polyethylenimine Search Results


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  • 99
    Millipore polyethylenimines
    Testing results of membrane and draw molecule performance. ( a ) The forward osmosis performance of various combinations of membranes and draw molecules. Performance metric is the average water flux over a 3 h period. Sample is 10 mL deionized water; draw osmolarity is 1 OsM. Overlay image depicts testing apparatus. ( b ) The MWCOs of each membrane and MW of each draw molecule is plotted together on the same graph to give insights on the size compatibility of each combination. Three membrane classes are represented: forward osmosis (FTS), nanofiltration (NFS, NFX, NFW, and NFG), and dialysis (0.1–0.5 kDa, 0.5–1.0 kDa, and 3.5–5.0 kDa). Draw molecules are NaCl, sucrose, and both low and high MW branched <t>polyethylenimine</t> (PEI).
    Polyethylenimines, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Polysciences inc polyethyleneimine
    Schematic synthesis of brevinin 2R (BR-2R)-linked <t>polyethylenimine</t> (PEI) by EDC/NHS conjugation method
    Polyethyleneimine, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 94/100, based on 2882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    PolyScience polyethyleneimine
    as an example) and on the thermally induced functionalization with oligomeric <t>polyethyleneimine</t> (PEI). For the cartoon on carbon dot, it is generally a small carbon nanoparticle core with attached and strongly adsorbed surface passivation molecules (a configuration similar to a soft corona).
    Polyethyleneimine, supplied by PolyScience, used in various techniques. Bioz Stars score: 92/100, based on 258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Polysciences inc polyethylenimine pei
    Recombinant TRIM25 co-purifies with nucleic acid. ( a ) Affinity purification scheme for Strep/FLAG-tagged TRIM25 expressed in insect cells. ( b ) Coomassie-stained SDS-PAGE gels of TRIM25 fractions after initial affinity chromatography step. ( c ) UV absorbance spectra of pooled protein fractions. Vertical dashed lines indicate the peak wavelength. ( d ) Visualization by agarose gel electrophoresis and SYBR-green staining of the co-purifying nucleic acid (−PEI) and its removal by <t>polyethyleneimine</t> treatment <t>(+PEI).</t>
    Polyethylenimine Pei, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 94/100, based on 1789 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    PolyScience polyethylenimine pei
    Recombinant TRIM25 co-purifies with nucleic acid. ( a ) Affinity purification scheme for Strep/FLAG-tagged TRIM25 expressed in insect cells. ( b ) Coomassie-stained SDS-PAGE gels of TRIM25 fractions after initial affinity chromatography step. ( c ) UV absorbance spectra of pooled protein fractions. Vertical dashed lines indicate the peak wavelength. ( d ) Visualization by agarose gel electrophoresis and SYBR-green staining of the co-purifying nucleic acid (−PEI) and its removal by <t>polyethyleneimine</t> treatment <t>(+PEI).</t>
    Polyethylenimine Pei, supplied by PolyScience, used in various techniques. Bioz Stars score: 92/100, based on 197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Poly Scientific Inc polyethylenimine
    Recombinant TRIM25 co-purifies with nucleic acid. ( a ) Affinity purification scheme for Strep/FLAG-tagged TRIM25 expressed in insect cells. ( b ) Coomassie-stained SDS-PAGE gels of TRIM25 fractions after initial affinity chromatography step. ( c ) UV absorbance spectra of pooled protein fractions. Vertical dashed lines indicate the peak wavelength. ( d ) Visualization by agarose gel electrophoresis and SYBR-green staining of the co-purifying nucleic acid (−PEI) and its removal by <t>polyethyleneimine</t> treatment <t>(+PEI).</t>
    Polyethylenimine, supplied by Poly Scientific Inc, used in various techniques. Bioz Stars score: 93/100, based on 801 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher polyethyleneimine
    Fluorescence images of organs and tumors in A549 tumor-bearing mice 24 h after intravenous injection of complex nanoparticles (DIR=50 µg/kg, siRNA Cy5 =2 mg/kg) (n=3). ( A ) Fluorescence image of DIR channel: (a) DIR; (b) PEI-PLA/DIR/siRNA Cy5 ; (c) PEI-PLA/DIR/siRNA Cy5 /PEG-PAsp. ( B ) Fluorescence image of Cy5 channel: (a) siRNA Cy5 ; (b) PEI-PLA/DIR/siRNA Cy5 ; (c) PEI-PLA/DIR/siRNA Cy5 /PEG-PAsp. Abbreviations: PEI-PLA, <t>polyethyleneimine-block-polylactic</t> acid; PEG-PAsp, poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt).
    Polyethyleneimine, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Poly Scientific Inc polyethylenimine pei
    Fluorescence images of organs and tumors in A549 tumor-bearing mice 24 h after intravenous injection of complex nanoparticles (DIR=50 µg/kg, siRNA Cy5 =2 mg/kg) (n=3). ( A ) Fluorescence image of DIR channel: (a) DIR; (b) PEI-PLA/DIR/siRNA Cy5 ; (c) PEI-PLA/DIR/siRNA Cy5 /PEG-PAsp. ( B ) Fluorescence image of Cy5 channel: (a) siRNA Cy5 ; (b) PEI-PLA/DIR/siRNA Cy5 ; (c) PEI-PLA/DIR/siRNA Cy5 /PEG-PAsp. Abbreviations: PEI-PLA, <t>polyethyleneimine-block-polylactic</t> acid; PEG-PAsp, poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt).
    Polyethylenimine Pei, supplied by Poly Scientific Inc, used in various techniques. Bioz Stars score: 93/100, based on 263 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Merck & Co polyethyleneimine cellulose plates
