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  • 99
    ATCC transient transfection
    Effects of miR-101 on DU-145 and LNCaP cells . A and B. Effects of ectopic miR-101 on the proliferation and invasiveness of DU-145 cells. DU-145 cells were transfected by 120 nM of miR-cont or miR-101 mimics and cells were collected at 72 h <t>post-transfection.</t> Aliquots of the cells of each treatment were studied in triplicates by ( A ) WST-1 cell proliferation assay and ( B ) Matrigel invasion assay. Western blot analyses of these transfected cells are shown as an insert of
    Transient Transfection, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 2004 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore transient transfection
    Constitutive active FOXO1 up-regulates human Sell core promoter activity in a dose-dependent manner. Notes: Jurkat cells were transiently co-transfected with the combination of plasmids as labeled in the figure. Luciferase activity normalized to that of Renilla activity was analyzed 30 hours after <t>transfection.</t> Data were presented as mean ± SD of at least three independent experiments in triplicate on each transfection. Data were graphed as fold increase relative to that of pcDNA3 transfected Jurkat cells, which was set as 1.
    Transient Transfection, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2095 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher transient transfection
    Relative change in TAG- or CE-specific intracellular lipid storage upon expression of various Plin proteins. AML12 cells were transiently transfected with the indicated GFP–Plin-expressing constructs and cultured overnight in the presence of oleic acid and cholesterol. <t>Transfection</t> efficiencies were confirmed by visualizing GFP fluorescence. Untransfected cells, cultured with/without exogenous oleic acid and cholesterol, were grown in parallel. LSDs were isolated by centrifugation and lipids were extracted, separated by TLC in parallel with lipid markers, and TAG and CE detected after staining with iodine vapor. Relative TAG/CE ratios were quantified in cells transfected with each specific Plin-expressing construct and analyzed in parallel with identically grown untransfected cells for normalization and TLC background correction. The TAG/CE ratio for untransfected lipid-loaded cells (controls) was set to 0.5. The relative TAG/CE-ratio of Plin1a- and Plin1c-expressing cells were always analyzed in parallel and normalized to those of control cells, and then secondarily compared to results determined for its Plin-expressing counterpart. Values > 0.5 indicate a proportional increase in TAG lipid bias, whereas values
    Transient Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 30295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Mirus Bio transient transfection
    IRE1 activation increases the surface expression of α1(A322D) subunit of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or XBP1-s (spliced XBP1) plasmids. Forty-eight hrs post <t>transfection,</t> cells were lysed, and total proteins were extracted. The cell lysates are then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B C ) (n = 5 for α1 and n = 3 for BiP, paired t-test). ( D ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface protein expression levels of α1 is shown in ( E ) (n = 5, paired t-test). ( F ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or XBP-s or transfected with XBP-s and treated with lactacystin (2.5 μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( G ) (n = 3, one-way ANOVA followed by Fisher test, *, p
    Transient Transfection, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 93/100, based on 1306 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Addgene inc transient transfections
    Vpu-mediated control of IFN-I production by pDCs involves engagement and activation of ILT7 by BST2. (A-C) Vpu-mediated BST2 antagonism enhances activation of ILT7. ILT7+ NFAT-GFP reporter cells were co-cultured with HEK293T (mock) or BST2-expressing HEK293T cells mock-transfected or transfected with the indicated pNL4.3 constructs (WT or dU). (A) Representative example of ILT7 activation as determined by the percentage of NFAT-GFP positive cells measured by flow cytometry. (B) Percentage of ILT7 activation after co-culture with the indicated HIV/BST2 expressing HEK 293T cells relative to WT HIV-producing cells (100%) (n = 4). (C) Percentage of BST2 surface expression in HEK293T cells after <t>co-transfection</t> of BST2 with the indicated HIV provirus relative to dU HIV-producing cells (100%) (n = 4). (D-F) Effect of ILT7 depletion on IFN-I production by pDCs. (D) Non-pDC fraction (BDCA-2-) and siRNA-treated enriched pDCs (CD14-/BDCA-2+) were stained using anti-ILT7 Abs as indicated. A representative example of absolute levels (E) or relative percentages (F) of IFN-I produced after co-culture of control pDCs (pDC-siCTRL) or ILT7-depleted pDCs (pDC-siILT7) with the indicated infected MT4 cells are shown. The amount of IFN-I released by pDC siCTRL in contact with dU HIV-infected cells was set at 100% (n = 4). (G-I) Effect of recombinant soluble ILT7 on IFN-I production by pDCs. (G) Expression of a HA-tagged soluble ILT7 (soILT7-HA) in HEK 293T cells. Cells and supernatants (sup) were analyzed by Western blot (WB) using anti-HA Abs. Purity of secreted soILT7-HA was confirmed by Coomassie staining (sup Coom). A representative example of (H) absolute levels or (I) relative percentages of IFN-I production after co-culture of PBMCs with the indicated infected MT4 cells pre-treated with control (CTRL) or soILT7-HA-containing supernatants are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected cells in presence of CTRL supernatant was set at 100% (n = 6). Two-tailed paired t -test was used in B-C while repeated measures ANOVA with Bonferroni’s multiple comparison test was used in F and I (*** p
    Transient Transfections, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery transient transfection
    Outline of pharmacological manipulation of CK1 functions and its effects on p53 signalling pathway, cell viability, and CK1α post-translational modifications. ( A ) H1299 cells were transfected with STReP-tagged CK1α sp1 or 2, FLAG-tagged peptide 35 and His-Ubiquitin or His-Nedd8, using Attractene. Eighteen hours after <t>transfection</t> cells were treated with 50 µM MG132 for 4 hours. Whole cell lysates were analysed for transfected CK1α levels, and His-ubiquitin/NEDD8 conjugates were purified by metal affinity chromatography and analyzed by 4%–12% NuPAGE/immunoblots with an anti-CK1α antibody. Changes highlighted were representative of two independent experiments. ( B ) Three approaches for manipulation of signal transduction pathways. Genetic depletion of CK1α using siRNA or the CK1 kinase attenuation using the ATP-competitive inhibitor D4476 can alter the p53 response and cell viability, as indicated. The bioactive peptide from the high affinity MDM2-CK1α interface (peptide 35) highlights a novel peptide lead for disrupting signalling in cancers. All three approaches gave rise to overlapping but distinct effects on signalling. Specific siRNA against CK1α led to p53 induction and growth arrest accompanied with slight sub-G1 increase, whereas non-specific CK1 drugs such as D4476 mediated p53 induction and significant apoptosis. Low levels of the bioactive peptide 35 did not lead to p53 protein induction but to a growth arrest in G0-G1 and reduced cell viability. These data suggest that peptide 35 may function in a pharmacologically novel manner compared to the ATP-active site inhibitor or CK1α siRNA and highlights the general utility of targeting protein-protein interactions as approaches for developing therapeutic strategies that target signalling mechanisms in cancer.
    Transient Transfection, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 93/100, based on 662 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Amaxa transient transfection
    YFP-N300 downregulates MHC class I expression in M553(R240)tpsn cells M553(R240)Tpsn cells were transiently transfected with YFP-N300 or co-transfected with empty vector and pmaxGFP. Twenty-four hours post <t>transfection,</t> MHC class I expression was analyzed by flow cytometry using a PE directly conjugated W6/32 (MHC class I) antibody. Mean fluorescent intensity was determined for W6/32 staining on populations of cells gated on forward and side scatter (R1) and for the negative or positive expression of GFP/YFP (R2, GFP/YFP negative) and R3, (GFP/YFP positive).
    Transient Transfection, supplied by Amaxa, used in various techniques. Bioz Stars score: 92/100, based on 995 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Polyplus Transfection transient transfection
    Methylation-sensitive binding of CTCF to the first exon of hTERT . (A) In vivo binding of CTCF to the first exon of hTERT in telomerase-positive and negative cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments ( hTERT and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (B) 5′-end-labeled control (F1) and SssI methylase-treated (F1-met) fragments were digested with methylation-sensitive BstUI and analyzed on polyacrylamide gels to verify efficiency of in vitro methylation. ( C ) Control unmethylated (F1) or SssI-methylated (F1-met) fragments were analyzed by gel-shift assay (EMSA). F, free probe; B, CTCF-bound probe. ( D ) Representation of the hTERT sequence cloned into the pTERT-297/ex2/FRT. Arrows represent the localization of the primers used for hTERT methylation analysis of stable transfectant. ( E ) The methylation status of the stable transfectants was verified by MS-SSCA. Unmethylated and fully methylated controls were obtained from plasmids used for stable <t>transfection.</t> UT and MT represent, respectively, the unmethylated and methylated plasmids stably transfected into HeLa cells, and after 30 population doublings. ( F ) Binding of CTCF to the first exon of hTERT in stably transfected cell line was analyzed by ChIP assay using anti-CTCF antibody.
    Transient Transfection, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 94/100, based on 1003 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology transient transfection
    Dose effect of ENZ fragment on BAR efficiency. ( A ) Schematic representation of the Gal4-tagged (gray–blue square) ORF2 fragments. ENZ490Δ fragment was generated from wild-type ORF2 sequence, while Δ348RTCys fragment was generated using codon optimized ORF2 sequence. ( B ) BAR output using different amounts (0.4–0.05 μg) of the wild-type ENZ expression plasmid with 0.4 μg of the RTCys expression plasmid. Empty vector used as filler to ensure equal DNA amounts in <t>transfections.</t> Error bars denote standard deviation ( n = 3). Statistical significance assessed using Student's t -test for paired samples with denoted by *. Representative flasks are shown above corresponding graph bars.
    Transient Transfection, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 666 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company transient transfection
    TIGAR expression is increased in high metastatic lung cancer cells, affecting cell migration, invasion and adhesion in vitro. ( a - b ) Wound healing assays and Trans-well analysis to determine the migration and invasion of A549, H1299, H1650, H1975 and PC9, respectively. ( c - d ) Statistical analysis was to quantify the migratory and invasive ability of five NSCLC cells. ( e ) Real-time PCR and Western blot analysis to determine endogenous TIGAR expression of five NSCLC cells. ( f ) Real-time PCR and Western blot analysis to respectively quantify mRNA and protein expression of TIGAR after <t>transfection</t> with siTIGAR (or siNC as control) for 48h. ( g - h ) Cells were transfected with siTIGAR (or siNC as control) for 48h were collected to determine the migration and invasion capability by wound-healing assays and Trans-well assay, respectively. ( i - j ) Statistical analysis was to quantify cells migratory and invasive ability upon TIGAR knockdown. ( k ) H1299 and A549 cells were infected with lentivirus with shTIGAR and Green fluorescent protein. To get more effective knockdown, monoclonal cell was to screen in A549. Monoclonal cells A549-shTIGAR#7 was the A549-shTIGAR in following assays. ( l ) Wound-healing assay for H1299 and A549 cells stably knockdown TIGAR or negative control. ( m ) Trans-well invasion assay for A549 cells stably knockdown TIGAR or negative control. ( n ) The effect of TIGAR on cell adhesion activity was assessed in A549
    Transient Transfection, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 92/100, based on 638 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene transient transfection
    Site-directed mutagenesis for luciferase activity assay and effect of miR-7 silencing over MAFG expression. (A) Chromosomal localization of miR-7 predicted binding sites at 3'UTR of MAFG. Regions 2 and 8 were identified by six or more bioinformatical algorithms. Sanger sequencing showed that the seed sequence of miR-7 was fully mutated at regions 2 and 8 of the 3' UTR of MAFG. (B) <t>Co-transfection</t> of mimic miR-7 (miR-7) or mimic control (miR-NC) with the 3' UTR of MAFG WT, mutated on region 2 (MAFG 2*) and mutated on region 8 (MAFG 8*). Experiments were performed at 15nM and data was analyzed after 24h of co-transfection. (Upper panel) Relative luciferase activity. The figures represent the mean ± SD of at least 3 independent experiments after data normalization with Renilla and the data from the negative control 3'-UTR; p
    Transient Transfection, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 607 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene transient transfections
    LMP2A augments activation of the TOPFlash luciferase reporter by β-catenin. (A) 293 cells were transfected with SV-β-galactosidase and either TOPFlash or FOPFlash plus vector, β-catenin, LMP2A, or β-catenin with LMP2A. TOPFlash contains four consensus TCF-4 binding sites in a promoter driving expression of the luciferase gene, and FOPFlash is an analogous construct with the four TCF-4 sites mutated. <t>Transfections</t> and assays were performed in triplicate in two separate experiments, and luciferase activity was normalized to β-galactosidase activity to control for transfection efficiency. The data are presented graphically as TOPFlash activity normalized to FOPFlash activity for each of the indicated conditions. (B) Immunoblot analyses were performed on the lysates used in the luciferase assay described above. Antibodies against HA confirm the presence of transfected HA-tagged LMP2A. Transfected and endogenous β-catenin levels were revealed with an anti-β-catenin antibody, and immunoblot with an antibody against TCF-4 attested to equal levels of this transcription factor in the transfected 293 cells.
    Transient Transfections, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ribobio transient transfection
    miR-320a suppresses migration and invasion of TSCC cells by targeting Suz12 in vitro A. Wound healing and transwell assays of UM1 after transient <t>transfection.</t> B. Wound healing and transwell assays of Cal27 after transient transfection. C. Western blot of Suz12 in stable transfected Cal27 and UM1 cell lines. D. Suz12 mRNA in stable transfected Cal27 and UM1 cell lines. E. Dual-luciferase reporter gene array. F. Western blot assays of Cal27 after co-transfection of Suz12 siRNA. G. Wound healing of Cal27 after co-transfection of Suz12 siRNA. * P
    Transient Transfection, supplied by Ribobio, used in various techniques. Bioz Stars score: 93/100, based on 313 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 293fectin transfection reagent
    miR-320a suppresses migration and invasion of TSCC cells by targeting Suz12 in vitro A. Wound healing and transwell assays of UM1 after transient <t>transfection.</t> B. Wound healing and transwell assays of Cal27 after transient transfection. C. Western blot of Suz12 in stable transfected Cal27 and UM1 cell lines. D. Suz12 mRNA in stable transfected Cal27 and UM1 cell lines. E. Dual-luciferase reporter gene array. F. Western blot assays of Cal27 after co-transfection of Suz12 siRNA. G. Wound healing of Cal27 after co-transfection of Suz12 siRNA. * P
    293fectin Transfection Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 845 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson transient transfection
    Blimp1 in Foxp3 + regulatory T (Treg) cells limits generation of follicular regulatory T (Tfr) cells. (a) Expression of surface markers CD4 + CD25 + in spleen cells of C57BL/6 after magnetic bead isolation. The purity of CD4 + CD25 + Treg cells was 91 ± 2%. (b) Microscopy observation of small interfering RNA (siRNA) ‐transfected cells. The <t>transfection</t> efficiency of siRNA in Treg cells was over 90%. (c, d) Transcription and expression verification of Blimp1 was significantly decreased in siRNA‐Blimp1‐infected cells. (e) Quantification of Tfr cells (CD4 + CXCR5 + ICOS + Foxp3 + ]
    Transient Transfection, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 333 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen transient transfection
    Inhibition of encystation by AcAtg3-siRNA. Cells transfected with Superfect <t>Transfection</t> reagent, nonspecific negative siRNA, or AcAtg3-siRNA were incubated in encystment media for 3 days and the number of mature cysts scored. The formation of mature cysts was almost inhibited in AcAtg3-siRNA transfected cells.
    Transient Transfection, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 3504 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Corning Life Sciences transient transfections
    TAp63 α and TAp73 α can activate the TIGAR-hBS2 reporter. ( a ) Representative iRFP reporter assay scan of HCT116 p53 −/− cells 24 h after <t>co-transfection</t> with TIGAR-hBS2 iRFP reporter along with human p53, HA-tagged TAp63 α or HA-tagged TAp73 α . ( b ) Western blot analysis of HCT116 p53 −/− cells with transfected p53, HA-tagged TAp63 α or HA-tagged TAp73 α . ( c ) Quantification of iRFP reporter scans. ( d ) Representative iRFP reporter assay scan of HCT116 p53 −/− cells 24 h after co-transfection with TIGAR-hBS2 iRFP reporter along with TAp73 α , TAp73 β , TAp73 γ or ΔNp73 α . ( e ) Western blot analysis of HCT116 p53 −/− cells with transfected HA-tagged TAp73 α , HA-tagged TAp73 β , HA-tagged TAp73 γ or HA-tagged ΔNp73 α . ( f ) Quantification of iRFP reporter scans on human (hBS2) and mouse (mBS1 and mBS2) TIGAR promoter-binding sites. Values represent mean±S.E.M. of three independent experiments. * P
    Transient Transfections, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega transient transfection
    Stimulation-induced processing and degradation of p105 is not affected by mutating lysine residues 18 (683) to 30 (967) in the C-terminal domain of the molecule. (A) COS-7 cells were transiently transfected with either a cDNA coding for p105-WT (lanes 2 to 5) or p105-K18-30R (lanes 6 to 9) as indicated. Control cells (lane 1) were transfected with an empty vector. Where indicated, a cDNA coding for constitutively active IKKβ was cotransfected. Twenty-four hours after <t>transfection</t> cells were harvested, and nuclear and cytosolic fractions were isolated as described in Materials and Methods. Aliquots of cytosolic and nuclear extracts representing an equal number of cells were resolved via SDS-10% PAGE and were blotted onto nitrocellulose paper, and proteins were visualized by using anti-p50 antibody and ECL as described in Materials and Methods. C, cytosolic fraction; N, nuclear fraction. (B) COS-7 cells were transiently transfected with a cDNA coding for either p105-WT (lanes 1 to 4), p105-K18-30R (lanes 5 to 8), or p105-Δ917-932 (lanes 9 to 12). Where indicated, cDNA coding for a constitutively active IKKβ was cotransfected. Twenty-four hours after transfection cells were pulse labeled with [ 35 S]methionine (0; pulse). Following removal and dilution of the labeled amino acid and further incubation for 2 h (2; chase), the labeled proteins were immunoprecipitated by using anti-p50 antibody, resolved by SDS-10% PAGE, and visualized by phosphorimaging as described in Materials and Methods.
    Transient Transfection, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 3443 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC transient transfection hela cells
    POTE over-expression induces apoptosis in <t>Hela</t> cells. a Hela cells transfected with pEGFP, pPOTE-2 α -actin-EGFP or pPOTE-2 γ C-EGFP. At 2 or 3 days <t>post-transfection,</t> cells were incubated with RED-VAD-FMK for 1 h before fixation with 2% PFA. Cells were visualized with confocal microscopy. b Quantitative analysis of POTE induced apoptosis. Transfected cells were stained with RED-VAD-FMK for 1 h and red-staining cells and total transfected cells were counted under microscopy. Three separate experiments were repeated and each time about 100 transfected cells were counted
    Transient Transfection Hela Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery transient sirna transfections
    USP7 depletion results in stabilization of cyclin B (cycB) in a p53-independent manner. Control or USP7 stably depleted HEp2 cell extracts were generated from cells arrested in Taxol ( a ) or Nocodazole ( b ) collected by mitotic shake-off. Extracts were supplemented with non-destructible cycB fragment, an energy-regenerating system, and UbcH10 where indicated. Endogenous cycB was monitored over time. Right panels: Relative quantization of cycB protein levels in USP7-depleted cell extracts using actin as internal control and normalized over cycB/actin protein levels in control shRNA extracts. CycB is stabilized in USP7-depleted cell extracts. Western blot analysis of cycB stability in HEp2 ( c ) or H1299 ( d ) cells synchronized by DTB and simultaneously transfected with either control or USP7 siRNAs. Samples were taken 72 h <t>post-transfection</t> at 0, 7, 9 and 11 h after DTB release to allow cells to progress through mitosis. Right panels: Relative quantification of cycB protein levels using actin as internal control for each time point. Data are normalized over cycB/actin protein levels in control <t>siRNA-transfected</t> cells. Histone H3 phosphorylated at serine 10 is shown as control for mitotic progression. CycB is stabilized in USP7-depleted cells in a p53-independent manner. Data show representative experiments out of three
    Transient Sirna Transfections, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 89/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher transient transfection lipofectamine 2000
    USP7 depletion results in stabilization of cyclin B (cycB) in a p53-independent manner. Control or USP7 stably depleted HEp2 cell extracts were generated from cells arrested in Taxol ( a ) or Nocodazole ( b ) collected by mitotic shake-off. Extracts were supplemented with non-destructible cycB fragment, an energy-regenerating system, and UbcH10 where indicated. Endogenous cycB was monitored over time. Right panels: Relative quantization of cycB protein levels in USP7-depleted cell extracts using actin as internal control and normalized over cycB/actin protein levels in control shRNA extracts. CycB is stabilized in USP7-depleted cell extracts. Western blot analysis of cycB stability in HEp2 ( c ) or H1299 ( d ) cells synchronized by DTB and simultaneously transfected with either control or USP7 siRNAs. Samples were taken 72 h <t>post-transfection</t> at 0, 7, 9 and 11 h after DTB release to allow cells to progress through mitosis. Right panels: Relative quantification of cycB protein levels using actin as internal control for each time point. Data are normalized over cycB/actin protein levels in control <t>siRNA-transfected</t> cells. Histone H3 phosphorylated at serine 10 is shown as control for mitotic progression. CycB is stabilized in USP7-depleted cells in a p53-independent manner. Data show representative experiments out of three
    Transient Transfection Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Polypus Transfection transient transfection
    miRNA profiling of p63-depleted HaCaT cells. siRNA-mediated knockdown of p63 was assessed at the mRNA level ( A ) and protein level ( B ) 48 hours <t>post-transfection.</t> The total miRNA extracted (three independent siP63 transfection and three independent miRNA extraction) was processed for miRNA profiling. Error bars represent the s.d. of triplicate experiments. *, p
    Transient Transfection, supplied by Polypus Transfection, used in various techniques. Bioz Stars score: 92/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Polyplus Transfection transient plasmid transfections
    miRNA profiling of p63-depleted HaCaT cells. siRNA-mediated knockdown of p63 was assessed at the mRNA level ( A ) and protein level ( B ) 48 hours <t>post-transfection.</t> The total miRNA extracted (three independent siP63 transfection and three independent miRNA extraction) was processed for miRNA profiling. Error bars represent the s.d. of triplicate experiments. *, p
    Transient Plasmid Transfections, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 92/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC transient transfection human embryonic kidney hek 293t cells
    Effect of bovine ADA binding on A 2A R. (A) Competition experiments of 2.2 nM [ 3 H]ZM 241385 binding in the presence of increasing concentrations of unlabeled ZM 241385, in the absence (○) or in the presence (●) of 1 μg/ml of ADA were performed using membranes from <t>HEK-293T</t> cells transfected with A 2A R-Nluc-spacer cDNA (1.5 μg). (B) Dose-response effect of ADA on 2.2 nM [ 3 H]ZM 241385 binding to membranes from HEK-293T cells transfected with cDNA (1.5 μg) corresponding to A 2A R-Nluc-spacer (■) or A 2A R (□). Data are mean ± SD from a representative experiment ( n = 3) performed in triplicate. (C) DMR assays were performed in HEK-293T cells transfected with A 2A R-Nluc-spacer cDNA (1.5 μg). Cells were stimulated with increasing CGS 21680 concentrations in the presence (white columns) or in the absence (black columns) of ADA (1 μg/ml). Values are mean ± SEM ( n = 4) and are expressed as shift at 3000 s of reflected light wavelength (pm) over basal obtained from the corresponding DMR curves. Statistical significance was calculated by one-way ANOVA followed by a Bonferroni multiple comparison post hoc test; ∗∗ p
    Transient Transfection Human Embryonic Kidney Hek 293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of miR-101 on DU-145 and LNCaP cells . A and B. Effects of ectopic miR-101 on the proliferation and invasiveness of DU-145 cells. DU-145 cells were transfected by 120 nM of miR-cont or miR-101 mimics and cells were collected at 72 h post-transfection. Aliquots of the cells of each treatment were studied in triplicates by ( A ) WST-1 cell proliferation assay and ( B ) Matrigel invasion assay. Western blot analyses of these transfected cells are shown as an insert of

