two-tailed Student’s t-test p

    Article Snippet: Total RNA (10 ng per 15 μL reaction) was converted to cDNA using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems).

    Techniques: Expressing, Selection, Two Tailed Test

    miR-198 functions to repress Cyclin T1 protein expression in primary monocytes. (A) Expression levels of Cyclin T1 and β-actin proteins in freshly isolated monocytes (d0) or macrophages allowed to differentiate for five days (d5) were examined in immunoblots for two donors. (B) Expression levels of Cyclin T1 mRNA relative to β-actin mRNA were measured by RT-real-time PCR assay; expression levels of miR-198 were measured using TaqMan MicroRNA assays. The relative abundance was calculated by normalizing of β-actin mRNA or miR-198 levels. Bars represent range in three measurements. (C) miR-198 inhibitor (anti-miR-198) or control inhibitor (anti-miR-Control) were transfected into freshly isolated monocytes from two donors (Donor 3, 4). miR-198 precursor (pre-miR-198) or control precursor (pre-miR-Control) were transfected into freshly isolated monocytes and differentiation was induced with GM-CSF treatment (Donor 5). Cells were harvested by direct lysis at times indicated after transfection and Cyclin T1 protein expression was examined in immunoblots. Quantification of Cyclin T1 protein levels is shown below each band (normalized to β-actin).

    Journal: PLoS Pathogens

    Article Title: miR-198 Inhibits HIV-1 Gene Expression and Replication in Monocytes and Its Mechanism of Action Appears To Involve Repression of Cyclin T1

    doi: 10.1371/journal.ppat.1000263

    Figure Lengend Snippet: miR-198 functions to repress Cyclin T1 protein expression in primary monocytes. (A) Expression levels of Cyclin T1 and β-actin proteins in freshly isolated monocytes (d0) or macrophages allowed to differentiate for five days (d5) were examined in immunoblots for two donors. (B) Expression levels of Cyclin T1 mRNA relative to β-actin mRNA were measured by RT-real-time PCR assay; expression levels of miR-198 were measured using TaqMan MicroRNA assays. The relative abundance was calculated by normalizing of β-actin mRNA or miR-198 levels. Bars represent range in three measurements. (C) miR-198 inhibitor (anti-miR-198) or control inhibitor (anti-miR-Control) were transfected into freshly isolated monocytes from two donors (Donor 3, 4). miR-198 precursor (pre-miR-198) or control precursor (pre-miR-Control) were transfected into freshly isolated monocytes and differentiation was induced with GM-CSF treatment (Donor 5). Cells were harvested by direct lysis at times indicated after transfection and Cyclin T1 protein expression was examined in immunoblots. Quantification of Cyclin T1 protein levels is shown below each band (normalized to β-actin).

    Article Snippet: As quantified by TaqMan MicroRNA assays (Applied Biosystems), miR-198 was expressed > 400-fold in cell pools expressing shmiR-198 relative to pools expressing shGFP.

    Techniques: Expressing, Isolation, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Lysis

    Effect of CtBP1 -siRNA on expression of MDR1 mRNA and P-gp a. MDR cell lines, NCI/ADR-RES and A2780DX, were treated with 100 nM of a CtBP1 -targeted siRNA (CtBP1siRNA), or non-targeting RNA, or mock, which only consisted of oligofectamine and OPTI-MEM ® I reduced serum medium. Seventy-two hours later, total RNA was extracted from the cells and the RT reaction was performed. MDR 1 and CtBP1 cDNA were amplified by PCR using the MDR 1 primer (5′-ATATCAGCAGCCCACATCAT-3′; 5′-GAAGCACTGGGATGTCCGGT-3′) and CtBP1 primer (5′-TACCACACCATCACTCTCAC-3′; 5′-CTCTGGACTCGTGTGCCCTC-3′), respectively. GAPDH was used as an internal control. Aliquots of PCR products were electrophoresed on 1.5% agarose gels, and PCR fragments were visualized by ethidium bromide staining. b. NCI/ADR-RES and A2780DX cells were treated with 100 nM of a CtBP1 siRNA (CtBP1siRNA), or non-targeting RNA, or mock, and cell lysates were prepared from the treated cells. Equal amounts (50 μg proteins) of cell lysates were separated by 6% SDS-polyacrylamide gel electrophoresis, and then transferred onto nitrocellulose membrane. The membranes were immunoblotted with monoclonal anti-P-gp, anti-CtBP1, or anti-tubulin antibodies. Detections of proteins were performed using enzyme-linked chemiluminescence. Protein bands were quantified by the Molecular Analyst software. Results are the representative of three similar experiments. c. NCI/ADR-RES and A2780DX cells were treated with 100 nM of CtBP1siRNA-2, and the effect of the siRNA on MDR1 mRNA expression was determined as described above. d. HCT-15 colon carcinoma cells were treated with 100 nM of a CtBP1 siRNA (CtBP1siRNA) or non-targeting RNA, and the effects of the siRNA on MDR1 mRNA and P-gp expression were determined as described above. Results are the representative of two similar experiments.

    Journal: Biochemical pharmacology

    Article Title: Involvement of CtBP1 in the Transcriptional Activation of the MDR1 Gene in Human Multidrug Resistant Cancer Cells

    doi: 10.1016/j.bcp.2007.06.017

    Figure Lengend Snippet: Effect of CtBP1 -siRNA on expression of MDR1 mRNA and P-gp a. MDR cell lines, NCI/ADR-RES and A2780DX, were treated with 100 nM of a CtBP1 -targeted siRNA (CtBP1siRNA), or non-targeting RNA, or mock, which only consisted of oligofectamine and OPTI-MEM ® I reduced serum medium. Seventy-two hours later, total RNA was extracted from the cells and the RT reaction was performed. MDR 1 and CtBP1 cDNA were amplified by PCR using the MDR 1 primer (5′-ATATCAGCAGCCCACATCAT-3′; 5′-GAAGCACTGGGATGTCCGGT-3′) and CtBP1 primer (5′-TACCACACCATCACTCTCAC-3′; 5′-CTCTGGACTCGTGTGCCCTC-3′), respectively. GAPDH was used as an internal control. Aliquots of PCR products were electrophoresed on 1.5% agarose gels, and PCR fragments were visualized by ethidium bromide staining. b. NCI/ADR-RES and A2780DX cells were treated with 100 nM of a CtBP1 siRNA (CtBP1siRNA), or non-targeting RNA, or mock, and cell lysates were prepared from the treated cells. Equal amounts (50 μg proteins) of cell lysates were separated by 6% SDS-polyacrylamide gel electrophoresis, and then transferred onto nitrocellulose membrane. The membranes were immunoblotted with monoclonal anti-P-gp, anti-CtBP1, or anti-tubulin antibodies. Detections of proteins were performed using enzyme-linked chemiluminescence. Protein bands were quantified by the Molecular Analyst software. Results are the representative of three similar experiments. c. NCI/ADR-RES and A2780DX cells were treated with 100 nM of CtBP1siRNA-2, and the effect of the siRNA on MDR1 mRNA expression was determined as described above. d. HCT-15 colon carcinoma cells were treated with 100 nM of a CtBP1 siRNA (CtBP1siRNA) or non-targeting RNA, and the effects of the siRNA on MDR1 mRNA and P-gp expression were determined as described above. Results are the representative of two similar experiments.