    In vitro GAP activity of Git2-short toward ARF1. ARF1 (0.5 μg) preloaded with [α- 32 P]GTP were incubated for 5 min at 30°C with buffer alone (lane 1), with 2.5 μg of Git2-short (lane 2), or the CA mutant (lane 3). The nucleotides were then extracted and separated on a <t>polyethyleneimine-cellulose</t> plate and subjected to autoradiography. Positions of GTP and GDP were identified using authentic nucleotides.
    Polyethyleneimine Cellulose Plates, supplied by Merck & Co, used in various techniques. Bioz Stars score: 90/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Polysciences inc 25 kda linear polyethyleneimine
    In vitro GAP activity of Git2-short toward ARF1. ARF1 (0.5 μg) preloaded with [α- 32 P]GTP were incubated for 5 min at 30°C with buffer alone (lane 1), with 2.5 μg of Git2-short (lane 2), or the CA mutant (lane 3). The nucleotides were then extracted and separated on a <t>polyethyleneimine-cellulose</t> plate and subjected to autoradiography. Positions of GTP and GDP were identified using authentic nucleotides.
    25 Kda Linear Polyethyleneimine, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 88/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Polyplus Transfection polyethyleneimine
    In vitro GAP activity of Git2-short toward ARF1. ARF1 (0.5 μg) preloaded with [α- 32 P]GTP were incubated for 5 min at 30°C with buffer alone (lane 1), with 2.5 μg of Git2-short (lane 2), or the CA mutant (lane 3). The nucleotides were then extracted and separated on a <t>polyethyleneimine-cellulose</t> plate and subjected to autoradiography. Positions of GTP and GDP were identified using authentic nucleotides.
    Polyethyleneimine, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 92/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore polyethyleneimine reagent
    Confocal laser scanning microscopy analysis. Left panels: Human gingival mesenchymal stem cells (hGMSCs) anti-ANA positive nuclei in a 3D-PLA + hGMSCs, b 3D-PLA + extracellular vesicles (EVs) + hGMSCs, and c 3D-PLA + <t>polyethyleneimine</t> (PEI)-EVs + hGMSCs (green fluorescent). Middle panels: Confocal contrast image of 3D-PLA (gray) grafted in mice calvaria after 6 weeks of implantation. Right panels: Merged images of the two abovementioned channels. Scale bars = 10 μm. *, scaffold; C, rat calvaria
    Polyethyleneimine Reagent, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Polysciences inc polyethylenimine pei transfection reagent
    Confocal laser scanning microscopy analysis. Left panels: Human gingival mesenchymal stem cells (hGMSCs) anti-ANA positive nuclei in a 3D-PLA + hGMSCs, b 3D-PLA + extracellular vesicles (EVs) + hGMSCs, and c 3D-PLA + <t>polyethyleneimine</t> (PEI)-EVs + hGMSCs (green fluorescent). Middle panels: Confocal contrast image of 3D-PLA (gray) grafted in mice calvaria after 6 weeks of implantation. Right panels: Merged images of the two abovementioned channels. Scale bars = 10 μm. *, scaffold; C, rat calvaria
    Polyethylenimine Pei Transfection Reagent, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 89/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Polysciences inc polyethylenimine reagent
    Confocal laser scanning microscopy analysis. Left panels: Human gingival mesenchymal stem cells (hGMSCs) anti-ANA positive nuclei in a 3D-PLA + hGMSCs, b 3D-PLA + extracellular vesicles (EVs) + hGMSCs, and c 3D-PLA + <t>polyethyleneimine</t> (PEI)-EVs + hGMSCs (green fluorescent). Middle panels: Confocal contrast image of 3D-PLA (gray) grafted in mice calvaria after 6 weeks of implantation. Right panels: Merged images of the two abovementioned channels. Scale bars = 10 μm. *, scaffold; C, rat calvaria
    Polyethylenimine Reagent, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 89/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Polyplus Transfection polyethylenimine pei
    Confocal laser scanning microscopy analysis. Left panels: Human gingival mesenchymal stem cells (hGMSCs) anti-ANA positive nuclei in a 3D-PLA + hGMSCs, b 3D-PLA + extracellular vesicles (EVs) + hGMSCs, and c 3D-PLA + <t>polyethyleneimine</t> (PEI)-EVs + hGMSCs (green fluorescent). Middle panels: Confocal contrast image of 3D-PLA (gray) grafted in mice calvaria after 6 weeks of implantation. Right panels: Merged images of the two abovementioned channels. Scale bars = 10 μm. *, scaffold; C, rat calvaria
    Polyethylenimine Pei, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 92/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Testing results of membrane and draw molecule performance. ( a ) The forward osmosis performance of various combinations of membranes and draw molecules. Performance metric is the average water flux over a 3 h period. Sample is 10 mL deionized water; draw osmolarity is 1 OsM. Overlay image depicts testing apparatus. ( b ) The MWCOs of each membrane and MW of each draw molecule is plotted together on the same graph to give insights on the size compatibility of each combination. Three membrane classes are represented: forward osmosis (FTS), nanofiltration (NFS, NFX, NFW, and NFG), and dialysis (0.1–0.5 kDa, 0.5–1.0 kDa, and 3.5–5.0 kDa). Draw molecules are NaCl, sucrose, and both low and high MW branched polyethylenimine (PEI).