    Journal: Molecular Cancer

    Article Title: MicroRNA-101 negatively regulates Ezh2 and its expression is modulated by androgen receptor and HIF-1?/HIF-1?

    doi: 10.1186/1476-4598-9-108

    Figure Lengend Snippet: Effects of miR-101 on DU-145 and LNCaP cells . A and B. Effects of ectopic miR-101 on the proliferation and invasiveness of DU-145 cells. DU-145 cells were transfected by 120 nM of miR-cont or miR-101 mimics and cells were collected at 72 h post-transfection. Aliquots of the cells of each treatment were studied in triplicates by ( A ) WST-1 cell proliferation assay and ( B ) Matrigel invasion assay. Western blot analyses of these transfected cells are shown as an insert of " A " and representative images of invasion assay are also shown in " B " (right panel). C , and D . Effects of ectopic miR-101 on the morphology and proliferation of LNCaP cells. In C , the LNCaP cells at 72 h post transfection were imaged under microscope (20×). The black arrow heads indicate the rounding of the cell body, while the white arrow heads point the extensions of cell cytoplasmic portion. In D , the LNCaP cells were seeded in triplicate at a density of 6000 cells/well in 96-well plates and transfected with 120 nM of miR-cont or miR-101 mimics. Cell proliferation was studied by WST-1 reagent. E . Wound healing assay of LNCaP cells transfected with miRNA mimics (120 nM). Images were captured at the time points as indicated. F . Boyden chamber migration assay of LNCaP cells transfected with miRNA mimics (120 nM). The cell numbers were quantified after crystal violet staining with represented images shown below.

    Article Snippet: Cell culture, transient transfection and Lentiviral production PWR-1E, LNCaP, DU145 and PC-3 cell lines were obtained from the American Type Culture Collection (Manassas, VA).

    Techniques: Transfection, Proliferation Assay, Invasion Assay, Western Blot, Microscopy, Wound Healing Assay, Migration, Staining

    Negative effect of miRNAs, miR-489 and -27a, on osteogenesis in hMSC is, at least in part, mediated by repression of GCA gene expression. (A–G) Transfection with inhibitors of miR-489 and -27a results in up-regulation of GCA protein expression. (A–F). hMSC were transfected with control molecule, ic1 (25 nM) or with combination of inhibitors, i489+i27a (12.5 nM each). GCA protein expression was determined by immunofluorescence as described in Materials and Methods . Cells were stained with Hoechst 33342 (A–C) and GCA specific primary antibody, followed by a secondary antibody (E,F). Control wells were treated with secondary antibody only and demonstrate no specific staining (D). (G) Quantitation of GCA immunohystochemistry. Data shown represents three independent transfections, four fields each. (mean+/−SD). ** - Student's ttest p value between treated cells and corresponding control group, p

    Journal: PLoS ONE

    Article Title: Functional Profiling Reveals Critical Role for miRNA in Differentiation of Human Mesenchymal Stem Cells

    doi: 10.1371/journal.pone.0005605

    Figure Lengend Snippet: Negative effect of miRNAs, miR-489 and -27a, on osteogenesis in hMSC is, at least in part, mediated by repression of GCA gene expression. (A–G) Transfection with inhibitors of miR-489 and -27a results in up-regulation of GCA protein expression. (A–F). hMSC were transfected with control molecule, ic1 (25 nM) or with combination of inhibitors, i489+i27a (12.5 nM each). GCA protein expression was determined by immunofluorescence as described in Materials and Methods . Cells were stained with Hoechst 33342 (A–C) and GCA specific primary antibody, followed by a secondary antibody (E,F). Control wells were treated with secondary antibody only and demonstrate no specific staining (D). (G) Quantitation of GCA immunohystochemistry. Data shown represents three independent transfections, four fields each. (mean+/−SD). ** - Student's ttest p value between treated cells and corresponding control group, p

    Article Snippet: Transient transfection assays were conducted in HT1080 human fibrosarcoma cells (ATCC, Manassas, VA) in 96-well plates.