    Article Snippet: Cells in exponential phase of growth were plated in 6-well plates at 5 × 105 cells/well, grown for 24 h, and then transfected with CtBP1 -targeted siRNA or non-targeting siRNA at a final concentration of 100nM using oligofectamine and OPTI-MEM I reduced serum medium (Invitrogen Life Technologies, Inc., Carlsbad, CA), according to the manufacturer’s protocol.

    Techniques: Expressing, Amplification, Polymerase Chain Reaction, Staining, Polyacrylamide Gel Electrophoresis, Software

    Cell viability for each of the three methods: electroporation, Hiperfect, and Nanofectin. Determination of cell viability at 0 h and after 24 h for siRNA transfection experiments using electroporation, Hiperfect, and Nanofectin. The result was plotted as a histogram.

    Journal: Molecular Vision

    Article Title: Vascular endothelial growth factor receptor-1 (VEGFR-1) expression in human corneal fibroblast decreased with age

    doi:

    Figure Lengend Snippet: Cell viability for each of the three methods: electroporation, Hiperfect, and Nanofectin. Determination of cell viability at 0 h and after 24 h for siRNA transfection experiments using electroporation, Hiperfect, and Nanofectin. The result was plotted as a histogram.

    Article Snippet: Three microliters of HiPerfect transfection reagent (Qiagen) was added to the diluted siRNA and mixed by vortexing.

    Techniques: Electroporation, Transfection

    Interactions of ATF-2 with JunD in vitro as measured by glutathionine S -transferase (GST)-ATF-2 pull-down assays. A : human ATF-2 amino acid sequence. The basic region and leucine-zipper area were indicated by green and red colors, respectively. B : GST-ATF-2 fusion proteins: schematic diagram depicting various GST-ATF-2 constructs( a ); and GST-ATF-2 fusion proteins as measured by Coomassie blue staining assays( b ). Constructs were transformed into Eschericia coli BL21, and their expression was induced by treatment with isopropyl-b-d-thiogalactopyranoside (IPTG) at the concentration of 0.5 mM. Expressed GST (without ATF-2) or GST-ATF-2 fusion proteins were harvested and purified by equilibrated MagneGST particles. These fusion proteins were monitored by SDS-PAGE analysis and shown by Coomassie blue staining. C : ATF-2 association with JunD in cells overexpressing JunD. Cells were transfected by using the expression vector containing human junD cDNA by LipofectAMINE technique; whole cell lysates were harvested 48 h after the transfection. The magnetic particles bound to GST or GST-ATF-2 fusion proteins were incubated with cell lysate for 30 min, dissolved in 1× SDS loading buffer, and then subjected to SDS-PAGE. Levels of JunD in the complexes pull-down by using GST or GST-ATF-2 fusion proteins were measured by Western blot analysis with the antibody against JunD ( top ), whereas input GST or GST-ATF-2 fusion proteins were examined by using anti-GST antibody ( bottom ). Three experiments were performed that showed similar results. D : levels of JunD protein in the complexes pull-down by GST-ATF-2 fusion proteins GST-505 ( a ) and GST-176 ( b ) from control cells and cells treated with DFMO alone or DFMO plus Put for 6 days.

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Induced ATF-2 represses CDK4 transcription through dimerization with JunD inhibiting intestinal epithelial cell growth after polyamine depletion

    doi: 10.1152/ajpcell.00021.2010

    Figure Lengend Snippet: Interactions of ATF-2 with JunD in vitro as measured by glutathionine S -transferase (GST)-ATF-2 pull-down assays. A : human ATF-2 amino acid sequence. The basic region and leucine-zipper area were indicated by green and red colors, respectively. B : GST-ATF-2 fusion proteins: schematic diagram depicting various GST-ATF-2 constructs( a ); and GST-ATF-2 fusion proteins as measured by Coomassie blue staining assays( b ). Constructs were transformed into Eschericia coli BL21, and their expression was induced by treatment with isopropyl-b-d-thiogalactopyranoside (IPTG) at the concentration of 0.5 mM. Expressed GST (without ATF-2) or GST-ATF-2 fusion proteins were harvested and purified by equilibrated MagneGST particles. These fusion proteins were monitored by SDS-PAGE analysis and shown by Coomassie blue staining. C : ATF-2 association with JunD in cells overexpressing JunD. Cells were transfected by using the expression vector containing human junD cDNA by LipofectAMINE technique; whole cell lysates were harvested 48 h after the transfection. The magnetic particles bound to GST or GST-ATF-2 fusion proteins were incubated with cell lysate for 30 min, dissolved in 1× SDS loading buffer, and then subjected to SDS-PAGE. Levels of JunD in the complexes pull-down by using GST or GST-ATF-2 fusion proteins were measured by Western blot analysis with the antibody against JunD ( top ), whereas input GST or GST-ATF-2 fusion proteins were examined by using anti-GST antibody ( bottom ). Three experiments were performed that showed similar results. D : levels of JunD protein in the complexes pull-down by GST-ATF-2 fusion proteins GST-505 ( a ) and GST-176 ( b ) from control cells and cells treated with DFMO alone or DFMO plus Put for 6 days.

    Article Snippet: Transient transfection was performed with Lipofectamine Reagent from Invitrogen (Carlsbad, CA).