    Journal: PLoS ONE

    Article Title: Continuous, quantifiable, and simple osmotic preconcentration and sensing within microfluidic devices

    doi: 10.1371/journal.pone.0210286

    Figure Lengend Snippet: Testing results of membrane and draw molecule performance. ( a ) The forward osmosis performance of various combinations of membranes and draw molecules. Performance metric is the average water flux over a 3 h period. Sample is 10 mL deionized water; draw osmolarity is 1 OsM. Overlay image depicts testing apparatus. ( b ) The MWCOs of each membrane and MW of each draw molecule is plotted together on the same graph to give insights on the size compatibility of each combination. Three membrane classes are represented: forward osmosis (FTS), nanofiltration (NFS, NFX, NFW, and NFG), and dialysis (0.1–0.5 kDa, 0.5–1.0 kDa, and 3.5–5.0 kDa). Draw molecules are NaCl, sucrose, and both low and high MW branched polyethylenimine (PEI).

    Article Snippet: Branched polyethylenimines were used with both low (Mn ~600 Da; P/N: 408719, Sigma-Aldrich) and high (Mn ~60 kDa; P/N: 181978, Sigma-Aldrich) molecular weights.

    Techniques:

    General scheme of the whole-cell bioluminescence biosensor for detection of oligomerization. Note: Schematic representation explains split-luciferase strategy for monitoring of A53T αS oligomerization and using of this system for screening of GQDs effect on A53T αS oligomerization. Abbreviations: αS, alpha-synuclein; PEI, polyethylenimine; GQDs, graphene quantum dots.

    Journal: International Journal of Nanomedicine

    Article Title: Investigation of the effects of carbon-based nanomaterials on A53T alpha-synuclein aggregation using a whole-cell recombinant biosensor

    doi: 10.2147/IJN.S144764

    Figure Lengend Snippet: General scheme of the whole-cell bioluminescence biosensor for detection of oligomerization. Note: Schematic representation explains split-luciferase strategy for monitoring of A53T αS oligomerization and using of this system for screening of GQDs effect on A53T αS oligomerization. Abbreviations: αS, alpha-synuclein; PEI, polyethylenimine; GQDs, graphene quantum dots.