    Techniques: Expressing, Transfection, Immunofluorescence, Staining, Quantitation Assay

    Alteration of the miRNAs activity rescues osteogenic potential in transfected hMSC with high passage number. (A) Bone marrow hMSC with high passage number demonstrate a decrease in basal level of AP activity and poor response to differentiation media. hMSC were plated in 96-well plates and incubated either in propagation (P, grey bar) or osteogenic (O, black bar) media for 6 days. Data normalized to relative AP activity values obtained from passage 21 hMSC incubated in propagation media. (B and C) Transfection with miRNA inhibitors and mimics restore differentiation in over-propagated hMSC incubated in propagation (B) or differentiation (C) media. hMSC propagated for certain number of passages (16, white bar, 18, grey bar or 21 , black bar) plated in 96-well plates and transfected with inhibitors and mimics (as indicated, all at 25 nM). Data are representative of three independent experiments performed in triplicate. (mean+/−SD). *, **: Student's ttest p value between treated cells and corresponding control group, * - p

    Journal: PLoS ONE

    Article Title: Functional Profiling Reveals Critical Role for miRNA in Differentiation of Human Mesenchymal Stem Cells

    doi: 10.1371/journal.pone.0005605

    Figure Lengend Snippet: Alteration of the miRNAs activity rescues osteogenic potential in transfected hMSC with high passage number. (A) Bone marrow hMSC with high passage number demonstrate a decrease in basal level of AP activity and poor response to differentiation media. hMSC were plated in 96-well plates and incubated either in propagation (P, grey bar) or osteogenic (O, black bar) media for 6 days. Data normalized to relative AP activity values obtained from passage 21 hMSC incubated in propagation media. (B and C) Transfection with miRNA inhibitors and mimics restore differentiation in over-propagated hMSC incubated in propagation (B) or differentiation (C) media. hMSC propagated for certain number of passages (16, white bar, 18, grey bar or 21 , black bar) plated in 96-well plates and transfected with inhibitors and mimics (as indicated, all at 25 nM). Data are representative of three independent experiments performed in triplicate. (mean+/−SD). *, **: Student's ttest p value between treated cells and corresponding control group, * - p

    Article Snippet: Transient transfection assays were conducted in HT1080 human fibrosarcoma cells (ATCC, Manassas, VA) in 96-well plates.

    Techniques: Activity Assay, Transfection, Incubation

    Transfection and library screening

    Journal: PLoS ONE

    Article Title: Functional Profiling Reveals Critical Role for miRNA in Differentiation of Human Mesenchymal Stem Cells

    doi: 10.1371/journal.pone.0005605

    Figure Lengend Snippet: Transfection and library screening

    Article Snippet: Transient transfection assays were conducted in HT1080 human fibrosarcoma cells (ATCC, Manassas, VA) in 96-well plates.

    Techniques: Transfection, Library Screening

    Constitutive active FOXO1 up-regulates human Sell core promoter activity in a dose-dependent manner. Notes: Jurkat cells were transiently co-transfected with the combination of plasmids as labeled in the figure. Luciferase activity normalized to that of Renilla activity was analyzed 30 hours after transfection. Data were presented as mean ± SD of at least three independent experiments in triplicate on each transfection. Data were graphed as fold increase relative to that of pcDNA3 transfected Jurkat cells, which was set as 1.

    Journal: Gene Regulation and Systems Biology

    Article Title: FOXO1 Up-Regulates Human L-selectin Expression Through Binding to a Consensus FOXO1 Motif

    doi: 10.4137/GRSB.S10343

    Figure Lengend Snippet: Constitutive active FOXO1 up-regulates human Sell core promoter activity in a dose-dependent manner. Notes: Jurkat cells were transiently co-transfected with the combination of plasmids as labeled in the figure. Luciferase activity normalized to that of Renilla activity was analyzed 30 hours after transfection. Data were presented as mean ± SD of at least three independent experiments in triplicate on each transfection. Data were graphed as fold increase relative to that of pcDNA3 transfected Jurkat cells, which was set as 1.

    Article Snippet: Western blot Thirty hours after transient transfection, cells were lysed in lysis buffer containing 1% (w/v) SDS, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM EDTA, and 1× protease inhibitor cocktail (Sigma, St. Louis, Missouri).

    Techniques: Activity Assay, Transfection, Labeling, Luciferase

    Locating the FOXO1 motif. Notes: Jurkat cells were transiently co-transfected with either pcDNA3 (open bars) or FOXO1-3A (solid bars) and one of the 5′ serial deletion mutants as labeled. Luciferase activity normalized to that of Renilla activity was analyzed 30 hours after the co-transfection. Data were presented as mean ± SD of at least three independent experiments in triplicate on each transfection. Data were graphed as fold increase relative to that of pcDNA3 co-transfected Jurkat cells, which was set as 1.

    Journal: Gene Regulation and Systems Biology

    Article Title: FOXO1 Up-Regulates Human L-selectin Expression Through Binding to a Consensus FOXO1 Motif

    doi: 10.4137/GRSB.S10343

    Figure Lengend Snippet: Locating the FOXO1 motif. Notes: Jurkat cells were transiently co-transfected with either pcDNA3 (open bars) or FOXO1-3A (solid bars) and one of the 5′ serial deletion mutants as labeled. Luciferase activity normalized to that of Renilla activity was analyzed 30 hours after the co-transfection. Data were presented as mean ± SD of at least three independent experiments in triplicate on each transfection. Data were graphed as fold increase relative to that of pcDNA3 co-transfected Jurkat cells, which was set as 1.

    Article Snippet: Western blot Thirty hours after transient transfection, cells were lysed in lysis buffer containing 1% (w/v) SDS, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM EDTA, and 1× protease inhibitor cocktail (Sigma, St. Louis, Missouri).

    Techniques: Transfection, Labeling, Luciferase, Activity Assay, Cotransfection

    Constitutive active FOXO1 up-regulates endogenous human Sell expression. Jurkat cells were transiently transfected with pcDNA3 (white bar), pcDNA3-FOXO1 (striped bar), or pcDNA3-FOXO1-3A (solid bar) for 30 hours. Of the two sets of transfected cells, one set was lysed with SDS buffer and equal amount of lysate from each treatment was analyzed for expression of FOXO1 by Western blot, where β-actin was used as loading control ( A ); the other set was used to analyze the expression of human Sell by real time PCR, which was normalized to that of GAPDH ( B ). Notes: Data were presented as mean ± SD of at least three independent experiments in triplicate on each transfection. Data were graphed as fold increase relative to that of pcDNA3 transfected Jurkat cells, which was set as 1.

    Journal: Gene Regulation and Systems Biology

    Article Title: FOXO1 Up-Regulates Human L-selectin Expression Through Binding to a Consensus FOXO1 Motif

    doi: 10.4137/GRSB.S10343

    Figure Lengend Snippet: Constitutive active FOXO1 up-regulates endogenous human Sell expression. Jurkat cells were transiently transfected with pcDNA3 (white bar), pcDNA3-FOXO1 (striped bar), or pcDNA3-FOXO1-3A (solid bar) for 30 hours. Of the two sets of transfected cells, one set was lysed with SDS buffer and equal amount of lysate from each treatment was analyzed for expression of FOXO1 by Western blot, where β-actin was used as loading control ( A ); the other set was used to analyze the expression of human Sell by real time PCR, which was normalized to that of GAPDH ( B ). Notes: Data were presented as mean ± SD of at least three independent experiments in triplicate on each transfection. Data were graphed as fold increase relative to that of pcDNA3 transfected Jurkat cells, which was set as 1.

    Article Snippet: Western blot Thirty hours after transient transfection, cells were lysed in lysis buffer containing 1% (w/v) SDS, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM EDTA, and 1× protease inhibitor cocktail (Sigma, St. Louis, Missouri).

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

    FOXO1 transactivates human Sell promoter through binding to the motif. ( A ) Alignment of the IRE with the FOXO1 motifs in the Sell promoter of both human and chimpanzee. ( B ) Jurkat cells were transiently co-transfected with the combination of plasmids, pcDNA3 and hSell108, FOXO1-3A and hSell108, or FOXO1-3A and hSellFmut, as labeled on the bottom. Notes: Luciferase activity was analyzed 30 hours after transfection. Luciferase activity expressed as fold increase relative to that of pcDNA3 transfected cells. Data shown are mean ± SD of three independent transfections in one experiment. Each experiment was repeated at least three times in triplicates.

    Journal: Gene Regulation and Systems Biology

    Article Title: FOXO1 Up-Regulates Human L-selectin Expression Through Binding to a Consensus FOXO1 Motif

    doi: 10.4137/GRSB.S10343

    Figure Lengend Snippet: FOXO1 transactivates human Sell promoter through binding to the motif. ( A ) Alignment of the IRE with the FOXO1 motifs in the Sell promoter of both human and chimpanzee. ( B ) Jurkat cells were transiently co-transfected with the combination of plasmids, pcDNA3 and hSell108, FOXO1-3A and hSell108, or FOXO1-3A and hSellFmut, as labeled on the bottom. Notes: Luciferase activity was analyzed 30 hours after transfection. Luciferase activity expressed as fold increase relative to that of pcDNA3 transfected cells. Data shown are mean ± SD of three independent transfections in one experiment. Each experiment was repeated at least three times in triplicates.

    Article Snippet: Western blot Thirty hours after transient transfection, cells were lysed in lysis buffer containing 1% (w/v) SDS, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM EDTA, and 1× protease inhibitor cocktail (Sigma, St. Louis, Missouri).

    Techniques: Binding Assay, Transfection, Labeling, Luciferase, Activity Assay

    Mapping the core promoter region of human Sell gene. ( A ) 5′ serial deletion mutants shown on the left side were transiently transfected into Jurkat (stripped bars), EL4 (open bars), or HeLa cells (solid bars) and Luciferase activity shown on the right side was analyzed 30 hours after transfection. ( B ) Jurkat cells were co-transfected with core promoter construct, hSell288, with plasmids expressing Sp1, Mzf1, Klf2, Irf1, Ets1. Notes: Luciferase activity was analyzed 30 hours after the co-transfection. Luciferase activity was expressed as percentage of that of pGL3-Promoter in 2A and as fold changes relative to that of pGL3 vector transfected Jurkat cells in 2B. Data shown are mean ± SD of three independent transfections in one experiment. Each experiment was repeated at least three times.

    Journal: Gene Regulation and Systems Biology

    Article Title: FOXO1 Up-Regulates Human L-selectin Expression Through Binding to a Consensus FOXO1 Motif

    doi: 10.4137/GRSB.S10343

    Figure Lengend Snippet: Mapping the core promoter region of human Sell gene. ( A ) 5′ serial deletion mutants shown on the left side were transiently transfected into Jurkat (stripped bars), EL4 (open bars), or HeLa cells (solid bars) and Luciferase activity shown on the right side was analyzed 30 hours after transfection. ( B ) Jurkat cells were co-transfected with core promoter construct, hSell288, with plasmids expressing Sp1, Mzf1, Klf2, Irf1, Ets1. Notes: Luciferase activity was analyzed 30 hours after the co-transfection. Luciferase activity was expressed as percentage of that of pGL3-Promoter in 2A and as fold changes relative to that of pGL3 vector transfected Jurkat cells in 2B. Data shown are mean ± SD of three independent transfections in one experiment. Each experiment was repeated at least three times.

    Article Snippet: Western blot Thirty hours after transient transfection, cells were lysed in lysis buffer containing 1% (w/v) SDS, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM EDTA, and 1× protease inhibitor cocktail (Sigma, St. Louis, Missouri).

    Techniques: Transfection, Luciferase, Activity Assay, Construct, Expressing, Cotransfection, Plasmid Preparation

    Relative change in TAG- or CE-specific intracellular lipid storage upon expression of various Plin proteins. AML12 cells were transiently transfected with the indicated GFP–Plin-expressing constructs and cultured overnight in the presence of oleic acid and cholesterol. Transfection efficiencies were confirmed by visualizing GFP fluorescence. Untransfected cells, cultured with/without exogenous oleic acid and cholesterol, were grown in parallel. LSDs were isolated by centrifugation and lipids were extracted, separated by TLC in parallel with lipid markers, and TAG and CE detected after staining with iodine vapor. Relative TAG/CE ratios were quantified in cells transfected with each specific Plin-expressing construct and analyzed in parallel with identically grown untransfected cells for normalization and TLC background correction. The TAG/CE ratio for untransfected lipid-loaded cells (controls) was set to 0.5. The relative TAG/CE-ratio of Plin1a- and Plin1c-expressing cells were always analyzed in parallel and normalized to those of control cells, and then secondarily compared to results determined for its Plin-expressing counterpart. Values > 0.5 indicate a proportional increase in TAG lipid bias, whereas values

    Journal: Journal of Cell Science

    Article Title: Perilipin family members preferentially sequester to either triacylglycerol-specific or cholesteryl-ester-specific intracellular lipid storage droplets

    doi: 10.1242/jcs.104943

    Figure Lengend Snippet: Relative change in TAG- or CE-specific intracellular lipid storage upon expression of various Plin proteins. AML12 cells were transiently transfected with the indicated GFP–Plin-expressing constructs and cultured overnight in the presence of oleic acid and cholesterol. Transfection efficiencies were confirmed by visualizing GFP fluorescence. Untransfected cells, cultured with/without exogenous oleic acid and cholesterol, were grown in parallel. LSDs were isolated by centrifugation and lipids were extracted, separated by TLC in parallel with lipid markers, and TAG and CE detected after staining with iodine vapor. Relative TAG/CE ratios were quantified in cells transfected with each specific Plin-expressing construct and analyzed in parallel with identically grown untransfected cells for normalization and TLC background correction. The TAG/CE ratio for untransfected lipid-loaded cells (controls) was set to 0.5. The relative TAG/CE-ratio of Plin1a- and Plin1c-expressing cells were always analyzed in parallel and normalized to those of control cells, and then secondarily compared to results determined for its Plin-expressing counterpart. Values > 0.5 indicate a proportional increase in TAG lipid bias, whereas values

    Article Snippet: Transient transfection was carried out following the manufacturer's instructions (Invitrogen) using Lipofectamine LTX reagent.

    Techniques: Expressing, Transfection, Construct, Cell Culture, Fluorescence, Isolation, Centrifugation, Thin Layer Chromatography, Staining

    IRE1 activation increases the surface expression of α1(A322D) subunit of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or XBP1-s (spliced XBP1) plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. The cell lysates are then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B C ) (n = 5 for α1 and n = 3 for BiP, paired t-test). ( D ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface protein expression levels of α1 is shown in ( E ) (n = 5, paired t-test). ( F ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or XBP-s or transfected with XBP-s and treated with lactacystin (2.5 μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( G ) (n = 3, one-way ANOVA followed by Fisher test, *, p

    Journal: PLoS ONE

    Article Title: Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors

    doi: 10.1371/journal.pone.0207948

    Figure Lengend Snippet: IRE1 activation increases the surface expression of α1(A322D) subunit of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or XBP1-s (spliced XBP1) plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. The cell lysates are then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B C ) (n = 5 for α1 and n = 3 for BiP, paired t-test). ( D ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface protein expression levels of α1 is shown in ( E ) (n = 5, paired t-test). ( F ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or XBP-s or transfected with XBP-s and treated with lactacystin (2.5 μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( G ) (n = 3, one-way ANOVA followed by Fisher test, *, p

    Article Snippet: Cell lines that stably expressing α1β2γ2 and α1(A322D)β2γ2 receptors were generated by transient transfection with α1:β2:γ2 (1:1:1) and α1(A322D):β2:γ2 (1:1:1) plasmids.