    Techniques: In Vitro, Sequencing, Construct, Staining, Transformation Assay, Expressing, Concentration Assay, Purification, SDS Page, Transfection, Plasmid Preparation, Incubation, Western Blot

    Flow cytometric analysis of RFP expressing PAM212 cells that were stained with either MitoTracker or Sytox red nucleic acid stain. Cells were transfected with either 18-3-18 GL-NPs or Lipofectamine Plus reagent. a A comparison between the proportion of live and dead PAM212 cells represented by either MitoTracker or Sytox red fluorescent signal. b The density plots show representative data from one of three separate experiments

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: Flow cytometric analysis of RFP expressing PAM212 cells that were stained with either MitoTracker or Sytox red nucleic acid stain. Cells were transfected with either 18-3-18 GL-NPs or Lipofectamine Plus reagent. a A comparison between the proportion of live and dead PAM212 cells represented by either MitoTracker or Sytox red fluorescent signal. b The density plots show representative data from one of three separate experiments

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Flow Cytometry, Expressing, Staining, Transfection

    Evaluating SSC intensity as a parameter of NPs internalization into the cells using flow cytometry. a The SSC intensity of 18-3-18 GL-NPs treated cells was higher than Lipofectamine Plus treated and untreated cells (R1). b RFP expression in SSC high (R3) and low cell population (R4). Each dot plot represents 10,000 events

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: Evaluating SSC intensity as a parameter of NPs internalization into the cells using flow cytometry. a The SSC intensity of 18-3-18 GL-NPs treated cells was higher than Lipofectamine Plus treated and untreated cells (R1). b RFP expression in SSC high (R3) and low cell population (R4). Each dot plot represents 10,000 events

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Flow Cytometry, Cytometry, Expressing

    RFP expression in PAM212 cells with different status of cell membrane integrity. a The status of cell membrane integrity was determined based on the intensity of Sytox red DNA stain using flow cytometry; R1 highly-porated cells, R2 partially-porated cells, R3 intact cells, R4 debris and cell fragments. b Stacked bar graph presents the membrane status of cells expressing RFP after transfection by Lipofectamine Plus or 18-3-18 GL-NPs

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: RFP expression in PAM212 cells with different status of cell membrane integrity. a The status of cell membrane integrity was determined based on the intensity of Sytox red DNA stain using flow cytometry; R1 highly-porated cells, R2 partially-porated cells, R3 intact cells, R4 debris and cell fragments. b Stacked bar graph presents the membrane status of cells expressing RFP after transfection by Lipofectamine Plus or 18-3-18 GL-NPs

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry, Transfection

    Effect of GL-NPs on the viability of PAM212 cells measured by MitoTracker staining and flow cytometry. Results are expressed as the mean of cell viability index ± standard deviation compared to the untreated control (as 100 %). Asterisks represent LSD post hoc statistical significance compared to Lipofectamine Plus (P

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: Effect of GL-NPs on the viability of PAM212 cells measured by MitoTracker staining and flow cytometry. Results are expressed as the mean of cell viability index ± standard deviation compared to the untreated control (as 100 %). Asterisks represent LSD post hoc statistical significance compared to Lipofectamine Plus (P

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Staining, Flow Cytometry, Cytometry, Standard Deviation

    RFP expression in PAM212 cells, transfected GL-NPs and Lipofectamine Plus reagent measured by flow cytometry. Results are expressed as the mean percentage of RFP positive cells ± standard deviation. Results are expressed as mean measurements ± SD (n = 4). Asterisks represent LSD post hoc statistical significance compared to Lipofectamine Plus (P

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: RFP expression in PAM212 cells, transfected GL-NPs and Lipofectamine Plus reagent measured by flow cytometry. Results are expressed as the mean percentage of RFP positive cells ± standard deviation. Results are expressed as mean measurements ± SD (n = 4). Asterisks represent LSD post hoc statistical significance compared to Lipofectamine Plus (P

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Standard Deviation

    Median fluorescence intensity (MFI) of RFP positive PAM212 cells transfected by Lipofectamine Plus or 18-3-18 GL-NPs. MFI for RFP in cells transfected by 18-3-18 GL-NPs was 1.6-fold higher than MFI for RFP in Lipofectamine Plus treated cells. The black and red peaks represent control negative and test respectively

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: Median fluorescence intensity (MFI) of RFP positive PAM212 cells transfected by Lipofectamine Plus or 18-3-18 GL-NPs. MFI for RFP in cells transfected by 18-3-18 GL-NPs was 1.6-fold higher than MFI for RFP in Lipofectamine Plus treated cells. The black and red peaks represent control negative and test respectively

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Fluorescence, Transfection

    Confocal microscopic images of PAM212 cells treated with pDNA complexed to Lipofectamine Plus or GL-NPs, prepared using gemini surfactant series 12, 16, and 18. The expression of the tdTomato RFP is shown in red and nuclei were stained with DRAQ-5 and are shown in blue . Magnification ×20

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: Confocal microscopic images of PAM212 cells treated with pDNA complexed to Lipofectamine Plus or GL-NPs, prepared using gemini surfactant series 12, 16, and 18. The expression of the tdTomato RFP is shown in red and nuclei were stained with DRAQ-5 and are shown in blue . Magnification ×20

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Expressing, Staining

    SOD2 knockdown inhibits both the migration and invasion abilities of TSCC To characterize the role of SOD2 in aiding metastasis, the plasmid containing SOD2 shRNA was transiently transfected into UM1 cells using Lipofectamine Plus reagent. Cells were tested 24 h post-transfection. (A) Significant reduction of SOD2 protein levels and activities were observed in the SOD2 shRNA-transfected UM1 cells compared to the vector control transfected cells. *: P

    Journal: Free radical biology & medicine

    Article Title: Manganese superoxide dismutase induces migration and invasion of tongue squamous cell carcinoma via H2O2-dependent Snail signaling

    doi: 10.1016/j.freeradbiomed.2012.04.031

    Figure Lengend Snippet: SOD2 knockdown inhibits both the migration and invasion abilities of TSCC To characterize the role of SOD2 in aiding metastasis, the plasmid containing SOD2 shRNA was transiently transfected into UM1 cells using Lipofectamine Plus reagent. Cells were tested 24 h post-transfection. (A) Significant reduction of SOD2 protein levels and activities were observed in the SOD2 shRNA-transfected UM1 cells compared to the vector control transfected cells. *: P

    Article Snippet: The constructed plasmids were transiently transfected into UM1 cells using Lipofectamine Plus reagent (Invitrogen, CA, USA), according to the manufacturer's instructions[ ].