    Article Snippet: Then, the plasmid mixtures were prepared at an N:P ratio of 12 with branched 25 kDa polyethylenimine (PEI) (Sigma-Aldrich, St Louis, MO, USA), and transfection mixture was applied dropwise to each well and incubated with 5% CO2 for 4 hours at 37°C.

    Techniques: Luciferase

    Schematic synthesis of brevinin 2R (BR-2R)-linked polyethylenimine (PEI) by EDC/NHS conjugation method

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: Brevinin-2R-linked polyethylenimine as a promising hybrid nano-gene-delivery vector

    doi: 10.22038/ijbms.2019.37125.8842

    Figure Lengend Snippet: Schematic synthesis of brevinin 2R (BR-2R)-linked polyethylenimine (PEI) by EDC/NHS conjugation method

    Article Snippet: Branched polyethyleneimine (b-PEI 10 kDa) has been procured from Polysciences Inc. (Warrington, PA, USA).

    Techniques: Conjugation Assay

    Schematic synthesis of brevinin 2R (BR-2R)-linked polyethylenimine (PEI) by 6-bromohexanoic acid linker

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: Brevinin-2R-linked polyethylenimine as a promising hybrid nano-gene-delivery vector

    doi: 10.22038/ijbms.2019.37125.8842

    Figure Lengend Snippet: Schematic synthesis of brevinin 2R (BR-2R)-linked polyethylenimine (PEI) by 6-bromohexanoic acid linker

    Article Snippet: Branched polyethyleneimine (b-PEI 10 kDa) has been procured from Polysciences Inc. (Warrington, PA, USA).

    Techniques:

    Schematic synthesis of brevinin 2R (BR-2R)-linked polyethylenimine (PEI) by SPDP conjugation method

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: Brevinin-2R-linked polyethylenimine as a promising hybrid nano-gene-delivery vector

    doi: 10.22038/ijbms.2019.37125.8842

    Figure Lengend Snippet: Schematic synthesis of brevinin 2R (BR-2R)-linked polyethylenimine (PEI) by SPDP conjugation method

    Article Snippet: Branched polyethyleneimine (b-PEI 10 kDa) has been procured from Polysciences Inc. (Warrington, PA, USA).

    Techniques: Conjugation Assay

    Synthesis of crosslinked linear polyethylenimine using NHS/EDC chemistry with a diacid. For degradable crosslinks that break down by disulfide oxidation R contains a disulfide group.

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    Article Title: Crosslinked Linear Polyethyleneimine Enhances Delivery of DNA to the Cytoplasm

    doi: 10.1016/j.jconrel.2012.09.004

    Figure Lengend Snippet: Synthesis of crosslinked linear polyethylenimine using NHS/EDC chemistry with a diacid. For degradable crosslinks that break down by disulfide oxidation R contains a disulfide group.

    Article Snippet: Linear polyethylenimine (lPEI, Mw = 2.5k, 25k) was purchased from Polysciences (Warrington, PA).

    Techniques:

    HeLa cell growth in the presence of polyethylenimine (PEI)-coated magnetic nanoparticles (MNPs) at 300 μg/mL and with MNP/antibody complexes added at 100, 200 and 300 μg/mL. The control did not include these treatments. Cell numbers were normalized by the number of control cells. Cell numbers decreased with increasing complex doses.

    Journal: Nanomaterials

    Article Title: Hyperthermia Using Antibody-Conjugated Magnetic Nanoparticles and Its Enhanced Effect with Cryptotanshinone

    doi: 10.3390/nano4020319

    Figure Lengend Snippet: HeLa cell growth in the presence of polyethylenimine (PEI)-coated magnetic nanoparticles (MNPs) at 300 μg/mL and with MNP/antibody complexes added at 100, 200 and 300 μg/mL. The control did not include these treatments. Cell numbers were normalized by the number of control cells. Cell numbers decreased with increasing complex doses.

    Article Snippet: Materials and Reagents Fe3 O4 nanoparticles (primary diameter, 20–30 nm) were obtained from Nanostructured and Amorphous Materials, Inc. Polyethylenimine max (PEI; mw 40,000) was from Polysciences, Inc.

