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  • 99
    Qiagen attractene transfection reagent
    Evaluation of <t>transfection</t> efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes. (a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from <t>Attractene</t> treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P
    Attractene Transfection Reagent, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 4661 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/attractene transfection reagent/product/Qiagen
    Average 99 stars, based on 4661 article reviews
    Price from $9.99 to $1999.99
    attractene transfection reagent - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    Promega attractene transfection reagent
    Evaluation of <t>transfection</t> efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes. (a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from <t>Attractene</t> treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P
    Attractene Transfection Reagent, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/attractene transfection reagent/product/Promega
    Average 93 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    attractene transfection reagent - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    91
    Thermo Fisher attractene
    Evaluation of <t>transfection</t> efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes. (a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from <t>Attractene</t> treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P
    Attractene, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/attractene/product/Thermo Fisher
    Average 91 stars, based on 58 article reviews
    Price from $9.99 to $1999.99
    attractene - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    93
    Thermo Fisher attractene transfection reagent kit
    Evaluation of <t>transfection</t> efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes. (a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from <t>Attractene</t> treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P
    Attractene Transfection Reagent Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/attractene transfection reagent kit/product/Thermo Fisher
    Average 93 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    attractene transfection reagent kit - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    92
    Promega attractene
    Evaluation of <t>transfection</t> efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes. (a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from <t>Attractene</t> treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P
    Attractene, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/attractene/product/Promega
    Average 92 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    attractene - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    Evaluation of transfection efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes. (a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from Attractene treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P

    Journal: PLoS ONE

    Article Title: Prenyl Ammonium Salts – New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model

    doi: 10.1371/journal.pone.0153633

    Figure Lengend Snippet: Evaluation of transfection efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes. (a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from Attractene treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P

    Article Snippet: Control experiments with Attractene Transfection Reagent (Qiagen) and Lipofectamine3000 Transfection Reagent (Life Technologies) were conducted according to the manufacturer’s recommendations.

    Techniques: Transfection, FACS, Activity Assay

    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: Cell Treatments and Fractionation D4476 (Calbiochem) was transfected using Attractene (Qiagen) into A375 cells as recommended by the supplier.

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

    Treatment of immune cells with filtered and unfiltered Ebola-transfected cell supernatants. 293T cells were transfected with either VP40, GP, or NP plasmids with Attractene Transfection Reagent (Qiagen) as per the manufacturer’s instructions and incubated for 2 days. Transfection complexes were removed and cells were treated with antibiotics for plasmid selection for 14 days. Transfection supernatants were either left unfiltered or passed through a 0.22 micron filter. Control 293T cells were not transfected nor treated with antibiotics (Hygromycin B, Zeocin, or Geneticin). Seventy-five microliters (0.45 mU AChE) of both filtered and unfiltered supernatants were used to treat CEM (A) , Jurkat (B) and U937 (C) cells, which were subsequently assayed for cell viability via CellTiter-Glo after 4 days of incubation. Statistical significance was determined using 2-tailed student’s t -test, with filtered or unfiltered experimental values compared to the Control values of like type. (D) U937 cells were treated with PMA (50 nM) for 5 days to stimulate differentiation into macrophages. On day 5 media was replaced and differentiated U937 macrophages were treated with either E. coli -purified free VP40 protein, or supernatants from VP40-transfected 293T cells (filtered and unfiltered). Untreated macrophages received no external supernatant treatment. Cells were incubated for an additional 5 days and cell viability was assayed with CellTiter-Glo. (E) Primary monocytes from 3 healthy donors were treated with PMA (10 nM) for 2 days to stimulate differentiation into macrophages. Primary macrophages were then treated with supernatants from either VP40-transfected 293T cells (filtered and unfiltered), or from VP40-resistant clone EVTR2C cells (filtered). Control primary macrophages were treated with filtered supernatant from normal 293T cells. Cells were incubated for 2 days before being assayed for viability with CellTiter-Glo. Statistical significance was determined using 2-tailed student’s t -test, with filtered or unfiltered experimental groups compared to the Control groups of like type. MØ, Macrophage; RLU, Relative Luminescent Units. ∗ p