    Techniques: Migration, Plasmid Preparation, shRNA, Transfection

    Snail signaling contributes to SOD2-induced migration and invasion of TSCC To characterize the role of Snail signaling in SOD2-induced metastasis of TSCC, western blot analysis was used with actin as the loading control. Plasmids containing SOD2 shRNA were transiently transfected into UM1 cells using Lipofectamine Plus reagent. Cells were tested 24 h post-transfection. The UM2 cells or SOD2 shRNA-transfected UM1 cells were treated with 100μM H 2 O 2 for 24 h. (A) UM1 cells displayed an increase in Snai1, Snai2, MMP-1, ERK1/2 and pERK1/2 protein levels, and decreased protein levels of E-cadtherin compared to UM2 cells. (B) UM1 cells displayed decreased snai1, snai2, MMP-1, ERK1/2 and pERK1/2 protein levels and increased E-cadtherin protein levels upon SOD2 knockdown. (C, D) The addition of H 2 O 2 increased the protein levels of Snai1, Snai2, MMP-1, ERK1/2 and pERK1/2 and decreased protein levels of E-cadtherin in both UM2 cells and the SOD2 shRNA-transfected UMl cells.

    Journal: Free radical biology & medicine

    Article Title: Manganese superoxide dismutase induces migration and invasion of tongue squamous cell carcinoma via H2O2-dependent Snail signaling

    doi: 10.1016/j.freeradbiomed.2012.04.031

    Figure Lengend Snippet: Snail signaling contributes to SOD2-induced migration and invasion of TSCC To characterize the role of Snail signaling in SOD2-induced metastasis of TSCC, western blot analysis was used with actin as the loading control. Plasmids containing SOD2 shRNA were transiently transfected into UM1 cells using Lipofectamine Plus reagent. Cells were tested 24 h post-transfection. The UM2 cells or SOD2 shRNA-transfected UM1 cells were treated with 100μM H 2 O 2 for 24 h. (A) UM1 cells displayed an increase in Snai1, Snai2, MMP-1, ERK1/2 and pERK1/2 protein levels, and decreased protein levels of E-cadtherin compared to UM2 cells. (B) UM1 cells displayed decreased snai1, snai2, MMP-1, ERK1/2 and pERK1/2 protein levels and increased E-cadtherin protein levels upon SOD2 knockdown. (C, D) The addition of H 2 O 2 increased the protein levels of Snai1, Snai2, MMP-1, ERK1/2 and pERK1/2 and decreased protein levels of E-cadtherin in both UM2 cells and the SOD2 shRNA-transfected UMl cells.

    Article Snippet: The constructed plasmids were transiently transfected into UM1 cells using Lipofectamine Plus reagent (Invitrogen, CA, USA), according to the manufacturer's instructions[ ].

    Techniques: Migration, Western Blot, shRNA, Transfection

    BTK expression is induced in HIV-1 infected cells (A) To assess BTK expression in a series of HIV-1 matched cell targets, 50 µg of whole cell extracts from B cells (BJAB), uninfected T cells (CEM and Jurkat), uninfected monocytic cells (U937 and U937-derived macrophage model cells, mΦ), HIV-1 infected T cells (ACH2 and J1.1), and infected monocytic cells (U1 and U1 mΦ) were run on a 4–20% SDS-PAGE and immunoblotted against BTK and β-actin. (B) Jurkat and U937 cells were transfected with the HIV-1 clone pNL4-3 using Effectene transfection reagent, harvested at 48 hr and lysed in Laemmli sample buffer. Twenty microgram of whole cell extract was assayed by western blot using α-BTK and α-β-actin antibodies. Phosphorylated BTK (p-BTK) is suggestive of BTK activation. (C) Cytosolic and nuclear extracts were prepared from Jurkat and J1.1 cells as described in Materials and Methods. The extracts were analyzed by western blot using anti-BTK antibody. Lamin A was used as a nuclear control. (D) Total cell lysates of both uninfected U937 and infected U1 cells were loaded onto a size-exclusion chromatography column and proteins separated in the presence of high salt buffer (500 mM NaCl) to minimize non-specific binding. No detergent was used during fractionation. A sampling (250 µl) of every 5 th fraction from 10 – 55 was acetone precipitated and resuspended in 30 µl of Laemmli buffer, and then 15 µl were run on a gel for western blotting using BTK and β-actin antibody. More soluble actin was present in the U1 fractions as compared to uninfected U937 cells.

    Journal: Journal of neurovirology

    Article Title: Role of Bruton’s Tyrosine Kinase inhibitors in HIV-1 infected cells

    doi: 10.1007/s13365-015-0323-5

    Figure Lengend Snippet: BTK expression is induced in HIV-1 infected cells (A) To assess BTK expression in a series of HIV-1 matched cell targets, 50 µg of whole cell extracts from B cells (BJAB), uninfected T cells (CEM and Jurkat), uninfected monocytic cells (U937 and U937-derived macrophage model cells, mΦ), HIV-1 infected T cells (ACH2 and J1.1), and infected monocytic cells (U1 and U1 mΦ) were run on a 4–20% SDS-PAGE and immunoblotted against BTK and β-actin. (B) Jurkat and U937 cells were transfected with the HIV-1 clone pNL4-3 using Effectene transfection reagent, harvested at 48 hr and lysed in Laemmli sample buffer. Twenty microgram of whole cell extract was assayed by western blot using α-BTK and α-β-actin antibodies. Phosphorylated BTK (p-BTK) is suggestive of BTK activation. (C) Cytosolic and nuclear extracts were prepared from Jurkat and J1.1 cells as described in Materials and Methods. The extracts were analyzed by western blot using anti-BTK antibody. Lamin A was used as a nuclear control. (D) Total cell lysates of both uninfected U937 and infected U1 cells were loaded onto a size-exclusion chromatography column and proteins separated in the presence of high salt buffer (500 mM NaCl) to minimize non-specific binding. No detergent was used during fractionation. A sampling (250 µl) of every 5 th fraction from 10 – 55 was acetone precipitated and resuspended in 30 µl of Laemmli buffer, and then 15 µl were run on a gel for western blotting using BTK and β-actin antibody. More soluble actin was present in the U1 fractions as compared to uninfected U937 cells.

    Article Snippet: HIV-1 proviral DNA construct pNL4.3 (5 µg) was also transfected in U937 and Jurkat cells using Effectene (Qiagen) according to the manufacturer's instructions ( ).