    Techniques:

    as an example) and on the thermally induced functionalization with oligomeric polyethyleneimine (PEI). For the cartoon on carbon dot, it is generally a small carbon nanoparticle core with attached and strongly adsorbed surface passivation molecules (a configuration similar to a soft corona).

    Journal: Journal of materials chemistry. C, Materials for optical and electronic devices

    Article Title: Photoexcited State Properties of Carbon Dots from Thermally Induced Functionalization of Carbon Nanoparticles

    doi: 10.1039/C6TC03666J

    Figure Lengend Snippet: as an example) and on the thermally induced functionalization with oligomeric polyethyleneimine (PEI). For the cartoon on carbon dot, it is generally a small carbon nanoparticle core with attached and strongly adsorbed surface passivation molecules (a configuration similar to a soft corona).

    Article Snippet: The carbon nanopowder sample was purchased from US Research Nanomaterials, Inc., polyethyleneimine (PEI, branched, average molecular weight ~1,200) from Polyscience, Inc., and silicon carbide (120 Grit) from Panadyne Abrasives.

    Techniques:

    Recombinant TRIM25 co-purifies with nucleic acid. ( a ) Affinity purification scheme for Strep/FLAG-tagged TRIM25 expressed in insect cells. ( b ) Coomassie-stained SDS-PAGE gels of TRIM25 fractions after initial affinity chromatography step. ( c ) UV absorbance spectra of pooled protein fractions. Vertical dashed lines indicate the peak wavelength. ( d ) Visualization by agarose gel electrophoresis and SYBR-green staining of the co-purifying nucleic acid (−PEI) and its removal by polyethyleneimine treatment (+PEI).

    Journal: Journal of molecular biology

    Article Title: TRIM25 binds RNA to modulate cellular anti-viral defense

    doi: 10.1016/j.jmb.2018.10.003

    Figure Lengend Snippet: Recombinant TRIM25 co-purifies with nucleic acid. ( a ) Affinity purification scheme for Strep/FLAG-tagged TRIM25 expressed in insect cells. ( b ) Coomassie-stained SDS-PAGE gels of TRIM25 fractions after initial affinity chromatography step. ( c ) UV absorbance spectra of pooled protein fractions. Vertical dashed lines indicate the peak wavelength. ( d ) Visualization by agarose gel electrophoresis and SYBR-green staining of the co-purifying nucleic acid (−PEI) and its removal by polyethyleneimine treatment (+PEI).

    Article Snippet: TRIM25 -KO HEK 293T cells [ ], seeded into 12-well plates (~5 × 105 cells/well), were transfected with 2 μg of pCMV empty vector or the indicated FLAG-tagged TRIM25 constructs using linear polyethylenimine (PEI) (1 mg/mL solution in 20 mM Tris pH 6.8; Polysciences), according to the manufacturer’s instructions.

    Techniques: Recombinant, Affinity Purification, Staining, SDS Page, Affinity Chromatography, Agarose Gel Electrophoresis, SYBR Green Assay

    Fluorescence images of organs and tumors in A549 tumor-bearing mice 24 h after intravenous injection of complex nanoparticles (DIR=50 µg/kg, siRNA Cy5 =2 mg/kg) (n=3). ( A ) Fluorescence image of DIR channel: (a) DIR; (b) PEI-PLA/DIR/siRNA Cy5 ; (c) PEI-PLA/DIR/siRNA Cy5 /PEG-PAsp. ( B ) Fluorescence image of Cy5 channel: (a) siRNA Cy5 ; (b) PEI-PLA/DIR/siRNA Cy5 ; (c) PEI-PLA/DIR/siRNA Cy5 /PEG-PAsp. Abbreviations: PEI-PLA, polyethyleneimine-block-polylactic acid; PEG-PAsp, poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt).

    Journal: International Journal of Nanomedicine

    Article Title: Smart polymeric nanoparticles with pH-responsive and PEG-detachable properties for co-delivering paclitaxel and survivin siRNA to enhance antitumor outcomes

    doi: 10.2147/IJN.S161426

    Figure Lengend Snippet: Fluorescence images of organs and tumors in A549 tumor-bearing mice 24 h after intravenous injection of complex nanoparticles (DIR=50 µg/kg, siRNA Cy5 =2 mg/kg) (n=3). ( A ) Fluorescence image of DIR channel: (a) DIR; (b) PEI-PLA/DIR/siRNA Cy5 ; (c) PEI-PLA/DIR/siRNA Cy5 /PEG-PAsp. ( B ) Fluorescence image of Cy5 channel: (a) siRNA Cy5 ; (b) PEI-PLA/DIR/siRNA Cy5 ; (c) PEI-PLA/DIR/siRNA Cy5 /PEG-PAsp. Abbreviations: PEI-PLA, polyethyleneimine-block-polylactic acid; PEG-PAsp, poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt).