    Journal: Frontiers in Microbiology

    Article Title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

    doi: 10.3389/fmicb.2016.01765

    Figure Lengend Snippet: Treatment of immune cells with filtered and unfiltered Ebola-transfected cell supernatants. 293T cells were transfected with either VP40, GP, or NP plasmids with Attractene Transfection Reagent (Qiagen) as per the manufacturer’s instructions and incubated for 2 days. Transfection complexes were removed and cells were treated with antibiotics for plasmid selection for 14 days. Transfection supernatants were either left unfiltered or passed through a 0.22 micron filter. Control 293T cells were not transfected nor treated with antibiotics (Hygromycin B, Zeocin, or Geneticin). Seventy-five microliters (0.45 mU AChE) of both filtered and unfiltered supernatants were used to treat CEM (A) , Jurkat (B) and U937 (C) cells, which were subsequently assayed for cell viability via CellTiter-Glo after 4 days of incubation. Statistical significance was determined using 2-tailed student’s t -test, with filtered or unfiltered experimental values compared to the Control values of like type. (D) U937 cells were treated with PMA (50 nM) for 5 days to stimulate differentiation into macrophages. On day 5 media was replaced and differentiated U937 macrophages were treated with either E. coli -purified free VP40 protein, or supernatants from VP40-transfected 293T cells (filtered and unfiltered). Untreated macrophages received no external supernatant treatment. Cells were incubated for an additional 5 days and cell viability was assayed with CellTiter-Glo. (E) Primary monocytes from 3 healthy donors were treated with PMA (10 nM) for 2 days to stimulate differentiation into macrophages. Primary macrophages were then treated with supernatants from either VP40-transfected 293T cells (filtered and unfiltered), or from VP40-resistant clone EVTR2C cells (filtered). Control primary macrophages were treated with filtered supernatant from normal 293T cells. Cells were incubated for 2 days before being assayed for viability with CellTiter-Glo. Statistical significance was determined using 2-tailed student’s t -test, with filtered or unfiltered experimental groups compared to the Control groups of like type. MØ, Macrophage; RLU, Relative Luminescent Units. ∗ p

    Article Snippet: Twenty microgram of Escherichia coli -purified DNA was transfected into 293T cells using either Attractene reagent (Qiagen, Chatsworth, CA, USA) according to the manufacturer’s instructions, or by electroporation as previously described ( ).

    Techniques: Transfection, Incubation, Plasmid Preparation, Selection, Purification

    ( a ) Exogenous stimulation of S. gordonii lipoproteins augment TLR2 mRNA expressions in hDPCs, and were maintained after calcium hydroxide (CH) or HBD3-C15 treatment. ( b ) S. gordonii lipoprotein-stimulated NF-κB are mediated by TLR2. HEK-TLR2 cells (2.5 × 10 5 cells/mL) were transfected with an NF-κB luciferase reporter plasmid using Attractene transfection reagent. After 16 h, the cells were stimulated with lipoprotein purified from S. gordonii , and then treated with either of CH or HBD3-C15. After the cells were lysed, a luciferease assay was conducted.

    Journal: International Journal of Molecular Sciences

    Article Title: Synthetic Human β Defensin-3-C15 Peptide in Endodontics: Potential Therapeutic Agent in Streptococcus gordonii Lipoprotein-Stimulated Human Dental Pulp-Derived Cells

    doi: 10.3390/ijms21010071

    Figure Lengend Snippet: ( a ) Exogenous stimulation of S. gordonii lipoproteins augment TLR2 mRNA expressions in hDPCs, and were maintained after calcium hydroxide (CH) or HBD3-C15 treatment. ( b ) S. gordonii lipoprotein-stimulated NF-κB are mediated by TLR2. HEK-TLR2 cells (2.5 × 10 5 cells/mL) were transfected with an NF-κB luciferase reporter plasmid using Attractene transfection reagent. After 16 h, the cells were stimulated with lipoprotein purified from S. gordonii , and then treated with either of CH or HBD3-C15. After the cells were lysed, a luciferease assay was conducted.