    Techniques: Expressing, Infection, Derivative Assay, SDS Page, Transfection, Western Blot, Activation Assay, Size-exclusion Chromatography, Binding Assay, Fractionation, Sampling

    siRNA mediated BTK knockdown induces apoptosis in HIV-1 infected cells (A) Latently infected HLM-1 cells were used for selective depletion with siLuc (control) or two different siRNAs targeting BTK (siBTK #1 and siBTK #2). Log phase growing cells (5×10 5 /ml) were transfected with either siLuc or siBTK (10 nm) using Effectene. Cells were harvested at 24, 48 and 72 h post-transfection, and 20 µg of whole cell lysates were resolved by SDS-PAGE and immunoblotted against BTK to assay for effective siRNA-mediated BTK downregulation. (B) Similar to panel A, where cell lysates were further probed with α-caspase 3 and α-PARP antibodies as markers of program cell death. Western blots against β-actin were performed as a loading control. (C) Similar to panel A, where HLM-1 cells were transfected with siLuc, siBTK #1 and siBTK #2 (50 nM). Viability was assessed using CellTiter-Glo assay 72 hours post-treatment. Results are shown as mean of three independent experiments ± SD.

    Journal: Journal of neurovirology

    Article Title: Role of Bruton’s Tyrosine Kinase inhibitors in HIV-1 infected cells

    doi: 10.1007/s13365-015-0323-5

    Figure Lengend Snippet: siRNA mediated BTK knockdown induces apoptosis in HIV-1 infected cells (A) Latently infected HLM-1 cells were used for selective depletion with siLuc (control) or two different siRNAs targeting BTK (siBTK #1 and siBTK #2). Log phase growing cells (5×10 5 /ml) were transfected with either siLuc or siBTK (10 nm) using Effectene. Cells were harvested at 24, 48 and 72 h post-transfection, and 20 µg of whole cell lysates were resolved by SDS-PAGE and immunoblotted against BTK to assay for effective siRNA-mediated BTK downregulation. (B) Similar to panel A, where cell lysates were further probed with α-caspase 3 and α-PARP antibodies as markers of program cell death. Western blots against β-actin were performed as a loading control. (C) Similar to panel A, where HLM-1 cells were transfected with siLuc, siBTK #1 and siBTK #2 (50 nM). Viability was assessed using CellTiter-Glo assay 72 hours post-treatment. Results are shown as mean of three independent experiments ± SD.

    Article Snippet: HIV-1 proviral DNA construct pNL4.3 (5 µg) was also transfected in U937 and Jurkat cells using Effectene (Qiagen) according to the manufacturer's instructions ( ).

    Techniques: Infection, Transfection, SDS Page, Western Blot, Glo Assay

    Luciferase reporter gene assay. NuLi-1 cells, MS-1 cells, A549 cells, and LK-2 cells were plated onto 24-well plates and incubated for 20 h at 37°C in 5% CO2. The control pGL3 vector DNA (0.2 μg) or similar amounts of Luciferase-NF-κB reporter vectors, was cotransfected into cells with the pRL-SV40 vector using the effectene transfection reagent. Cells were then treated with DMSO (0.016%, v/v), TNF (10 ng/ml), 17-DMAG (0.05 μM), or TNF (10 ng/ml) plus 17-DMAG (0.05 μM) for 24 h. Luciferase activity was measured in cell lysates. Each experiment was repeated thrice with similar results. *, it is considered as a significant difference, when P

    Journal: Diagnostic Pathology

    Article Title: Combined effects of 17-DMAG and TNF on cells through a mechanism related to the NF-kappaB pathway

    doi: 10.1186/1746-1596-8-70

    Figure Lengend Snippet: Luciferase reporter gene assay. NuLi-1 cells, MS-1 cells, A549 cells, and LK-2 cells were plated onto 24-well plates and incubated for 20 h at 37°C in 5% CO2. The control pGL3 vector DNA (0.2 μg) or similar amounts of Luciferase-NF-κB reporter vectors, was cotransfected into cells with the pRL-SV40 vector using the effectene transfection reagent. Cells were then treated with DMSO (0.016%, v/v), TNF (10 ng/ml), 17-DMAG (0.05 μM), or TNF (10 ng/ml) plus 17-DMAG (0.05 μM) for 24 h. Luciferase activity was measured in cell lysates. Each experiment was repeated thrice with similar results. *, it is considered as a significant difference, when P

    Article Snippet: The control pGL3 vector DNA (0.2 μg) (purchased from Clontech Co.) or similar amounts of Luciferase-NF-κB reporter vectors (Clontech Co., USA), was cotransfected into cells with the pRL-SV40 vector using the effectene transfection reagent (Qiagen, USA) for 20 h. Cells were then treated with DMSO (0.016%, v/v), TNF (10 ng/ml), 17-DMAG (0.05 μM), or TNF (10 ng/ml) plus 17-DMAG (0.05 μM) for 24 h. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer's protocol.

    Techniques: Luciferase, Reporter Gene Assay, Mass Spectrometry, Incubation, Plasmid Preparation, Transfection, Activity Assay

    GBP1 is recruited to the bacterial surface and binds LPS through electrostatic interactions. a Size exclusion chromatograms of recombinant His-tagged GBP1 incubated with E. coli LPS, or with E. coli LPS pre-treated with CaCl 2 (5 mM), Polymyxin B (10 µg/mL) or with alkaline phosphatase. Curves were corrected by subtracting the respective LPS-specific absorbance at 280 nm. Black curves representing control condition were overlayed. b Release of LDH from IFNγ-primed HeLa 5 h after transfection with E. coli LPS, or after transfection with LPS previously treated with alkaline phosphatase. c 3D structure of human GBP1 (PDB 1f5n), highlighting five different negatively charged patches (A to E). Residues comprising patch E are only visible in PDB 6k1z. For each patch, the indicated residues were all mutated to alanines and analyzed for GBP1-LPS interaction by size exclusion chromatography. Purple indicates GTPase domain, green indicates helical domain. d Size exclusion chromatograms of different His-tagged GBP1 mutants incubated with E. coli LPS. Curves were corrected by subtracting LPS-specific absorbance at 280 nm. e Fluorescence confocal microscopy of naive HeLa expressing eGFP-GBP1 wt , eGFP-GBP1 KKK61-63AAA or eGFP-GBP1 KK87-88AA and infected with Salmonella -dsRed for 1 h. DNA was stained with Hoechst (blue). Representative confocal images are shown and scale bar corresponds to 5 µm. f Percentage of eGFP-GBP1 positive Salmonella at 1 h p.i., as quantified by counting between 100–200 bacteria per coverslip. Graphs show the mean ± SD, and data are pooled from three independent experiments performed in duplicate ( f ), four independent experiments performed in triplicate ( b ), or are representative from three ( a , d , e ) independent experiments. *** P