    Techniques: Activation Assay, Expressing, Transfection, SDS Page, Western Blot, Affinity Purification, Quantitation Assay

    ATF6 activation promotes the forward trafficking of α1(A322D) subunit of GABA A receptors. (A) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or HA-tagged full-length ATF6α plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Endo H resistant v1 subunit bands (top arrow, lane 4) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 3 and 4) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B ) and ( C ) (n = 5 for α1 and n = 4 for BiP, paired t-test). Quantification of the ratio of endo H resistant α1 / total α1 is shown in ( D ) (n = 3, paired t-test). ( E ) Cells were treated as in ( A ). Forty-eight hrs post transfection, the nuclear fractions were extracted and subject to SDS-PAGE. ATF6 (N) is the cleaved, activated N-terminal ATF6 in the nucleus. Matrin-3 serves as a nuclear protein loading control. ( F ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels is shown in ( G ) (n = 6, paired t-test). ( H ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or ATF6, or transfected with ATF6 and treated with lactacystin (2.5μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( I ) (n = 5, one-way ANOVA followed by Fisher test, *, p

    Journal: PLoS ONE

    Article Title: Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors

    doi: 10.1371/journal.pone.0207948

    Figure Lengend Snippet: ATF6 activation promotes the forward trafficking of α1(A322D) subunit of GABA A receptors. (A) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or HA-tagged full-length ATF6α plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Endo H resistant v1 subunit bands (top arrow, lane 4) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 3 and 4) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B ) and ( C ) (n = 5 for α1 and n = 4 for BiP, paired t-test). Quantification of the ratio of endo H resistant α1 / total α1 is shown in ( D ) (n = 3, paired t-test). ( E ) Cells were treated as in ( A ). Forty-eight hrs post transfection, the nuclear fractions were extracted and subject to SDS-PAGE. ATF6 (N) is the cleaved, activated N-terminal ATF6 in the nucleus. Matrin-3 serves as a nuclear protein loading control. ( F ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels is shown in ( G ) (n = 6, paired t-test). ( H ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or ATF6, or transfected with ATF6 and treated with lactacystin (2.5μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( I ) (n = 5, one-way ANOVA followed by Fisher test, *, p

    Article Snippet: Cell lines that stably expressing α1β2γ2 and α1(A322D)β2γ2 receptors were generated by transient transfection with α1:β2:γ2 (1:1:1) and α1(A322D):β2:γ2 (1:1:1) plasmids.

    Techniques: Activation Assay, Expressing, Transfection, Incubation, SDS Page, Western Blot, Affinity Purification, Quantitation Assay

    Vpu-mediated control of IFN-I production by pDCs involves engagement and activation of ILT7 by BST2. (A-C) Vpu-mediated BST2 antagonism enhances activation of ILT7. ILT7+ NFAT-GFP reporter cells were co-cultured with HEK293T (mock) or BST2-expressing HEK293T cells mock-transfected or transfected with the indicated pNL4.3 constructs (WT or dU). (A) Representative example of ILT7 activation as determined by the percentage of NFAT-GFP positive cells measured by flow cytometry. (B) Percentage of ILT7 activation after co-culture with the indicated HIV/BST2 expressing HEK 293T cells relative to WT HIV-producing cells (100%) (n = 4). (C) Percentage of BST2 surface expression in HEK293T cells after co-transfection of BST2 with the indicated HIV provirus relative to dU HIV-producing cells (100%) (n = 4). (D-F) Effect of ILT7 depletion on IFN-I production by pDCs. (D) Non-pDC fraction (BDCA-2-) and siRNA-treated enriched pDCs (CD14-/BDCA-2+) were stained using anti-ILT7 Abs as indicated. A representative example of absolute levels (E) or relative percentages (F) of IFN-I produced after co-culture of control pDCs (pDC-siCTRL) or ILT7-depleted pDCs (pDC-siILT7) with the indicated infected MT4 cells are shown. The amount of IFN-I released by pDC siCTRL in contact with dU HIV-infected cells was set at 100% (n = 4). (G-I) Effect of recombinant soluble ILT7 on IFN-I production by pDCs. (G) Expression of a HA-tagged soluble ILT7 (soILT7-HA) in HEK 293T cells. Cells and supernatants (sup) were analyzed by Western blot (WB) using anti-HA Abs. Purity of secreted soILT7-HA was confirmed by Coomassie staining (sup Coom). A representative example of (H) absolute levels or (I) relative percentages of IFN-I production after co-culture of PBMCs with the indicated infected MT4 cells pre-treated with control (CTRL) or soILT7-HA-containing supernatants are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected cells in presence of CTRL supernatant was set at 100% (n = 6). Two-tailed paired t -test was used in B-C while repeated measures ANOVA with Bonferroni’s multiple comparison test was used in F and I (*** p

    Journal: PLoS Pathogens

    Article Title: Vpu Exploits the Cross-Talk between BST2 and the ILT7 Receptor to Suppress Anti-HIV-1 Responses by Plasmacytoid Dendritic Cells

    doi: 10.1371/journal.ppat.1005024

    Figure Lengend Snippet: Vpu-mediated control of IFN-I production by pDCs involves engagement and activation of ILT7 by BST2. (A-C) Vpu-mediated BST2 antagonism enhances activation of ILT7. ILT7+ NFAT-GFP reporter cells were co-cultured with HEK293T (mock) or BST2-expressing HEK293T cells mock-transfected or transfected with the indicated pNL4.3 constructs (WT or dU). (A) Representative example of ILT7 activation as determined by the percentage of NFAT-GFP positive cells measured by flow cytometry. (B) Percentage of ILT7 activation after co-culture with the indicated HIV/BST2 expressing HEK 293T cells relative to WT HIV-producing cells (100%) (n = 4). (C) Percentage of BST2 surface expression in HEK293T cells after co-transfection of BST2 with the indicated HIV provirus relative to dU HIV-producing cells (100%) (n = 4). (D-F) Effect of ILT7 depletion on IFN-I production by pDCs. (D) Non-pDC fraction (BDCA-2-) and siRNA-treated enriched pDCs (CD14-/BDCA-2+) were stained using anti-ILT7 Abs as indicated. A representative example of absolute levels (E) or relative percentages (F) of IFN-I produced after co-culture of control pDCs (pDC-siCTRL) or ILT7-depleted pDCs (pDC-siILT7) with the indicated infected MT4 cells are shown. The amount of IFN-I released by pDC siCTRL in contact with dU HIV-infected cells was set at 100% (n = 4). (G-I) Effect of recombinant soluble ILT7 on IFN-I production by pDCs. (G) Expression of a HA-tagged soluble ILT7 (soILT7-HA) in HEK 293T cells. Cells and supernatants (sup) were analyzed by Western blot (WB) using anti-HA Abs. Purity of secreted soILT7-HA was confirmed by Coomassie staining (sup Coom). A representative example of (H) absolute levels or (I) relative percentages of IFN-I production after co-culture of PBMCs with the indicated infected MT4 cells pre-treated with control (CTRL) or soILT7-HA-containing supernatants are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected cells in presence of CTRL supernatant was set at 100% (n = 6). Two-tailed paired t -test was used in B-C while repeated measures ANOVA with Bonferroni’s multiple comparison test was used in F and I (*** p

    Article Snippet: WT BST2, BST2-dGPI, short BST2 and long BST2 open reading frames were cloned into the pcDNA3.1 backbone for transient transfections and in pLenti-CMV/TO_Puro_DEST (Addgene) for the generation of stable cell lines.

    Techniques: Activation Assay, Cell Culture, Expressing, Transfection, Construct, Flow Cytometry, Cytometry, Co-Culture Assay, Cotransfection, Staining, Produced, Infection, Recombinant, Western Blot, Two Tailed Test

    Effect of a BST2 GPI anchor mutant on Vpu-mediated control of IFN-I production by pDCs. (A-B) HEK293T cells were transfected with either an empty plasmid or plasmids expressing BST2 or BST2-dGPI 48 h prior to co-culture with PBMCs. (A) Surface expression of BST2 was evaluated 48 h post transfection by flow cytometry in controls cells (shaded grey histogram), as well as in cell expressing BST2 (solid black histogram), or BST2-dGPI (dashed grey histogram). Mean fluorescence intensity (MFI) values are indicated for each sample (B) After 6 h of co-culture, samples were untreated or treated with Imiquimod (TLR7 agonist) and levels of bioactive IFN-I in supernatants were measured 18 h later. The amount of IFN-I released by PBMCs in contact with HEK293T cells transfected with the empty plasmid in presence of the TLR 7 agonist was set at 100% (n = 4). As a control, transfected HEK293T cells were treated with TLR7 agonist without PBMCs. (C) Percentage of IFN-I released after co-culture of infected SupT1-Empty with PBMCs normalized to the value obtained with dU HIV-infected SuptT1 cells (100%) (n = 4). (D) A representative example of absolute levels of IFN-I produced after co-culture of mock or infected-SupT1,-SupT1-BST2 or-SupT1-BST2-dGPI cells with PBMCs is shown. (E) Relative percentages of IFN-I produced after co-culture of infected-SupT1-BST2 or SupT1-BST2-dGPI cells with PBMCs are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected SupT1-BST2 cells was set at 100% (n = 4). Two-tailed paired t -test was used in B and C (* p

    Journal: PLoS Pathogens

    Article Title: Vpu Exploits the Cross-Talk between BST2 and the ILT7 Receptor to Suppress Anti-HIV-1 Responses by Plasmacytoid Dendritic Cells

    doi: 10.1371/journal.ppat.1005024

    Figure Lengend Snippet: Effect of a BST2 GPI anchor mutant on Vpu-mediated control of IFN-I production by pDCs. (A-B) HEK293T cells were transfected with either an empty plasmid or plasmids expressing BST2 or BST2-dGPI 48 h prior to co-culture with PBMCs. (A) Surface expression of BST2 was evaluated 48 h post transfection by flow cytometry in controls cells (shaded grey histogram), as well as in cell expressing BST2 (solid black histogram), or BST2-dGPI (dashed grey histogram). Mean fluorescence intensity (MFI) values are indicated for each sample (B) After 6 h of co-culture, samples were untreated or treated with Imiquimod (TLR7 agonist) and levels of bioactive IFN-I in supernatants were measured 18 h later. The amount of IFN-I released by PBMCs in contact with HEK293T cells transfected with the empty plasmid in presence of the TLR 7 agonist was set at 100% (n = 4). As a control, transfected HEK293T cells were treated with TLR7 agonist without PBMCs. (C) Percentage of IFN-I released after co-culture of infected SupT1-Empty with PBMCs normalized to the value obtained with dU HIV-infected SuptT1 cells (100%) (n = 4). (D) A representative example of absolute levels of IFN-I produced after co-culture of mock or infected-SupT1,-SupT1-BST2 or-SupT1-BST2-dGPI cells with PBMCs is shown. (E) Relative percentages of IFN-I produced after co-culture of infected-SupT1-BST2 or SupT1-BST2-dGPI cells with PBMCs are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected SupT1-BST2 cells was set at 100% (n = 4). Two-tailed paired t -test was used in B and C (* p

    Article Snippet: WT BST2, BST2-dGPI, short BST2 and long BST2 open reading frames were cloned into the pcDNA3.1 backbone for transient transfections and in pLenti-CMV/TO_Puro_DEST (Addgene) for the generation of stable cell lines.

    Techniques: Mutagenesis, Transfection, Plasmid Preparation, Expressing, Co-Culture Assay, Flow Cytometry, Cytometry, Fluorescence, Infection, Produced, Two Tailed Test

    Outline of pharmacological manipulation of CK1 functions and its effects on p53 signalling pathway, cell viability, and CK1α post-translational modifications. ( A ) H1299 cells were transfected with STReP-tagged CK1α sp1 or 2, FLAG-tagged peptide 35 and His-Ubiquitin or His-Nedd8, using Attractene. Eighteen hours after transfection cells were treated with 50 µM MG132 for 4 hours. Whole cell lysates were analysed for transfected CK1α levels, and His-ubiquitin/NEDD8 conjugates were purified by metal affinity chromatography and analyzed by 4%–12% NuPAGE/immunoblots with an anti-CK1α antibody. Changes highlighted were representative of two independent experiments. ( B ) Three approaches for manipulation of signal transduction pathways. Genetic depletion of CK1α using siRNA or the CK1 kinase attenuation using the ATP-competitive inhibitor D4476 can alter the p53 response and cell viability, as indicated. The bioactive peptide from the high affinity MDM2-CK1α interface (peptide 35) highlights a novel peptide lead for disrupting signalling in cancers. All three approaches gave rise to overlapping but distinct effects on signalling. Specific siRNA against CK1α led to p53 induction and growth arrest accompanied with slight sub-G1 increase, whereas non-specific CK1 drugs such as D4476 mediated p53 induction and significant apoptosis. Low levels of the bioactive peptide 35 did not lead to p53 protein induction but to a growth arrest in G0-G1 and reduced cell viability. These data suggest that peptide 35 may function in a pharmacologically novel manner compared to the ATP-active site inhibitor or CK1α siRNA and highlights the general utility of targeting protein-protein interactions as approaches for developing therapeutic strategies that target signalling mechanisms in cancer.

    Journal: PLoS ONE

    Article Title: Exploiting the MDM2-CK1? Protein-Protein Interface to Develop Novel Biologics That Induce UBL-Kinase-Modification and Inhibit Cell Growth

    doi: 10.1371/journal.pone.0043391

    Figure Lengend Snippet: Outline of pharmacological manipulation of CK1 functions and its effects on p53 signalling pathway, cell viability, and CK1α post-translational modifications. ( A ) H1299 cells were transfected with STReP-tagged CK1α sp1 or 2, FLAG-tagged peptide 35 and His-Ubiquitin or His-Nedd8, using Attractene. Eighteen hours after transfection cells were treated with 50 µM MG132 for 4 hours. Whole cell lysates were analysed for transfected CK1α levels, and His-ubiquitin/NEDD8 conjugates were purified by metal affinity chromatography and analyzed by 4%–12% NuPAGE/immunoblots with an anti-CK1α antibody. Changes highlighted were representative of two independent experiments. ( B ) Three approaches for manipulation of signal transduction pathways. Genetic depletion of CK1α using siRNA or the CK1 kinase attenuation using the ATP-competitive inhibitor D4476 can alter the p53 response and cell viability, as indicated. The bioactive peptide from the high affinity MDM2-CK1α interface (peptide 35) highlights a novel peptide lead for disrupting signalling in cancers. All three approaches gave rise to overlapping but distinct effects on signalling. Specific siRNA against CK1α led to p53 induction and growth arrest accompanied with slight sub-G1 increase, whereas non-specific CK1 drugs such as D4476 mediated p53 induction and significant apoptosis. Low levels of the bioactive peptide 35 did not lead to p53 protein induction but to a growth arrest in G0-G1 and reduced cell viability. These data suggest that peptide 35 may function in a pharmacologically novel manner compared to the ATP-active site inhibitor or CK1α siRNA and highlights the general utility of targeting protein-protein interactions as approaches for developing therapeutic strategies that target signalling mechanisms in cancer.