    Article Snippet: Transfections were performed in Opti-MEM using Attractene transfection reagent for 16 h (Qiagen, Germantown, MD, USA).

    Techniques: Transfection, Luciferase, Plasmid Preparation, Purification

    Study of miR-33a-3p-targets by gain and loss of function assays; hMSCs after 24 h of transfection with specific mimic and antimiR were analyzed for the gene expression modulation by qRT-PCR and protein Western blot analysis. ( A ) EMT markers: SNAIL, SLUG, TWIST, TGF-β; ( B ) Slug; ( C ) ALPL. OsteoImage assay was performed to quantify the quantitative in vitro mineralization nodules, and data are shown as RFU (492/520 nm) ( D ) Western blot analysis for Runx-2 ( E ) protein was performed on total cell extract after miR-33a-5p and 3p over-expression or inhibition. Quantitative RT-PCR data are expressed as fold of change (FOI) in gene expression (2 −ΔΔCt ) that occurred in mimic or antimiR vs. scramble groups. Student t test: * p

    Journal: Cells

    Article Title: MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation

    doi: 10.3390/cells8121495

    Figure Lengend Snippet: Study of miR-33a-3p-targets by gain and loss of function assays; hMSCs after 24 h of transfection with specific mimic and antimiR were analyzed for the gene expression modulation by qRT-PCR and protein Western blot analysis. ( A ) EMT markers: SNAIL, SLUG, TWIST, TGF-β; ( B ) Slug; ( C ) ALPL. OsteoImage assay was performed to quantify the quantitative in vitro mineralization nodules, and data are shown as RFU (492/520 nm) ( D ) Western blot analysis for Runx-2 ( E ) protein was performed on total cell extract after miR-33a-5p and 3p over-expression or inhibition. Quantitative RT-PCR data are expressed as fold of change (FOI) in gene expression (2 −ΔΔCt ) that occurred in mimic or antimiR vs. scramble groups. Student t test: * p

    Article Snippet: Cell Transfection For cell transfection, Attractene Transfection Reagent (cat. number 1051531, Qiagen Srl, Milan, Italy) was used following the manufacturer’s indication.

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, In Vitro, Over Expression, Inhibition

    Analysis of Yes-associated protein (YAP) and PDZ-binding motif (TAZ) gene expression on hMSCs after gain and loss function studies. Bioinformatic analysis through TargetScanPrediction Software on miR-33a predicted target. ( A ) Predicted sequences of YAP. qRT-PCR was performed after 24 h of specific transfection. YAP and TAZ expression levels were evaluated on hMSCs after miR-33a-5p over-expression or inhibition ( B ) or after miR-33a-3p over-expression or inhibition ( D ) or after miR-33a-5p over-expression or inhibition on hMSCs maintained in OB ( F ) Western blot analysis for YAP/TAZ ( C – D ) proteins was performed on total cell extract after miR-33a-5p and 3p over-expression or inhibition. Quantitative RT-PCR data are expressed as fold of change (FOI) in gene expression (2 −ΔΔCt ) that occurred in mimic or antimiR vs. scramble groups. Student t test: * p