    Journal: Nature Communications

    Article Title: Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria

    doi: 10.1038/s41467-020-16889-z

    Figure Lengend Snippet: GBP1 is recruited to the bacterial surface and binds LPS through electrostatic interactions. a Size exclusion chromatograms of recombinant His-tagged GBP1 incubated with E. coli LPS, or with E. coli LPS pre-treated with CaCl 2 (5 mM), Polymyxin B (10 µg/mL) or with alkaline phosphatase. Curves were corrected by subtracting the respective LPS-specific absorbance at 280 nm. Black curves representing control condition were overlayed. b Release of LDH from IFNγ-primed HeLa 5 h after transfection with E. coli LPS, or after transfection with LPS previously treated with alkaline phosphatase. c 3D structure of human GBP1 (PDB 1f5n), highlighting five different negatively charged patches (A to E). Residues comprising patch E are only visible in PDB 6k1z. For each patch, the indicated residues were all mutated to alanines and analyzed for GBP1-LPS interaction by size exclusion chromatography. Purple indicates GTPase domain, green indicates helical domain. d Size exclusion chromatograms of different His-tagged GBP1 mutants incubated with E. coli LPS. Curves were corrected by subtracting LPS-specific absorbance at 280 nm. e Fluorescence confocal microscopy of naive HeLa expressing eGFP-GBP1 wt , eGFP-GBP1 KKK61-63AAA or eGFP-GBP1 KK87-88AA and infected with Salmonella -dsRed for 1 h. DNA was stained with Hoechst (blue). Representative confocal images are shown and scale bar corresponds to 5 µm. f Percentage of eGFP-GBP1 positive Salmonella at 1 h p.i., as quantified by counting between 100–200 bacteria per coverslip. Graphs show the mean ± SD, and data are pooled from three independent experiments performed in duplicate ( f ), four independent experiments performed in triplicate ( b ), or are representative from three ( a , d , e ) independent experiments. *** P

    Article Snippet: For electroporation of HeLa or HBEC3-KT cells, the Neon Transfection System (Life Technologies) was used.

    Techniques: Recombinant, Incubation, Transfection, Size-exclusion Chromatography, Fluorescence, Confocal Microscopy, Expressing, Infection, Staining

    IFNγ priming is required for LPS-induced caspase-4 activation in human epithelial cells. a – c Intracellular bacterial fold-replication ( a ) and release of LDH ( b , c ) in naive or IFNγ-primed wild-type, CASP4 –/– or GSDMD –/– HeLa cells, at 1 or 6-h post-infection (p.i.) with Salmonella . Cells in 96-well plates were infected for 30 min, washed and gentamicin was added to kill extracellular bacteria. At the indicated time points supernatant was collected to determine the release of LDH, and then cells were lysed and the number of viable intracellular bacteria was determined by counting colony forming units (CFUs). The bacterial fold-replication was calculated relative to 1 h p.i. d Percentage of CHQ-resistant cytosolic Salmonella in naive or IFNγ-primed HeLa at 1.5 h p.i. Cells were infected for 30 min as in ( a ) and then treated with gentamicin ± CHQ for an additional 1 h before cells were lysed and bacteria counted by CFUs. The percentage of cytosolic bacteria was calculated as the ratio of (CHQ + gentamicin resistant / gentamicin resistant ). e – g Release of LDH from naive or IFNγ-primed cells, 5 h after transfection with LPS (2.5 µg/50,000 cells) or 3–4 h after electroporation with LPS (300 ng/50,0000 cells). h Western blot analysis of full length (p43) and cleaved (p32) caspase-4 in the supernatants and cell lysates from naive or IFNγ-primed HaCaT cells, upon transfection with E. coli LPS LPS (2.5 µg/50,000 cells). i Streptavidin pull-down assay of the binding of biotin-conjugated LPS to endogenous caspase-4 from the lysates of naive or IFNγ-primed HBEC3-KT. Cells in 6-well plates were transfected with LPS-biotin (10 µg) or left untransfected, and biotinylated substrate was pulled down using equal amounts of streptavidin magnetic beads, which were then eluted in equal volumes of SDS-PAGE reducing sample buffer. Streptavidin-bound and -unbound fractions were analyzed by immunoblotting for caspase-4. Graphs show the mean ± SD, and data are pooled from two to six independent experiments performed in triplicate ( a – g ) or representative of two ( h , i ) independent experiments. *** P

    Journal: Nature Communications

    Article Title: Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria

    doi: 10.1038/s41467-020-16889-z

    Figure Lengend Snippet: IFNγ priming is required for LPS-induced caspase-4 activation in human epithelial cells. a – c Intracellular bacterial fold-replication ( a ) and release of LDH ( b , c ) in naive or IFNγ-primed wild-type, CASP4 –/– or GSDMD –/– HeLa cells, at 1 or 6-h post-infection (p.i.) with Salmonella . Cells in 96-well plates were infected for 30 min, washed and gentamicin was added to kill extracellular bacteria. At the indicated time points supernatant was collected to determine the release of LDH, and then cells were lysed and the number of viable intracellular bacteria was determined by counting colony forming units (CFUs). The bacterial fold-replication was calculated relative to 1 h p.i. d Percentage of CHQ-resistant cytosolic Salmonella in naive or IFNγ-primed HeLa at 1.5 h p.i. Cells were infected for 30 min as in ( a ) and then treated with gentamicin ± CHQ for an additional 1 h before cells were lysed and bacteria counted by CFUs. The percentage of cytosolic bacteria was calculated as the ratio of (CHQ + gentamicin resistant / gentamicin resistant ). e – g Release of LDH from naive or IFNγ-primed cells, 5 h after transfection with LPS (2.5 µg/50,000 cells) or 3–4 h after electroporation with LPS (300 ng/50,0000 cells). h Western blot analysis of full length (p43) and cleaved (p32) caspase-4 in the supernatants and cell lysates from naive or IFNγ-primed HaCaT cells, upon transfection with E. coli LPS LPS (2.5 µg/50,000 cells). i Streptavidin pull-down assay of the binding of biotin-conjugated LPS to endogenous caspase-4 from the lysates of naive or IFNγ-primed HBEC3-KT. Cells in 6-well plates were transfected with LPS-biotin (10 µg) or left untransfected, and biotinylated substrate was pulled down using equal amounts of streptavidin magnetic beads, which were then eluted in equal volumes of SDS-PAGE reducing sample buffer. Streptavidin-bound and -unbound fractions were analyzed by immunoblotting for caspase-4. Graphs show the mean ± SD, and data are pooled from two to six independent experiments performed in triplicate ( a – g ) or representative of two ( h , i ) independent experiments. *** P

    Article Snippet: For electroporation of HeLa or HBEC3-KT cells, the Neon Transfection System (Life Technologies) was used.