    Article Snippet: Transient Transfection of Small Interfering RNA (siRNA) siRNA to CK1α gene was obtained from Dharmacon (siGENOME SMARTpool against Human CSNK1A1 (NM_001892)). siCONTROL non-targeting siRNA Pool#2 was used as a control (siRNA control).

    Techniques: Transfection, Purification, Affinity Chromatography, Western Blot, Transduction

    CK1α peptide derived from its dominant MDM2 binding site triggers G0-G1 arrest and cell death in a p53-independent manner. CK1α peptide 35 was transfected into HCT116 cells using Nucleofectin reagent and Nucleofector device II. A DMSO control was included. ( A ) Protein levels were assessed 18 hours after peptide transfection by Western blotting. ( B ) Images of cells were captured 16 hours after transfection. ( C ) The number of non-viable cells was counted after treatment using Trypan Blue. The percentage of non-viable cells is expressed relative to each respective total cell count. ( D ) After treatment, the cells were harvested then fixed in ethanol followed by staining with propidium iodide. DNA content was determined by FACS and analyzed with FlowJo7 software.

    Journal: PLoS ONE

    Article Title: Exploiting the MDM2-CK1? Protein-Protein Interface to Develop Novel Biologics That Induce UBL-Kinase-Modification and Inhibit Cell Growth

    doi: 10.1371/journal.pone.0043391

    Figure Lengend Snippet: CK1α peptide derived from its dominant MDM2 binding site triggers G0-G1 arrest and cell death in a p53-independent manner. CK1α peptide 35 was transfected into HCT116 cells using Nucleofectin reagent and Nucleofector device II. A DMSO control was included. ( A ) Protein levels were assessed 18 hours after peptide transfection by Western blotting. ( B ) Images of cells were captured 16 hours after transfection. ( C ) The number of non-viable cells was counted after treatment using Trypan Blue. The percentage of non-viable cells is expressed relative to each respective total cell count. ( D ) After treatment, the cells were harvested then fixed in ethanol followed by staining with propidium iodide. DNA content was determined by FACS and analyzed with FlowJo7 software.

    Article Snippet: Transient Transfection of Small Interfering RNA (siRNA) siRNA to CK1α gene was obtained from Dharmacon (siGENOME SMARTpool against Human CSNK1A1 (NM_001892)). siCONTROL non-targeting siRNA Pool#2 was used as a control (siRNA control).

    Techniques: Derivative Assay, Binding Assay, Transfection, Western Blot, Cell Counting, Staining, FACS, Software

    The CK1α peptide derived from its dominant MDM2 binding site triggers G0-G1 arrest and cell death in A375 cells. CK1α peptide 35 was transfected into A375 cells using Nucleofectin reagent and Nucleofector device II. A DMSO control was included. ( A ) Protein levels were assessed 18 hours after peptide transfection by Western blotting. ( B ) Images of cells were captured 16 hours after transfection. ( C ) The number of non-viable cells was counted after treatment using Trypan Blue. Total cells are shown in percent relative to the DMSO control and percent of non-viable cells are expressed relative to each respective total cell count. ( D ) After treatment, the cells were harvested then fixed in ethanol followed by staining with propidium iodide. DNA content was determined by FACS and analyzed with FlowJo7 software.

    Journal: PLoS ONE

    Article Title: Exploiting the MDM2-CK1? Protein-Protein Interface to Develop Novel Biologics That Induce UBL-Kinase-Modification and Inhibit Cell Growth

    doi: 10.1371/journal.pone.0043391

    Figure Lengend Snippet: The CK1α peptide derived from its dominant MDM2 binding site triggers G0-G1 arrest and cell death in A375 cells. CK1α peptide 35 was transfected into A375 cells using Nucleofectin reagent and Nucleofector device II. A DMSO control was included. ( A ) Protein levels were assessed 18 hours after peptide transfection by Western blotting. ( B ) Images of cells were captured 16 hours after transfection. ( C ) The number of non-viable cells was counted after treatment using Trypan Blue. Total cells are shown in percent relative to the DMSO control and percent of non-viable cells are expressed relative to each respective total cell count. ( D ) After treatment, the cells were harvested then fixed in ethanol followed by staining with propidium iodide. DNA content was determined by FACS and analyzed with FlowJo7 software.

    Article Snippet: Transient Transfection of Small Interfering RNA (siRNA) siRNA to CK1α gene was obtained from Dharmacon (siGENOME SMARTpool against Human CSNK1A1 (NM_001892)). siCONTROL non-targeting siRNA Pool#2 was used as a control (siRNA control).

    Techniques: Derivative Assay, Binding Assay, Transfection, Western Blot, Cell Counting, Staining, FACS, Software

    YFP-N300 downregulates MHC class I expression in M553(R240)tpsn cells M553(R240)Tpsn cells were transiently transfected with YFP-N300 or co-transfected with empty vector and pmaxGFP. Twenty-four hours post transfection, MHC class I expression was analyzed by flow cytometry using a PE directly conjugated W6/32 (MHC class I) antibody. Mean fluorescent intensity was determined for W6/32 staining on populations of cells gated on forward and side scatter (R1) and for the negative or positive expression of GFP/YFP (R2, GFP/YFP negative) and R3, (GFP/YFP positive).

    Journal: Human immunology

    Article Title: Identification of an Alternate Splice Form of Tapasin in Human Melanoma

    doi: 10.1016/j.humimm.2010.05.019

    Figure Lengend Snippet: YFP-N300 downregulates MHC class I expression in M553(R240)tpsn cells M553(R240)Tpsn cells were transiently transfected with YFP-N300 or co-transfected with empty vector and pmaxGFP. Twenty-four hours post transfection, MHC class I expression was analyzed by flow cytometry using a PE directly conjugated W6/32 (MHC class I) antibody. Mean fluorescent intensity was determined for W6/32 staining on populations of cells gated on forward and side scatter (R1) and for the negative or positive expression of GFP/YFP (R2, GFP/YFP negative) and R3, (GFP/YFP positive).

    Article Snippet: For transient transfection experiments, 100 ng of the pmaxGFP plasmid which encodes an enhanced GFP protein driven by the CMV promoter (Amaxa, Gaithersburg, MD) and 2 micrograms of the tpsnΔEx3 cDNA in pCDNA3.1(−)PURO or empty pCDNA3.1(−)puro plasmid were transfected into 1 million cells using an Amaxa Nucleoporator II instrument (solution V, program T-030) according to manufacturers recommendations.

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

    Transient expression of truncated tapasin molecules does not reduce MHC class I expression in M553(R240)Tpsn cells A . Transient co-transfection of M553(R240)Tpsn cells with pmaxGFP and the indicated tapasin variants followed by W6/32 staining 24 hours post transfection. W6/32 staining on populations of cells gated on forward and side scatter (R1) and for the negative or positive expression of GFP (R2 vs. R3, respectively). B . The transient transfectants from A. were analyzed by western blot for the ratio of wild type (R240)tpsn to tpsnΔEx3 or tpsnΔN50 using TO-3 antibody.

    Journal: Human immunology

    Article Title: Identification of an Alternate Splice Form of Tapasin in Human Melanoma

    doi: 10.1016/j.humimm.2010.05.019

    Figure Lengend Snippet: Transient expression of truncated tapasin molecules does not reduce MHC class I expression in M553(R240)Tpsn cells A . Transient co-transfection of M553(R240)Tpsn cells with pmaxGFP and the indicated tapasin variants followed by W6/32 staining 24 hours post transfection. W6/32 staining on populations of cells gated on forward and side scatter (R1) and for the negative or positive expression of GFP (R2 vs. R3, respectively). B . The transient transfectants from A. were analyzed by western blot for the ratio of wild type (R240)tpsn to tpsnΔEx3 or tpsnΔN50 using TO-3 antibody.

    Article Snippet: For transient transfection experiments, 100 ng of the pmaxGFP plasmid which encodes an enhanced GFP protein driven by the CMV promoter (Amaxa, Gaithersburg, MD) and 2 micrograms of the tpsnΔEx3 cDNA in pCDNA3.1(−)PURO or empty pCDNA3.1(−)puro plasmid were transfected into 1 million cells using an Amaxa Nucleoporator II instrument (solution V, program T-030) according to manufacturers recommendations.

    Techniques: Expressing, Cotransfection, Staining, Transfection, Western Blot

    Methylation-sensitive binding of CTCF to the first exon of hTERT . (A) In vivo binding of CTCF to the first exon of hTERT in telomerase-positive and negative cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments ( hTERT and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (B) 5′-end-labeled control (F1) and SssI methylase-treated (F1-met) fragments were digested with methylation-sensitive BstUI and analyzed on polyacrylamide gels to verify efficiency of in vitro methylation. ( C ) Control unmethylated (F1) or SssI-methylated (F1-met) fragments were analyzed by gel-shift assay (EMSA). F, free probe; B, CTCF-bound probe. ( D ) Representation of the hTERT sequence cloned into the pTERT-297/ex2/FRT. Arrows represent the localization of the primers used for hTERT methylation analysis of stable transfectant. ( E ) The methylation status of the stable transfectants was verified by MS-SSCA. Unmethylated and fully methylated controls were obtained from plasmids used for stable transfection. UT and MT represent, respectively, the unmethylated and methylated plasmids stably transfected into HeLa cells, and after 30 population doublings. ( F ) Binding of CTCF to the first exon of hTERT in stably transfected cell line was analyzed by ChIP assay using anti-CTCF antibody.

    Journal: Nucleic Acids Research

    Article Title: Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene

    doi: 10.1093/nar/gkl1125

    Figure Lengend Snippet: Methylation-sensitive binding of CTCF to the first exon of hTERT . (A) In vivo binding of CTCF to the first exon of hTERT in telomerase-positive and negative cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments ( hTERT and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (B) 5′-end-labeled control (F1) and SssI methylase-treated (F1-met) fragments were digested with methylation-sensitive BstUI and analyzed on polyacrylamide gels to verify efficiency of in vitro methylation. ( C ) Control unmethylated (F1) or SssI-methylated (F1-met) fragments were analyzed by gel-shift assay (EMSA). F, free probe; B, CTCF-bound probe. ( D ) Representation of the hTERT sequence cloned into the pTERT-297/ex2/FRT. Arrows represent the localization of the primers used for hTERT methylation analysis of stable transfectant. ( E ) The methylation status of the stable transfectants was verified by MS-SSCA. Unmethylated and fully methylated controls were obtained from plasmids used for stable transfection. UT and MT represent, respectively, the unmethylated and methylated plasmids stably transfected into HeLa cells, and after 30 population doublings. ( F ) Binding of CTCF to the first exon of hTERT in stably transfected cell line was analyzed by ChIP assay using anti-CTCF antibody.

    Article Snippet: Transient transfection of reporter plasmids (0.75 μg/well) was carried out in triplicate using JetPEI Cationic Polymer Transfection reagent (4 μl/well) (Polyplus-transfection, Illkirch, France).

    Techniques: Methylation, Binding Assay, In Vivo, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Labeling, In Vitro, Electrophoretic Mobility Shift Assay, Sequencing, Clone Assay, Transfection, Mass Spectrometry, Stable Transfection

    Dose effect of ENZ fragment on BAR efficiency. ( A ) Schematic representation of the Gal4-tagged (gray–blue square) ORF2 fragments. ENZ490Δ fragment was generated from wild-type ORF2 sequence, while Δ348RTCys fragment was generated using codon optimized ORF2 sequence. ( B ) BAR output using different amounts (0.4–0.05 μg) of the wild-type ENZ expression plasmid with 0.4 μg of the RTCys expression plasmid. Empty vector used as filler to ensure equal DNA amounts in transfections. Error bars denote standard deviation ( n = 3). Statistical significance assessed using Student's t -test for paired samples with denoted by *. Representative flasks are shown above corresponding graph bars.

    Journal: Nucleic Acids Research

    Article Title: Identification of L1 ORF2p sequence important to retrotransposition using Bipartile Alu retrotransposition (BAR)

    doi: 10.1093/nar/gkw277

    Figure Lengend Snippet: Dose effect of ENZ fragment on BAR efficiency. ( A ) Schematic representation of the Gal4-tagged (gray–blue square) ORF2 fragments. ENZ490Δ fragment was generated from wild-type ORF2 sequence, while Δ348RTCys fragment was generated using codon optimized ORF2 sequence. ( B ) BAR output using different amounts (0.4–0.05 μg) of the wild-type ENZ expression plasmid with 0.4 μg of the RTCys expression plasmid. Empty vector used as filler to ensure equal DNA amounts in transfections. Error bars denote standard deviation ( n = 3). Statistical significance assessed using Student's t -test for paired samples with denoted by *. Representative flasks are shown above corresponding graph bars.

    Article Snippet: All truncated ORF2p fragments were expressed in HeLa cells following transient transfection with respective expression constructs as detected via western blot analysis with anti-Gal4 antibodies (Santa Cruz) (Figure ).