    Journal: Cells

    Article Title: MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation

    doi: 10.3390/cells8121495

    Figure Lengend Snippet: Analysis of Yes-associated protein (YAP) and PDZ-binding motif (TAZ) gene expression on hMSCs after gain and loss function studies. Bioinformatic analysis through TargetScanPrediction Software on miR-33a predicted target. ( A ) Predicted sequences of YAP. qRT-PCR was performed after 24 h of specific transfection. YAP and TAZ expression levels were evaluated on hMSCs after miR-33a-5p over-expression or inhibition ( B ) or after miR-33a-3p over-expression or inhibition ( D ) or after miR-33a-5p over-expression or inhibition on hMSCs maintained in OB ( F ) Western blot analysis for YAP/TAZ ( C – D ) proteins was performed on total cell extract after miR-33a-5p and 3p over-expression or inhibition. Quantitative RT-PCR data are expressed as fold of change (FOI) in gene expression (2 −ΔΔCt ) that occurred in mimic or antimiR vs. scramble groups. Student t test: * p

    Article Snippet: Cell Transfection For cell transfection, Attractene Transfection Reagent (cat. number 1051531, Qiagen Srl, Milan, Italy) was used following the manufacturer’s indication.

    Techniques: Binding Assay, Expressing, Software, Quantitative RT-PCR, Transfection, Over Expression, Inhibition, Western Blot

    Analysis of gene expression on hMSCs after Gefitinib treatments (36 h) and antimiR-33a-3p transfection (24 h). qRT-PCR was performed after 36 h of treatments: ( A ) miR-33a-3p; ( B ) EGFR-2, EGF, ERK-2, ERK3, YAP, and TAZ. Quantitative RT-PCR data are expressed as fold of change (FOI) in gene expression (2 −ΔΔCt ) that occurred in hMSCs after treatments vs. scramble groups and hMSCs after antimiR-33a-3p transfection and Gefitinib treatments vs. Gefitnib groups. Student t test: * p

    Journal: Cells

    Article Title: MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation

    doi: 10.3390/cells8121495

    Figure Lengend Snippet: Analysis of gene expression on hMSCs after Gefitinib treatments (36 h) and antimiR-33a-3p transfection (24 h). qRT-PCR was performed after 36 h of treatments: ( A ) miR-33a-3p; ( B ) EGFR-2, EGF, ERK-2, ERK3, YAP, and TAZ. Quantitative RT-PCR data are expressed as fold of change (FOI) in gene expression (2 −ΔΔCt ) that occurred in hMSCs after treatments vs. scramble groups and hMSCs after antimiR-33a-3p transfection and Gefitinib treatments vs. Gefitnib groups. Student t test: * p

    Article Snippet: Cell Transfection For cell transfection, Attractene Transfection Reagent (cat. number 1051531, Qiagen Srl, Milan, Italy) was used following the manufacturer’s indication.

    Techniques: Expressing, Transfection, Quantitative RT-PCR

    Analysis of gene expression on hMSCs after EGF treatments and EGFR signaling gene expression investigation on Nh-Ost. qRT-PCR was performed after 24 h of specific transfection on hMSCs. EGFR and ERK3 expression levels were evaluated on hMSCs after miR-33a-3p over-expression or inhibition or after miR-33a-5p over-expression or inhibition ( A ) qRT-PCR was performed on Nh-Ost, EGF, EGFR, ERK-2, and ERK-3 ( B ) Quantitative RT-PCR data are expressed as fold of change (FOI) in gene expression (2 −ΔΔCt ) that occurred in hMSCs after treatments vs. scramble groups and in Nh-Ost vs. hMSCs cells. Student t test: * p

    Journal: Cells

    Article Title: MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation

    doi: 10.3390/cells8121495

    Figure Lengend Snippet: Analysis of gene expression on hMSCs after EGF treatments and EGFR signaling gene expression investigation on Nh-Ost. qRT-PCR was performed after 24 h of specific transfection on hMSCs. EGFR and ERK3 expression levels were evaluated on hMSCs after miR-33a-3p over-expression or inhibition or after miR-33a-5p over-expression or inhibition ( A ) qRT-PCR was performed on Nh-Ost, EGF, EGFR, ERK-2, and ERK-3 ( B ) Quantitative RT-PCR data are expressed as fold of change (FOI) in gene expression (2 −ΔΔCt ) that occurred in hMSCs after treatments vs. scramble groups and in Nh-Ost vs. hMSCs cells. Student t test: * p

    Article Snippet: Cell Transfection For cell transfection, Attractene Transfection Reagent (cat. number 1051531, Qiagen Srl, Milan, Italy) was used following the manufacturer’s indication.