    Techniques: Activation Assay, Infection, Transfection, Electroporation, Western Blot, Pull Down Assay, Binding Assay, Magnetic Beads, SDS Page

    GBP1 is required for Salmonella - and LPS-induced caspase-4 activation to induce pyroptosis in epithelial cells. a Immunoblots for GBP1, caspase-4 and GAPDH (loading control) in cell lysates from IFNγ-primed wild-type or GBP1 –/– HeLa. b , c Release of LDH from naive or IFNγ-primed wild-type or GBP1 –/– HeLa after Salmonella infection ( b ) or after 5 h transfection with E. coli LPS (2.5 µg / 50,000 cells) ( c ). d , e Immunoblots for full length (p43) and cleaved (p32) caspase-4 in combined supernatants and cell lysates from naive or IFNγ-primed wild-type and GBP1 –/– HeLa, upon transfection with E. coli LPS for 5 h ( d ) or Salmonella infection ( e ). f Release of LDH from IFNγ-primed wild-type or GBP1 –/– HeLa, 3 h after electroporation with LPS (300 ng/50,000 cells). g Release of LDH in IFNγ-primed HBEC3-KT or HaCaT cells treated with non-targeting control siRNA (NT) or with siRNAs targeting CASP4 , GSDMD or GBP1 , after E. coli LPS transfection. Cells were treated with siRNAs for 24 h and transfected with LPS (2.5 µg / 50,000 cells) for 5 h. Graphs show the mean ± SD, and data are pooled from two ( c , f ), three ( g ) or four ( b ) independent experiments performed in triplicate, or representative of three independent experiments ( d , e ). *** P

    Journal: Nature Communications

    Article Title: Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria

    doi: 10.1038/s41467-020-16889-z

    Figure Lengend Snippet: GBP1 is required for Salmonella - and LPS-induced caspase-4 activation to induce pyroptosis in epithelial cells. a Immunoblots for GBP1, caspase-4 and GAPDH (loading control) in cell lysates from IFNγ-primed wild-type or GBP1 –/– HeLa. b , c Release of LDH from naive or IFNγ-primed wild-type or GBP1 –/– HeLa after Salmonella infection ( b ) or after 5 h transfection with E. coli LPS (2.5 µg / 50,000 cells) ( c ). d , e Immunoblots for full length (p43) and cleaved (p32) caspase-4 in combined supernatants and cell lysates from naive or IFNγ-primed wild-type and GBP1 –/– HeLa, upon transfection with E. coli LPS for 5 h ( d ) or Salmonella infection ( e ). f Release of LDH from IFNγ-primed wild-type or GBP1 –/– HeLa, 3 h after electroporation with LPS (300 ng/50,000 cells). g Release of LDH in IFNγ-primed HBEC3-KT or HaCaT cells treated with non-targeting control siRNA (NT) or with siRNAs targeting CASP4 , GSDMD or GBP1 , after E. coli LPS transfection. Cells were treated with siRNAs for 24 h and transfected with LPS (2.5 µg / 50,000 cells) for 5 h. Graphs show the mean ± SD, and data are pooled from two ( c , f ), three ( g ) or four ( b ) independent experiments performed in triplicate, or representative of three independent experiments ( d , e ). *** P

    Article Snippet: For electroporation of HeLa or HBEC3-KT cells, the Neon Transfection System (Life Technologies) was used.

    Techniques: Activation Assay, Western Blot, Infection, Transfection, Electroporation

    Comparison of major commercially available transfection reagents using described flow cytometric method that quantifies transfection efficiency. 293T cells underwent chemical transfection using labeled or standard transfected nucleic acids (pNL4-3 or mCherry plasmids) and different commercially available transfection reagents as described in Methods. Gating on viable cells was performed as shown in Fig 1 . Each experiment was performed in triplicates and six independent times (6 per plasmid and 12 in total) with specific (un)labelled nucleic acids. Data in each experiment were normalized by the average of the experimental control (standard transfected nucleic acids) in each experiment and were then pooled together. This approach increases statistical power while taking into consideration the inherent differences in transfection efficiency among different transfection methods (different chemical transfection reagents and plasmids). The non-parametric statistical Mann-Whitney test was used for comparisons between groups. Median and interquartile range (IQR) are shown. The transfection efficiency was assessed by both amount of labelled DNA (A) and protein (B). The use of TransIT-X2 and Jet Prime gave the best (and comparable) transfection efficiencies, whereas the efficiency was reduced with Lipofectamine 2000 and Fugene HD. There was a major decrease in the amount of detectable labelled DNA with Lipofectamine 2000 and Fugene HD.

    Journal: PLoS ONE

    Article Title: A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells

    doi: 10.1371/journal.pone.0182941

    Figure Lengend Snippet: Comparison of major commercially available transfection reagents using described flow cytometric method that quantifies transfection efficiency. 293T cells underwent chemical transfection using labeled or standard transfected nucleic acids (pNL4-3 or mCherry plasmids) and different commercially available transfection reagents as described in Methods. Gating on viable cells was performed as shown in Fig 1 . Each experiment was performed in triplicates and six independent times (6 per plasmid and 12 in total) with specific (un)labelled nucleic acids. Data in each experiment were normalized by the average of the experimental control (standard transfected nucleic acids) in each experiment and were then pooled together. This approach increases statistical power while taking into consideration the inherent differences in transfection efficiency among different transfection methods (different chemical transfection reagents and plasmids). The non-parametric statistical Mann-Whitney test was used for comparisons between groups. Median and interquartile range (IQR) are shown. The transfection efficiency was assessed by both amount of labelled DNA (A) and protein (B). The use of TransIT-X2 and Jet Prime gave the best (and comparable) transfection efficiencies, whereas the efficiency was reduced with Lipofectamine 2000 and Fugene HD. There was a major decrease in the amount of detectable labelled DNA with Lipofectamine 2000 and Fugene HD.