    Techniques: Generated, Sequencing, Expressing, Plasmid Preparation, Transfection, Standard Deviation

    TIGAR expression is increased in high metastatic lung cancer cells, affecting cell migration, invasion and adhesion in vitro. ( a - b ) Wound healing assays and Trans-well analysis to determine the migration and invasion of A549, H1299, H1650, H1975 and PC9, respectively. ( c - d ) Statistical analysis was to quantify the migratory and invasive ability of five NSCLC cells. ( e ) Real-time PCR and Western blot analysis to determine endogenous TIGAR expression of five NSCLC cells. ( f ) Real-time PCR and Western blot analysis to respectively quantify mRNA and protein expression of TIGAR after transfection with siTIGAR (or siNC as control) for 48h. ( g - h ) Cells were transfected with siTIGAR (or siNC as control) for 48h were collected to determine the migration and invasion capability by wound-healing assays and Trans-well assay, respectively. ( i - j ) Statistical analysis was to quantify cells migratory and invasive ability upon TIGAR knockdown. ( k ) H1299 and A549 cells were infected with lentivirus with shTIGAR and Green fluorescent protein. To get more effective knockdown, monoclonal cell was to screen in A549. Monoclonal cells A549-shTIGAR#7 was the A549-shTIGAR in following assays. ( l ) Wound-healing assay for H1299 and A549 cells stably knockdown TIGAR or negative control. ( m ) Trans-well invasion assay for A549 cells stably knockdown TIGAR or negative control. ( n ) The effect of TIGAR on cell adhesion activity was assessed in A549

    Journal: Molecular Cancer

    Article Title: Met is involved in TIGAR-regulated metastasis of non-small-cell lung cancer

    doi: 10.1186/s12943-018-0839-4

    Figure Lengend Snippet: TIGAR expression is increased in high metastatic lung cancer cells, affecting cell migration, invasion and adhesion in vitro. ( a - b ) Wound healing assays and Trans-well analysis to determine the migration and invasion of A549, H1299, H1650, H1975 and PC9, respectively. ( c - d ) Statistical analysis was to quantify the migratory and invasive ability of five NSCLC cells. ( e ) Real-time PCR and Western blot analysis to determine endogenous TIGAR expression of five NSCLC cells. ( f ) Real-time PCR and Western blot analysis to respectively quantify mRNA and protein expression of TIGAR after transfection with siTIGAR (or siNC as control) for 48h. ( g - h ) Cells were transfected with siTIGAR (or siNC as control) for 48h were collected to determine the migration and invasion capability by wound-healing assays and Trans-well assay, respectively. ( i - j ) Statistical analysis was to quantify cells migratory and invasive ability upon TIGAR knockdown. ( k ) H1299 and A549 cells were infected with lentivirus with shTIGAR and Green fluorescent protein. To get more effective knockdown, monoclonal cell was to screen in A549. Monoclonal cells A549-shTIGAR#7 was the A549-shTIGAR in following assays. ( l ) Wound-healing assay for H1299 and A549 cells stably knockdown TIGAR or negative control. ( m ) Trans-well invasion assay for A549 cells stably knockdown TIGAR or negative control. ( n ) The effect of TIGAR on cell adhesion activity was assessed in A549

    Article Snippet: Oligonucleotide transfection SiRNA and negative control that used with transient transfection was designed and synthesized by GenePharma (shanghai, China).

    Techniques: Expressing, Migration, In Vitro, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Infection, Wound Healing Assay, Stable Transfection, Negative Control, Invasion Assay, Activity Assay

    Site-directed mutagenesis for luciferase activity assay and effect of miR-7 silencing over MAFG expression. (A) Chromosomal localization of miR-7 predicted binding sites at 3'UTR of MAFG. Regions 2 and 8 were identified by six or more bioinformatical algorithms. Sanger sequencing showed that the seed sequence of miR-7 was fully mutated at regions 2 and 8 of the 3' UTR of MAFG. (B) Co-transfection of mimic miR-7 (miR-7) or mimic control (miR-NC) with the 3' UTR of MAFG WT, mutated on region 2 (MAFG 2*) and mutated on region 8 (MAFG 8*). Experiments were performed at 15nM and data was analyzed after 24h of co-transfection. (Upper panel) Relative luciferase activity. The figures represent the mean ± SD of at least 3 independent experiments after data normalization with Renilla and the data from the negative control 3'-UTR; p

    Journal: Theranostics

    Article Title: DNA Methylation of miR-7 is a Mechanism Involved in Platinum Response through MAFG Overexpression in Cancer Cells

    doi: 10.7150/thno.20112

    Figure Lengend Snippet: Site-directed mutagenesis for luciferase activity assay and effect of miR-7 silencing over MAFG expression. (A) Chromosomal localization of miR-7 predicted binding sites at 3'UTR of MAFG. Regions 2 and 8 were identified by six or more bioinformatical algorithms. Sanger sequencing showed that the seed sequence of miR-7 was fully mutated at regions 2 and 8 of the 3' UTR of MAFG. (B) Co-transfection of mimic miR-7 (miR-7) or mimic control (miR-NC) with the 3' UTR of MAFG WT, mutated on region 2 (MAFG 2*) and mutated on region 8 (MAFG 8*). Experiments were performed at 15nM and data was analyzed after 24h of co-transfection. (Upper panel) Relative luciferase activity. The figures represent the mean ± SD of at least 3 independent experiments after data normalization with Renilla and the data from the negative control 3'-UTR; p

    Article Snippet: cDNA plasmids transfection A Myc-DDK-tagged ORF clone of MAFG , ELK-1 or ABCA1 and the negative control pCMV6 were used for in transient transfection (OriGene, USA).

    Techniques: Mutagenesis, Luciferase, Activity Assay, Expressing, Binding Assay, Sequencing, Cotransfection, Negative Control

    LMP2A augments activation of the TOPFlash luciferase reporter by β-catenin. (A) 293 cells were transfected with SV-β-galactosidase and either TOPFlash or FOPFlash plus vector, β-catenin, LMP2A, or β-catenin with LMP2A. TOPFlash contains four consensus TCF-4 binding sites in a promoter driving expression of the luciferase gene, and FOPFlash is an analogous construct with the four TCF-4 sites mutated. Transfections and assays were performed in triplicate in two separate experiments, and luciferase activity was normalized to β-galactosidase activity to control for transfection efficiency. The data are presented graphically as TOPFlash activity normalized to FOPFlash activity for each of the indicated conditions. (B) Immunoblot analyses were performed on the lysates used in the luciferase assay described above. Antibodies against HA confirm the presence of transfected HA-tagged LMP2A. Transfected and endogenous β-catenin levels were revealed with an anti-β-catenin antibody, and immunoblot with an antibody against TCF-4 attested to equal levels of this transcription factor in the transfected 293 cells.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Latent Membrane Protein 2A Activates ?-Catenin Signaling in Epithelial Cells

    doi: 10.1128/JVI.77.22.12276-12284.2003

    Figure Lengend Snippet: LMP2A augments activation of the TOPFlash luciferase reporter by β-catenin. (A) 293 cells were transfected with SV-β-galactosidase and either TOPFlash or FOPFlash plus vector, β-catenin, LMP2A, or β-catenin with LMP2A. TOPFlash contains four consensus TCF-4 binding sites in a promoter driving expression of the luciferase gene, and FOPFlash is an analogous construct with the four TCF-4 sites mutated. Transfections and assays were performed in triplicate in two separate experiments, and luciferase activity was normalized to β-galactosidase activity to control for transfection efficiency. The data are presented graphically as TOPFlash activity normalized to FOPFlash activity for each of the indicated conditions. (B) Immunoblot analyses were performed on the lysates used in the luciferase assay described above. Antibodies against HA confirm the presence of transfected HA-tagged LMP2A. Transfected and endogenous β-catenin levels were revealed with an anti-β-catenin antibody, and immunoblot with an antibody against TCF-4 attested to equal levels of this transcription factor in the transfected 293 cells.

    Article Snippet: In addition, in these stable cells, the level of LMP2A expression was much lower than that observed in transient transfections, and LMP2A protein was not detected in the nuclear fraction of these cells.

    Techniques: Activation Assay, Luciferase, Transfection, Plasmid Preparation, Binding Assay, Expressing, Construct, Activity Assay

    miR-320a suppresses migration and invasion of TSCC cells by targeting Suz12 in vitro A. Wound healing and transwell assays of UM1 after transient transfection. B. Wound healing and transwell assays of Cal27 after transient transfection. C. Western blot of Suz12 in stable transfected Cal27 and UM1 cell lines. D. Suz12 mRNA in stable transfected Cal27 and UM1 cell lines. E. Dual-luciferase reporter gene array. F. Western blot assays of Cal27 after co-transfection of Suz12 siRNA. G. Wound healing of Cal27 after co-transfection of Suz12 siRNA. * P

    Journal: Oncotarget

    Article Title: Decreased miR-320a promotes invasion and metastasis of tumor budding cells in tongue squamous cell carcinoma

    doi: 10.18632/oncotarget.11612

    Figure Lengend Snippet: miR-320a suppresses migration and invasion of TSCC cells by targeting Suz12 in vitro A. Wound healing and transwell assays of UM1 after transient transfection. B. Wound healing and transwell assays of Cal27 after transient transfection. C. Western blot of Suz12 in stable transfected Cal27 and UM1 cell lines. D. Suz12 mRNA in stable transfected Cal27 and UM1 cell lines. E. Dual-luciferase reporter gene array. F. Western blot assays of Cal27 after co-transfection of Suz12 siRNA. G. Wound healing of Cal27 after co-transfection of Suz12 siRNA. * P

    Article Snippet: Cell lines, transient transfection and lentiviral transfection TSCC cells were cultivated and inoculated as previously described [ ]. miR-320a mimics (50 nM, RiboBio), miR-320a inhibitor (100nM, RiboBio), Suz12 siRNA (100nM, RiboBio) and their negative controls were transfected into TSCC cells using Lipofectamine (Lipofectamine RNA iMAX Transfection Reagent, Thermo Fisher) for downstream arrays.

    Techniques: Migration, In Vitro, Transfection, Western Blot, Luciferase, Cotransfection

    Blimp1 in Foxp3 + regulatory T (Treg) cells limits generation of follicular regulatory T (Tfr) cells. (a) Expression of surface markers CD4 + CD25 + in spleen cells of C57BL/6 after magnetic bead isolation. The purity of CD4 + CD25 + Treg cells was 91 ± 2%. (b) Microscopy observation of small interfering RNA (siRNA) ‐transfected cells. The transfection efficiency of siRNA in Treg cells was over 90%. (c, d) Transcription and expression verification of Blimp1 was significantly decreased in siRNA‐Blimp1‐infected cells. (e) Quantification of Tfr cells (CD4 + CXCR5 + ICOS + Foxp3 + ]

    Journal: Immunology

    Article Title: Transcriptional repressor Blimp1 regulates follicular regulatory T‐cell homeostasis and function

    doi: 10.1111/imm.12815

    Figure Lengend Snippet: Blimp1 in Foxp3 + regulatory T (Treg) cells limits generation of follicular regulatory T (Tfr) cells. (a) Expression of surface markers CD4 + CD25 + in spleen cells of C57BL/6 after magnetic bead isolation. The purity of CD4 + CD25 + Treg cells was 91 ± 2%. (b) Microscopy observation of small interfering RNA (siRNA) ‐transfected cells. The transfection efficiency of siRNA in Treg cells was over 90%. (c, d) Transcription and expression verification of Blimp1 was significantly decreased in siRNA‐Blimp1‐infected cells. (e) Quantification of Tfr cells (CD4 + CXCR5 + ICOS + Foxp3 + ]

    Article Snippet: Twenty‐four hours after transient transfection of Tfr cells, B cells from EAMG mice were plated with Tfh cells and/or transfected Tfr cells pooled from wild‐type C57BL/6 mice and 2 μg/ml soluble anti‐CD3 and 5 μg/ml anti‐IgM (BD Bioscience).

    Techniques: Expressing, Isolation, Microscopy, Small Interfering RNA, Transfection, Infection

    Inhibition of encystation by AcAtg3-siRNA. Cells transfected with Superfect Transfection reagent, nonspecific negative siRNA, or AcAtg3-siRNA were incubated in encystment media for 3 days and the number of mature cysts scored. The formation of mature cysts was almost inhibited in AcAtg3-siRNA transfected cells.

    Journal: The Korean Journal of Parasitology

    Article Title: Atg3-Mediated Lipidation of Atg8 Is Involved in Encystation of Acanthamoeba

    doi: 10.3347/kjp.2011.49.2.103

    Figure Lengend Snippet: Inhibition of encystation by AcAtg3-siRNA. Cells transfected with Superfect Transfection reagent, nonspecific negative siRNA, or AcAtg3-siRNA were incubated in encystment media for 3 days and the number of mature cysts scored. The formation of mature cysts was almost inhibited in AcAtg3-siRNA transfected cells.

    Article Snippet: Transient transfection was performed by Superfect transfection reagent (Qiagen, Valencia, California, USA) as previously described [ ].

    Techniques: Inhibition, Transfection, Incubation

    Transfection of pUbAcAtg3g for the intracellular localization of Atg3. Expressed Atg3-EGFP showed dispersed fluorescence in the cytoplasm in the trophozoites (encystation-0 hr) and the early stage of encystation (encystation-20 hr). After incubation for 40 hr in encystment media, AcAtg3-EGFP localized around autophagosomal membrane (encystation-40 hr).

    Journal: The Korean Journal of Parasitology

    Article Title: Atg3-Mediated Lipidation of Atg8 Is Involved in Encystation of Acanthamoeba

    doi: 10.3347/kjp.2011.49.2.103

    Figure Lengend Snippet: Transfection of pUbAcAtg3g for the intracellular localization of Atg3. Expressed Atg3-EGFP showed dispersed fluorescence in the cytoplasm in the trophozoites (encystation-0 hr) and the early stage of encystation (encystation-20 hr). After incubation for 40 hr in encystment media, AcAtg3-EGFP localized around autophagosomal membrane (encystation-40 hr).

    Article Snippet: Transient transfection was performed by Superfect transfection reagent (Qiagen, Valencia, California, USA) as previously described [ ].

    Techniques: Transfection, Fluorescence, Incubation

    Inhibition of Atg8-PE conjugation by AcAtg3-siRNA. The formation of the Atg8-PE conjugate was inhibited by AcAtg3-siRNA transfection. Lane 1; trophozoite, lane 2; encysting cells for 1 day, lane 3; encysting cells for 2 days, lane 4; encysting cells for 1 day after transfection with AcAtg3-siRNA, and lane 5; encysting cells for 2 days after transfection with AcAtg3-siRNA.

    Journal: The Korean Journal of Parasitology

    Article Title: Atg3-Mediated Lipidation of Atg8 Is Involved in Encystation of Acanthamoeba

    doi: 10.3347/kjp.2011.49.2.103

    Figure Lengend Snippet: Inhibition of Atg8-PE conjugation by AcAtg3-siRNA. The formation of the Atg8-PE conjugate was inhibited by AcAtg3-siRNA transfection. Lane 1; trophozoite, lane 2; encysting cells for 1 day, lane 3; encysting cells for 2 days, lane 4; encysting cells for 1 day after transfection with AcAtg3-siRNA, and lane 5; encysting cells for 2 days after transfection with AcAtg3-siRNA.

    Article Snippet: Transient transfection was performed by Superfect transfection reagent (Qiagen, Valencia, California, USA) as previously described [ ].