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Over Expression, Inhibition

    Study of miR-33a-5p-targets by gain and loss function assays; hMSCs after 24 h of transfection with specific mimic and antimiR were analyzed for the gene expression modulation by qRT-PCR and protein Western blot analysis. ( A ) miR-33a-5p targets HMGA-2; ( B ) EMT markers: SNAIL, SLUG, TWIST, TGF-β; ( C ) Slug; ( D ) ALPL. Quantitative RT-PCR data are expressed as fold of change (FOI) in gene expression (2 −ΔΔCt ) that occurred in mimic or antimiR vs. scramble groups. OsteoImage assay was performed to quantify the in vitro mineralization nodules, and data are shown as relative fluorescence units (RFU,492/520 nm) ( E ) Western blot analysis for Runx-2 ( F ) protein was performed on total cell extract after miR-33a-5p and 3p over-expression or inhibition. Student t test: * p

    Journal: Cells

    Article Title: MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation

    doi: 10.3390/cells8121495

    Figure Lengend Snippet: Study of miR-33a-5p-targets by gain and loss function assays; hMSCs after 24 h of transfection with specific mimic and antimiR were analyzed for the gene expression modulation by qRT-PCR and protein Western blot analysis. ( A ) miR-33a-5p targets HMGA-2; ( B ) EMT markers: SNAIL, SLUG, TWIST, TGF-β; ( C ) Slug; ( D ) ALPL. Quantitative RT-PCR data are expressed as fold of change (FOI) in gene expression (2 −ΔΔCt ) that occurred in mimic or antimiR vs. scramble groups. OsteoImage assay was performed to quantify the in vitro mineralization nodules, and data are shown as relative fluorescence units (RFU,492/520 nm) ( E ) Western blot analysis for Runx-2 ( F ) protein was performed on total cell extract after miR-33a-5p and 3p over-expression or inhibition. Student t test: * p

    Article Snippet: Cell Transfection For cell transfection, Attractene Transfection Reagent (cat. number 1051531, Qiagen Srl, Milan, Italy) was used following the manufacturer’s indication.

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, In Vitro, Fluorescence, Over Expression, Inhibition

    3.4. Tissue culture and transfection

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Mammalian Two-Hybrid assays for studies of interaction of p300 with transcription factors

    doi: 10.1007/978-1-62703-284-1_26

    Figure Lengend Snippet: 3.4. Tissue culture and transfection

    Article Snippet: Human fetal osteoblastic cells – hFOB 1.19 (ATCC CRL-11372); Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12); Opti-MEM I Reduced Serum Media; Fetal bovine serum (FBS); 100× antibiotic/antimycotic solution (with 10,000 units penicillin, 10 mg streptomycin and 25 µg amphotericin B per ml) (Sigma-Aldrich); 100× MEM Non-Essential Amino Acids Solution 10 mM (NEAA) (Invitrogen); 10× trypsin-EDTA solution (Sigma-Aldrich); Phosphate buffered saline (pH 7.4): 8 g NaCl, 0.2 g KCl, 1.44 g Na2 HPO4 , 0.24 g KH2 PO4 , add double distilled H2 O to 1 liter; Transfection reagent: Attractene (Qiagen); 150 mm tissue culture plates; 48-well tissue culture plates 50 ml conical tubes; Water bath set at 37 °C; Inverted microscope; Hemacytometer with cover glass; Tissue culture incubator; Centrifuge; Biosafety cabinet.

    Techniques: Transfection