    Article Snippet: Transfection reagents The following commercially available transfection reagents were purchased: TransIT-X2 (Mirus, Madison, WI), Jet Prime (Polyplus, France), Lipofectamine 2000 (Thermo Fisher, Waltham, MA) and Fugene HD (Promega, Madison, WI).

    Techniques: Transfection, Flow Cytometry, Cytometry, Labeling, Plasmid Preparation, MANN-WHITNEY

    Efficiency of DAI knockdown in primary murine microglia and astrocytes by small-interfering RNA . Microglia (Mg) and astrocytes (Ast) were untreated (Con) or transfected with a combination of three different siRNA duplexes targeting murine DAI using FuGENE HD transfection reagent. At 72 hours post-transfection, untreated and transfected cells were exposed to HSV-1 (MOI of 10) for 24 hours and DAI knockdown was confirmed in whole cell lysates by immunoblot analysis. Expression of a non-specific protein band observed following Coomassie blue staining is shown as a loading control (lc). For comparison purposes, DAI expression in murine small intestine tissue is shown (+).

    Journal: Journal of Neuroinflammation

    Article Title: A role for DNA-dependent activator of interferon regulatory factor in the recognition of herpes simplex virus type 1 by glial cells

    doi: 10.1186/1742-2094-8-99

    Figure Lengend Snippet: Efficiency of DAI knockdown in primary murine microglia and astrocytes by small-interfering RNA . Microglia (Mg) and astrocytes (Ast) were untreated (Con) or transfected with a combination of three different siRNA duplexes targeting murine DAI using FuGENE HD transfection reagent. At 72 hours post-transfection, untreated and transfected cells were exposed to HSV-1 (MOI of 10) for 24 hours and DAI knockdown was confirmed in whole cell lysates by immunoblot analysis. Expression of a non-specific protein band observed following Coomassie blue staining is shown as a loading control (lc). For comparison purposes, DAI expression in murine small intestine tissue is shown (+).

    Article Snippet: In vitro stimulation of microglia and astrocytes with the DAI ligand, B-DNA Poly(dA:dT) double-stranded B-DNA (InvivoGen, San Diego, CA) was directly introduced into microglia and astrocytes at concentrations of 3 μg/ml and 6 μg/ml using FuGENE HD Transfection Reagent (Promega, Madison, WI) according to the manufacturer's instructions.

    Techniques: Small Interfering RNA, AST Assay, Transfection, Expressing, Staining

    Rb phosphorylation at S807 is required for association with Bax. Rb mutant plasmids were generated as described in the Materials and Methods section. Rb-negative C33A cells were transfected using Fugene (Promega). ( A ) Rb plasmids expressing either S807A or S811A alanine mutants were transfected into cells and 48 hours later cell lysates were utilized in GST-Bax fusion protein pull down assays. Proteins associated with GST-Bax were analyzed by immunoblotting with Rb antibodies. ( B ) Rb plasmids expressing either S807A or S807E mutants were transfected into C33A cells and 48 hours later co-immunoprecipitation with Bax antibodies was performed. Immunoprecipitates were analyzed by immunoblotting with Rb and Bax antibodies. Equivalent expression of the Rb mutant proteins in the C33A cells is verified by immunoblotting (lower panel). Data shown is representative of three independent experiments.

    Journal: Cell Cycle

    Article Title: Phosphorylation of the Retinoblastoma protein (Rb) on serine-807 is required for association with Bax

    doi: 10.4161/15384101.2014.964093

    Figure Lengend Snippet: Rb phosphorylation at S807 is required for association with Bax. Rb mutant plasmids were generated as described in the Materials and Methods section. Rb-negative C33A cells were transfected using Fugene (Promega). ( A ) Rb plasmids expressing either S807A or S811A alanine mutants were transfected into cells and 48 hours later cell lysates were utilized in GST-Bax fusion protein pull down assays. Proteins associated with GST-Bax were analyzed by immunoblotting with Rb antibodies. ( B ) Rb plasmids expressing either S807A or S807E mutants were transfected into C33A cells and 48 hours later co-immunoprecipitation with Bax antibodies was performed. Immunoprecipitates were analyzed by immunoblotting with Rb and Bax antibodies. Equivalent expression of the Rb mutant proteins in the C33A cells is verified by immunoblotting (lower panel). Data shown is representative of three independent experiments.

    Article Snippet: Plasmids were introduced into C33A cells using Fugene (Promega) performed according to the manufacturer's directions.

    Techniques: Mutagenesis, Generated, Transfection, Expressing, Immunoprecipitation

    Impact of MetAP2 anti-sense oligonucleotide on mesothelioma cell viability and nucleosome formation. A: Mesothelioma cells (HM) were incubated with increasing concentrations of either a specific MetAP2 anti-sense or a scrambled oligonucleotide. After 24 hours total RNA was extracted and blotted with a specific probe for MetAP2. MetAP2 protein levels were determined by Western blotting after 48 hours exposure to the oligonucleotides. β-actin was used as an internal control. B: HM cells were treated for different periods of time with either lipofectin alone (mock) or with 100 nmol/L of the MetAP2 anti-sense or of a scrambled oligonucleotide. At the end of the incubation, cell viability was assessed by trypan blue exclusion. Data points depict mean values ± SD from two experiments with quadruplicate determinations (*, P = 0.02; **, P

    Journal: The American Journal of Pathology

    Article Title: Methionine Aminopeptidase-2 Regulates Human Mesothelioma Cell Survival

    doi:

    Figure Lengend Snippet: Impact of MetAP2 anti-sense oligonucleotide on mesothelioma cell viability and nucleosome formation. A: Mesothelioma cells (HM) were incubated with increasing concentrations of either a specific MetAP2 anti-sense or a scrambled oligonucleotide. After 24 hours total RNA was extracted and blotted with a specific probe for MetAP2. MetAP2 protein levels were determined by Western blotting after 48 hours exposure to the oligonucleotides. β-actin was used as an internal control. B: HM cells were treated for different periods of time with either lipofectin alone (mock) or with 100 nmol/L of the MetAP2 anti-sense or of a scrambled oligonucleotide. At the end of the incubation, cell viability was assessed by trypan blue exclusion. Data points depict mean values ± SD from two experiments with quadruplicate determinations (*, P = 0.02; **, P

    Article Snippet: Briefly, cells were washed three times with prewarmed (37°C), serum-free RPMI and incubated with either a MetAP2 anti-sense or a scrambled oligonucleotide previously mixed with 10 μg/ml of lipofectin (Life Technologies, Inc.) in serum-free RPMI.

    Techniques: Incubation, Western Blot