    Techniques: Inhibition, Conjugation Assay, Transfection

    TAp63 α and TAp73 α can activate the TIGAR-hBS2 reporter. ( a ) Representative iRFP reporter assay scan of HCT116 p53 −/− cells 24 h after co-transfection with TIGAR-hBS2 iRFP reporter along with human p53, HA-tagged TAp63 α or HA-tagged TAp73 α . ( b ) Western blot analysis of HCT116 p53 −/− cells with transfected p53, HA-tagged TAp63 α or HA-tagged TAp73 α . ( c ) Quantification of iRFP reporter scans. ( d ) Representative iRFP reporter assay scan of HCT116 p53 −/− cells 24 h after co-transfection with TIGAR-hBS2 iRFP reporter along with TAp73 α , TAp73 β , TAp73 γ or ΔNp73 α . ( e ) Western blot analysis of HCT116 p53 −/− cells with transfected HA-tagged TAp73 α , HA-tagged TAp73 β , HA-tagged TAp73 γ or HA-tagged ΔNp73 α . ( f ) Quantification of iRFP reporter scans on human (hBS2) and mouse (mBS1 and mBS2) TIGAR promoter-binding sites. Values represent mean±S.E.M. of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: p53- and p73-independent activation of TIGAR expression in vivo

    doi: 10.1038/cddis.2015.205

    Figure Lengend Snippet: TAp63 α and TAp73 α can activate the TIGAR-hBS2 reporter. ( a ) Representative iRFP reporter assay scan of HCT116 p53 −/− cells 24 h after co-transfection with TIGAR-hBS2 iRFP reporter along with human p53, HA-tagged TAp63 α or HA-tagged TAp73 α . ( b ) Western blot analysis of HCT116 p53 −/− cells with transfected p53, HA-tagged TAp63 α or HA-tagged TAp73 α . ( c ) Quantification of iRFP reporter scans. ( d ) Representative iRFP reporter assay scan of HCT116 p53 −/− cells 24 h after co-transfection with TIGAR-hBS2 iRFP reporter along with TAp73 α , TAp73 β , TAp73 γ or ΔNp73 α . ( e ) Western blot analysis of HCT116 p53 −/− cells with transfected HA-tagged TAp73 α , HA-tagged TAp73 β , HA-tagged TAp73 γ or HA-tagged ΔNp73 α . ( f ) Quantification of iRFP reporter scans on human (hBS2) and mouse (mBS1 and mBS2) TIGAR promoter-binding sites. Values represent mean±S.E.M. of three independent experiments. * P

    Article Snippet: Transient transfections and irfp reporter assays Cells were seeded on 6-well plates for protein expression analysis or 96-well CellBIND clear bottom black microplates (Corning, Corning, NY, USA) for iRFP reporter assays and grown overnight prior to being transfected using GeneJuice (Merck Millipore) according to the manufacturer's manual.

    Techniques: Reporter Assay, Cotransfection, Western Blot, Transfection, Binding Assay

    Comparison of human and mouse p53-binding sites on the TIGAR promoter. ( a ) Possible p53-binding sites along human and mouse TIGAR . ( b ) Representative iRFP reporter assay scan of HCT116 p53 −/− cells 24 h after co-transfection with TIGAR-hBS2 or TIGAR-mBS1 iRFP reporter and increasing amounts of human p53 or mouse p53. ( c ) Western blot analysis of HCT116 p53 −/− cells transfected with increasing amounts of human p53 or mouse p53. ( d ) Quantification of iRFP reporter scans on human (hBS1 and hBS2) and mouse (mBS1 and mBS2) TIGAR promoter-binding sites with increasing levels of human or mouse p53. ( e ) Chromatin-immunoprecipitation (ChIP) was performed for p53 with quantitative PCR for mBS1 (−2062 bp), mBS2 (+263 bp), a p53 response element on the p21 promoter (−2400 bp) and non-specific (N/S) binding regions on the Tigar (−992 bp) and p21 promoter (−50 bp), using 3T3s treated with 50 μ M cisplatin for 24 h. Values represent mean±S.E.M. of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: p53- and p73-independent activation of TIGAR expression in vivo

    doi: 10.1038/cddis.2015.205

    Figure Lengend Snippet: Comparison of human and mouse p53-binding sites on the TIGAR promoter. ( a ) Possible p53-binding sites along human and mouse TIGAR . ( b ) Representative iRFP reporter assay scan of HCT116 p53 −/− cells 24 h after co-transfection with TIGAR-hBS2 or TIGAR-mBS1 iRFP reporter and increasing amounts of human p53 or mouse p53. ( c ) Western blot analysis of HCT116 p53 −/− cells transfected with increasing amounts of human p53 or mouse p53. ( d ) Quantification of iRFP reporter scans on human (hBS1 and hBS2) and mouse (mBS1 and mBS2) TIGAR promoter-binding sites with increasing levels of human or mouse p53. ( e ) Chromatin-immunoprecipitation (ChIP) was performed for p53 with quantitative PCR for mBS1 (−2062 bp), mBS2 (+263 bp), a p53 response element on the p21 promoter (−2400 bp) and non-specific (N/S) binding regions on the Tigar (−992 bp) and p21 promoter (−50 bp), using 3T3s treated with 50 μ M cisplatin for 24 h. Values represent mean±S.E.M. of three independent experiments. * P

    Article Snippet: Transient transfections and irfp reporter assays Cells were seeded on 6-well plates for protein expression analysis or 96-well CellBIND clear bottom black microplates (Corning, Corning, NY, USA) for iRFP reporter assays and grown overnight prior to being transfected using GeneJuice (Merck Millipore) according to the manufacturer's manual.

    Techniques: Binding Assay, Reporter Assay, Cotransfection, Western Blot, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Stimulation-induced processing and degradation of p105 is not affected by mutating lysine residues 18 (683) to 30 (967) in the C-terminal domain of the molecule. (A) COS-7 cells were transiently transfected with either a cDNA coding for p105-WT (lanes 2 to 5) or p105-K18-30R (lanes 6 to 9) as indicated. Control cells (lane 1) were transfected with an empty vector. Where indicated, a cDNA coding for constitutively active IKKβ was cotransfected. Twenty-four hours after transfection cells were harvested, and nuclear and cytosolic fractions were isolated as described in Materials and Methods. Aliquots of cytosolic and nuclear extracts representing an equal number of cells were resolved via SDS-10% PAGE and were blotted onto nitrocellulose paper, and proteins were visualized by using anti-p50 antibody and ECL as described in Materials and Methods. C, cytosolic fraction; N, nuclear fraction. (B) COS-7 cells were transiently transfected with a cDNA coding for either p105-WT (lanes 1 to 4), p105-K18-30R (lanes 5 to 8), or p105-Δ917-932 (lanes 9 to 12). Where indicated, cDNA coding for a constitutively active IKKβ was cotransfected. Twenty-four hours after transfection cells were pulse labeled with [ 35 S]methionine (0; pulse). Following removal and dilution of the labeled amino acid and further incubation for 2 h (2; chase), the labeled proteins were immunoprecipitated by using anti-p50 antibody, resolved by SDS-10% PAGE, and visualized by phosphorimaging as described in Materials and Methods.

    Journal: Molecular and Cellular Biology

    Article Title: Dual Effects of I?B Kinase ?-Mediated Phosphorylation on p105 Fate: SCFβ-TrCP-Dependent Degradation and SCFβ-TrCP-Independent Processing

    doi: 10.1128/MCB.24.1.475-486.2004

    Figure Lengend Snippet: Stimulation-induced processing and degradation of p105 is not affected by mutating lysine residues 18 (683) to 30 (967) in the C-terminal domain of the molecule. (A) COS-7 cells were transiently transfected with either a cDNA coding for p105-WT (lanes 2 to 5) or p105-K18-30R (lanes 6 to 9) as indicated. Control cells (lane 1) were transfected with an empty vector. Where indicated, a cDNA coding for constitutively active IKKβ was cotransfected. Twenty-four hours after transfection cells were harvested, and nuclear and cytosolic fractions were isolated as described in Materials and Methods. Aliquots of cytosolic and nuclear extracts representing an equal number of cells were resolved via SDS-10% PAGE and were blotted onto nitrocellulose paper, and proteins were visualized by using anti-p50 antibody and ECL as described in Materials and Methods. C, cytosolic fraction; N, nuclear fraction. (B) COS-7 cells were transiently transfected with a cDNA coding for either p105-WT (lanes 1 to 4), p105-K18-30R (lanes 5 to 8), or p105-Δ917-932 (lanes 9 to 12). Where indicated, cDNA coding for a constitutively active IKKβ was cotransfected. Twenty-four hours after transfection cells were pulse labeled with [ 35 S]methionine (0; pulse). Following removal and dilution of the labeled amino acid and further incubation for 2 h (2; chase), the labeled proteins were immunoprecipitated by using anti-p50 antibody, resolved by SDS-10% PAGE, and visualized by phosphorimaging as described in Materials and Methods.

    Article Snippet: The human p105-WT, p105-Δ917-933 (Fig. , respectively), and p105-TthIII 1 (p105 to 544) (the TthIII site is shown on the p105-WT scheme in Fig. ) cDNA constructs used for in vitro translation (in pT7β105) and for transient transfection (in pCI-neo; Promega) in cells were described previously ( - ) and served as a base for further manipulations and expression.

    Techniques: Transfection, Plasmid Preparation, Isolation, Polyacrylamide Gel Electrophoresis, Labeling, Incubation, Immunoprecipitation

    POTE over-expression induces apoptosis in Hela cells. a Hela cells transfected with pEGFP, pPOTE-2 α -actin-EGFP or pPOTE-2 γ C-EGFP. At 2 or 3 days post-transfection, cells were incubated with RED-VAD-FMK for 1 h before fixation with 2% PFA. Cells were visualized with confocal microscopy. b Quantitative analysis of POTE induced apoptosis. Transfected cells were stained with RED-VAD-FMK for 1 h and red-staining cells and total transfected cells were counted under microscopy. Three separate experiments were repeated and each time about 100 transfected cells were counted

    Journal: Apoptosis : an international journal on programmed cell death

    Article Title: A primate-specific POTE-actin fusion protein plays a role in apoptosis

    doi: 10.1007/s10495-009-0392-0

    Figure Lengend Snippet: POTE over-expression induces apoptosis in Hela cells. a Hela cells transfected with pEGFP, pPOTE-2 α -actin-EGFP or pPOTE-2 γ C-EGFP. At 2 or 3 days post-transfection, cells were incubated with RED-VAD-FMK for 1 h before fixation with 2% PFA. Cells were visualized with confocal microscopy. b Quantitative analysis of POTE induced apoptosis. Transfected cells were stained with RED-VAD-FMK for 1 h and red-staining cells and total transfected cells were counted under microscopy. Three separate experiments were repeated and each time about 100 transfected cells were counted

    Article Snippet: Cell culture and transient transfection Hela cells was purchased from ATCC (Manassas, VA).

    Techniques: Over Expression, Transfection, Incubation, Confocal Microscopy, Staining, Microscopy

    POTE-2 α -actin and POTE-2 γ C are associated with actin protein in Hela cells. a Hela cells were transfected with pPOTE-2 α -actin-EGFP, pPOTE-2 γ C-EGFP or pEGFP vector. After 24 h of transfection, cells were fixed with 4% PFA and stained with phalloidin-TRITC ( red ) and DAPI ( blue ). Yellow shows merged image of co-localization of endogenous actin and POTE-2 α -actin-EGFP or POTE-2 γ C-EGFP. b POTE localization is dependent on intact actin filaments. Hela cells transfected with POTE-2 α -actin-EGFP were treated Lat A for 30 min before PFA fixation. Cells were then stained with phalloidin-TRITC and DAPI for 5 min and analyzed by confocal microscopy. c Hela cells were lysed with hypotonic buffer as described in “Experimental procedures”. Equivalent volume of soluble and membrane fractions were analyzed by western blot with anti-POTE (PG5), anti-Fas (membrane marker), anti-actin or anti-GAPDH (soluble fraction marker) antibodies

    Journal: Apoptosis : an international journal on programmed cell death

    Article Title: A primate-specific POTE-actin fusion protein plays a role in apoptosis

    doi: 10.1007/s10495-009-0392-0

    Figure Lengend Snippet: POTE-2 α -actin and POTE-2 γ C are associated with actin protein in Hela cells. a Hela cells were transfected with pPOTE-2 α -actin-EGFP, pPOTE-2 γ C-EGFP or pEGFP vector. After 24 h of transfection, cells were fixed with 4% PFA and stained with phalloidin-TRITC ( red ) and DAPI ( blue ). Yellow shows merged image of co-localization of endogenous actin and POTE-2 α -actin-EGFP or POTE-2 γ C-EGFP. b POTE localization is dependent on intact actin filaments. Hela cells transfected with POTE-2 α -actin-EGFP were treated Lat A for 30 min before PFA fixation. Cells were then stained with phalloidin-TRITC and DAPI for 5 min and analyzed by confocal microscopy. c Hela cells were lysed with hypotonic buffer as described in “Experimental procedures”. Equivalent volume of soluble and membrane fractions were analyzed by western blot with anti-POTE (PG5), anti-Fas (membrane marker), anti-actin or anti-GAPDH (soluble fraction marker) antibodies

    Article Snippet: Cell culture and transient transfection Hela cells was purchased from ATCC (Manassas, VA).

    Techniques: Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Western Blot, Marker

    USP7 depletion results in stabilization of cyclin B (cycB) in a p53-independent manner. Control or USP7 stably depleted HEp2 cell extracts were generated from cells arrested in Taxol ( a ) or Nocodazole ( b ) collected by mitotic shake-off. Extracts were supplemented with non-destructible cycB fragment, an energy-regenerating system, and UbcH10 where indicated. Endogenous cycB was monitored over time. Right panels: Relative quantization of cycB protein levels in USP7-depleted cell extracts using actin as internal control and normalized over cycB/actin protein levels in control shRNA extracts. CycB is stabilized in USP7-depleted cell extracts. Western blot analysis of cycB stability in HEp2 ( c ) or H1299 ( d ) cells synchronized by DTB and simultaneously transfected with either control or USP7 siRNAs. Samples were taken 72 h post-transfection at 0, 7, 9 and 11 h after DTB release to allow cells to progress through mitosis. Right panels: Relative quantification of cycB protein levels using actin as internal control for each time point. Data are normalized over cycB/actin protein levels in control siRNA-transfected cells. Histone H3 phosphorylated at serine 10 is shown as control for mitotic progression. CycB is stabilized in USP7-depleted cells in a p53-independent manner. Data show representative experiments out of three

    Journal: Cell Death and Differentiation

    Article Title: USP7 and Daxx regulate mitosis progression and taxane sensitivity by affecting stability of Aurora-A kinase

    doi: 10.1038/cdd.2012.169

    Figure Lengend Snippet: USP7 depletion results in stabilization of cyclin B (cycB) in a p53-independent manner. Control or USP7 stably depleted HEp2 cell extracts were generated from cells arrested in Taxol ( a ) or Nocodazole ( b ) collected by mitotic shake-off. Extracts were supplemented with non-destructible cycB fragment, an energy-regenerating system, and UbcH10 where indicated. Endogenous cycB was monitored over time. Right panels: Relative quantization of cycB protein levels in USP7-depleted cell extracts using actin as internal control and normalized over cycB/actin protein levels in control shRNA extracts. CycB is stabilized in USP7-depleted cell extracts. Western blot analysis of cycB stability in HEp2 ( c ) or H1299 ( d ) cells synchronized by DTB and simultaneously transfected with either control or USP7 siRNAs. Samples were taken 72 h post-transfection at 0, 7, 9 and 11 h after DTB release to allow cells to progress through mitosis. Right panels: Relative quantification of cycB protein levels using actin as internal control for each time point. Data are normalized over cycB/actin protein levels in control siRNA-transfected cells. Histone H3 phosphorylated at serine 10 is shown as control for mitotic progression. CycB is stabilized in USP7-depleted cells in a p53-independent manner. Data show representative experiments out of three

    Article Snippet: For transient siRNA transfections (Dharmacon, Thermo Fisher Scientific, Waltham, MA, USA) were used according the manufacturer's instructions.

    Techniques: Stable Transfection, Generated, shRNA, Western Blot, Transfection

    miRNA profiling of p63-depleted HaCaT cells. siRNA-mediated knockdown of p63 was assessed at the mRNA level ( A ) and protein level ( B ) 48 hours post-transfection. The total miRNA extracted (three independent siP63 transfection and three independent miRNA extraction) was processed for miRNA profiling. Error bars represent the s.d. of triplicate experiments. *, p

    Journal: PLoS ONE

    Article Title: The miR-17 Family Links p63 Protein to MAPK Signaling to Promote the Onset of Human Keratinocyte Differentiation

    doi: 10.1371/journal.pone.0045761

    Figure Lengend Snippet: miRNA profiling of p63-depleted HaCaT cells. siRNA-mediated knockdown of p63 was assessed at the mRNA level ( A ) and protein level ( B ) 48 hours post-transfection. The total miRNA extracted (three independent siP63 transfection and three independent miRNA extraction) was processed for miRNA profiling. Error bars represent the s.d. of triplicate experiments. *, p

    Article Snippet: Transient Transfection and RNA Expression Analysis Transient siRNA and locked nucleic acid (LNA) transfections in HaCaT cells were performed with INTERFERin according to the manufacturer’s instructions (PolyPlus Transfection).

    Techniques: Transfection

    Effect of bovine ADA binding on A 2A R. (A) Competition experiments of 2.2 nM [ 3 H]ZM 241385 binding in the presence of increasing concentrations of unlabeled ZM 241385, in the absence (○) or in the presence (●) of 1 μg/ml of ADA were performed using membranes from HEK-293T cells transfected with A 2A R-Nluc-spacer cDNA (1.5 μg). (B) Dose-response effect of ADA on 2.2 nM [ 3 H]ZM 241385 binding to membranes from HEK-293T cells transfected with cDNA (1.5 μg) corresponding to A 2A R-Nluc-spacer (■) or A 2A R (□). Data are mean ± SD from a representative experiment ( n = 3) performed in triplicate. (C) DMR assays were performed in HEK-293T cells transfected with A 2A R-Nluc-spacer cDNA (1.5 μg). Cells were stimulated with increasing CGS 21680 concentrations in the presence (white columns) or in the absence (black columns) of ADA (1 μg/ml). Values are mean ± SEM ( n = 4) and are expressed as shift at 3000 s of reflected light wavelength (pm) over basal obtained from the corresponding DMR curves. Statistical significance was calculated by one-way ANOVA followed by a Bonferroni multiple comparison post hoc test; ∗∗ p

    Journal: Frontiers in Pharmacology

    Article Title: Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

    doi: 10.3389/fphar.2018.00106

    Figure Lengend Snippet: Effect of bovine ADA binding on A 2A R. (A) Competition experiments of 2.2 nM [ 3 H]ZM 241385 binding in the presence of increasing concentrations of unlabeled ZM 241385, in the absence (○) or in the presence (●) of 1 μg/ml of ADA were performed using membranes from HEK-293T cells transfected with A 2A R-Nluc-spacer cDNA (1.5 μg). (B) Dose-response effect of ADA on 2.2 nM [ 3 H]ZM 241385 binding to membranes from HEK-293T cells transfected with cDNA (1.5 μg) corresponding to A 2A R-Nluc-spacer (■) or A 2A R (□). Data are mean ± SD from a representative experiment ( n = 3) performed in triplicate. (C) DMR assays were performed in HEK-293T cells transfected with A 2A R-Nluc-spacer cDNA (1.5 μg). Cells were stimulated with increasing CGS 21680 concentrations in the presence (white columns) or in the absence (black columns) of ADA (1 μg/ml). Values are mean ± SEM ( n = 4) and are expressed as shift at 3000 s of reflected light wavelength (pm) over basal obtained from the corresponding DMR curves. Statistical significance was calculated by one-way ANOVA followed by a Bonferroni multiple comparison post hoc test; ∗∗ p

    Article Snippet: Cell Culture and Transient Transfection Human embryonic kidney (HEK-293T) cells obtained from ATCC were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 100 μg/ml sodium pyruvate, 2 mM L -glutamine, 100 U/ml penicillin/streptomycin, essential medium non-essential amino acids solution (1/100) and 5% (v/v) heat inactivated fetal bovine serum (all from Invitrogen, Paisley, United Kingdom) and were maintained at 37°C in an atmosphere with 5% CO2 .

    Techniques: Binding Assay, Transfection

    Functional characterization of A 2A R fusion proteins. (A–C) DMR assays were performed in HEK-293T cells transfected with cDNA (1.5 μg) corresponding to A 2A R-Nluc (A) or A 2A R-Nluc-spacer (B) both fused on the N-terminal end or A 2A R-Rluc fused on the C-terminal end (C) . Cells were stimulated with vehicle (basal) or with increasing concentrations of the A 2A R agonist CGS 21680. The resulting shifts of reflected light wavelength (pm) were monitored over time. Each panel is a representative experiment of n = 3 different experiments. Each curve is the mean of a representative optical trace experiment carried out in quadruplicates. (D,E) ERK1/2 phosphorylation (D) and cAMP production (E) were determined in cells transfected with the cDNA (1.5 μg) corresponding to A 2A R, A 2A R-Rluc fused on the C-terminal end or A 2A R-Nluc-spacer fused on the N-terminal end. Cells were stimulated with 100 nM CGS 21680 for 10 min. Results are given as percentage respect cells expressing only A 2A R. Values are expressed as means ± SEM ( n = 4). (D) A representative western blot is shown at the top of the panel and in (E) 100% represents 80–100 pmols of cAMP/10 6 cells.

    Journal: Frontiers in Pharmacology

    Article Title: Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

    doi: 10.3389/fphar.2018.00106

    Figure Lengend Snippet: Functional characterization of A 2A R fusion proteins. (A–C) DMR assays were performed in HEK-293T cells transfected with cDNA (1.5 μg) corresponding to A 2A R-Nluc (A) or A 2A R-Nluc-spacer (B) both fused on the N-terminal end or A 2A R-Rluc fused on the C-terminal end (C) . Cells were stimulated with vehicle (basal) or with increasing concentrations of the A 2A R agonist CGS 21680. The resulting shifts of reflected light wavelength (pm) were monitored over time. Each panel is a representative experiment of n = 3 different experiments. Each curve is the mean of a representative optical trace experiment carried out in quadruplicates. (D,E) ERK1/2 phosphorylation (D) and cAMP production (E) were determined in cells transfected with the cDNA (1.5 μg) corresponding to A 2A R, A 2A R-Rluc fused on the C-terminal end or A 2A R-Nluc-spacer fused on the N-terminal end. Cells were stimulated with 100 nM CGS 21680 for 10 min. Results are given as percentage respect cells expressing only A 2A R. Values are expressed as means ± SEM ( n = 4). (D) A representative western blot is shown at the top of the panel and in (E) 100% represents 80–100 pmols of cAMP/10 6 cells.

    Article Snippet: Cell Culture and Transient Transfection Human embryonic kidney (HEK-293T) cells obtained from ATCC were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 100 μg/ml sodium pyruvate, 2 mM L -glutamine, 100 U/ml penicillin/streptomycin, essential medium non-essential amino acids solution (1/100) and 5% (v/v) heat inactivated fetal bovine serum (all from Invitrogen, Paisley, United Kingdom) and were maintained at 37°C in an atmosphere with 5% CO2 .

    Techniques: Functional Assay, Transfection, Expressing, Western Blot

    NanoBRET between A 2A R-Nluc and CD26-YFP expressed in different cells. HEK-293T cells transfected with 1.5 μg of A 2A R-Nluc-spacer cDNA (A–D) or NMDAR1A-Nluc (C) were mixed with HEK-293T cells transfected with 2 μg of CD26-YFP cDNA (A–D) or 2 μg of NMDAR1A-YFP cDNA (C) . Cells were incubated 10 min without shaking with HBSS in the absence or in the presence of increasing concentrations of ADA (from 0.01 to 10 μg/ml) (A) , in the presence or in the absence of 1 μg/ml of bovine ADA (B,C) , bovine albumin (1 μg/ml) (B) , human-specific mAb against CD26, TA5.9-CC1-4C8 (0.3 μg/ml) (B) or in the presence or in the absence of human wild-type ADA, Leu58Ala mutant ADA or Leu62Ala mutant ADA, all at 1 μg/ml (D) , previously to BRET detection. Both fluorescence and luminescence of each sample were measured before every experiment to confirm similar donor expressions (approximately 120.000 bioluminescence units) and similar acceptor expression (25.000 fluorescence units). BRET is expressed as milliBRET units (mBU = net BRET × 1000) and is means ± SEM of 3–4 different experiments grouped as a function of the amount of BRET acceptor. Statistical significance was calculated by one way ANOVA followed by a Dunnett’s multiple comparison post hoc test; ∗∗∗ p

    Journal: Frontiers in Pharmacology

    Article Title: Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

    doi: 10.3389/fphar.2018.00106

    Figure Lengend Snippet: NanoBRET between A 2A R-Nluc and CD26-YFP expressed in different cells. HEK-293T cells transfected with 1.5 μg of A 2A R-Nluc-spacer cDNA (A–D) or NMDAR1A-Nluc (C) were mixed with HEK-293T cells transfected with 2 μg of CD26-YFP cDNA (A–D) or 2 μg of NMDAR1A-YFP cDNA (C) . Cells were incubated 10 min without shaking with HBSS in the absence or in the presence of increasing concentrations of ADA (from 0.01 to 10 μg/ml) (A) , in the presence or in the absence of 1 μg/ml of bovine ADA (B,C) , bovine albumin (1 μg/ml) (B) , human-specific mAb against CD26, TA5.9-CC1-4C8 (0.3 μg/ml) (B) or in the presence or in the absence of human wild-type ADA, Leu58Ala mutant ADA or Leu62Ala mutant ADA, all at 1 μg/ml (D) , previously to BRET detection. Both fluorescence and luminescence of each sample were measured before every experiment to confirm similar donor expressions (approximately 120.000 bioluminescence units) and similar acceptor expression (25.000 fluorescence units). BRET is expressed as milliBRET units (mBU = net BRET × 1000) and is means ± SEM of 3–4 different experiments grouped as a function of the amount of BRET acceptor. Statistical significance was calculated by one way ANOVA followed by a Dunnett’s multiple comparison post hoc test; ∗∗∗ p

    Article Snippet: Cell Culture and Transient Transfection Human embryonic kidney (HEK-293T) cells obtained from ATCC were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 100 μg/ml sodium pyruvate, 2 mM L -glutamine, 100 U/ml penicillin/streptomycin, essential medium non-essential amino acids solution (1/100) and 5% (v/v) heat inactivated fetal bovine serum (all from Invitrogen, Paisley, United Kingdom) and were maintained at 37°C in an atmosphere with 5% CO2 .

    Techniques: Transfection, Incubation, Mutagenesis, Bioluminescence Resonance Energy Transfer, Fluorescence, Expressing

    Expression of A 2A R and CD26 fusion proteins. (A) Bioluminescence assays were performed in HEK-293T cells transfected with increasing concentrations of fusion protein cDNA corresponding to A 2A R (red), A 2A R-Rluc fused on the C-terminal end (blue), A 2A R-Nluc fused on the N-terminal (green) or A 2A R-Nluc-spacer fused on the N-terminal end (black) on the N-terminal end. Results are given in relative bioluminescence units by subtracting the value of non-transfected cells and represent mean ± SEM ( n = 6). Statistical significance was calculated by one-way ANOVA followed by a Bonferroni multiple comparison post hoc test; ∗∗∗ p

    Journal: Frontiers in Pharmacology

    Article Title: Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

    doi: 10.3389/fphar.2018.00106

    Figure Lengend Snippet: Expression of A 2A R and CD26 fusion proteins. (A) Bioluminescence assays were performed in HEK-293T cells transfected with increasing concentrations of fusion protein cDNA corresponding to A 2A R (red), A 2A R-Rluc fused on the C-terminal end (blue), A 2A R-Nluc fused on the N-terminal (green) or A 2A R-Nluc-spacer fused on the N-terminal end (black) on the N-terminal end. Results are given in relative bioluminescence units by subtracting the value of non-transfected cells and represent mean ± SEM ( n = 6). Statistical significance was calculated by one-way ANOVA followed by a Bonferroni multiple comparison post hoc test; ∗∗∗ p

    Article Snippet: Cell Culture and Transient Transfection Human embryonic kidney (HEK-293T) cells obtained from ATCC were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 100 μg/ml sodium pyruvate, 2 mM L -glutamine, 100 U/ml penicillin/streptomycin, essential medium non-essential amino acids solution (1/100) and 5% (v/v) heat inactivated fetal bovine serum (all from Invitrogen, Paisley, United Kingdom) and were maintained at 37°C in an atmosphere with 5% CO2 .

    Techniques: Expressing, Transfection