    Article Snippet: Branched polyethyleneimine (MW=1.8 kDa, bPEI1.8k ) was purchased from Alfa Aesar (Ward Hill, MA, USA).

    Techniques: Fluorescence, Mouse Assay, Injection, Proximity Ligation Assay, Blocking Assay

    ( A ) PTX/siRNA-loaded layer-by-layer nanoparticle delivery system. ( B ) Schematic of the intracellular therapeutic mechanism of the PTX/siRNA-loaded layer-by-layer nanoparticles. Abbreviations: PTX, paclitaxel; PEI-PLA, polyethyleneimine-block-polylactic acid; PEG-PAsp, poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt); EPR, enhanced permeation and retention.

    Journal: International Journal of Nanomedicine

    Article Title: Smart polymeric nanoparticles with pH-responsive and PEG-detachable properties for co-delivering paclitaxel and survivin siRNA to enhance antitumor outcomes

    doi: 10.2147/IJN.S161426

    Figure Lengend Snippet: ( A ) PTX/siRNA-loaded layer-by-layer nanoparticle delivery system. ( B ) Schematic of the intracellular therapeutic mechanism of the PTX/siRNA-loaded layer-by-layer nanoparticles. Abbreviations: PTX, paclitaxel; PEI-PLA, polyethyleneimine-block-polylactic acid; PEG-PAsp, poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt); EPR, enhanced permeation and retention.

    Article Snippet: Branched polyethyleneimine (MW=1.8 kDa, bPEI1.8k ) was purchased from Alfa Aesar (Ward Hill, MA, USA).

    Techniques: Proximity Ligation Assay, Blocking Assay, Electron Paramagnetic Resonance

    Cellular uptake of siRNA or PTX in different formulations. A549 cells were analyzed after 4 h of incubation at the final concentrations of different NPs (Oregon Green PTX content 200 ng/mL, Cy3 siRNA 50 nM, N/P=30, C/N=1/5). ( A ) Quantitative analyses of siRNA and PTX uptake by flow cytometry. Fluorescence signals of Cy3 and Oregon Green PTX are presented by four-quadrant diagram and histogram (400×). (a) Mock; (b) siRNA; (c) PEI-PLA/siRNA/PEG-PAsp; (d) PTX; (e) PEI-PLA/PTX/PEG-PAsp; (f) PEI-PLA/siRNA/PTX; (g) PEI-PLA/siRNA/PTX/PEG-PAsp. ( B ) Confocal laser scanning microscope images of cells treated with different formulations of NPs: (a) siRNA; (b) PEI-PLA/siRNA/PEG-PAsp; (c) PTX; (d) PEI-PLA/PTX/PEG-PAsp; (e) PEI-PLA/siRNA/PTX/PEG-PAsp. For each column, from left to right: nuclei were stained by DAPI (blue); Oregon Green PTX fluorescence in cells (green); Cy3 signal in cells (red); merged with nucleus, Cy3-siRNA, and Oregon Green PTX. The yellow stains (arrows) in the cytoplasm indicates that the NPs successfully enters into the cell nucleus, and could easily escape from the endosomes via the proton sponge effect of PEI. Abbreviations: PTX, paclitaxel; NPs, nanoparticles; PEI-PLA, polyethyleneimine-block-polylactic acid; PEG-PAsp, poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt).

    Journal: International Journal of Nanomedicine

    Article Title: Smart polymeric nanoparticles with pH-responsive and PEG-detachable properties for co-delivering paclitaxel and survivin siRNA to enhance antitumor outcomes

    doi: 10.2147/IJN.S161426

    Figure Lengend Snippet: Cellular uptake of siRNA or PTX in different formulations. A549 cells were analyzed after 4 h of incubation at the final concentrations of different NPs (Oregon Green PTX content 200 ng/mL, Cy3 siRNA 50 nM, N/P=30, C/N=1/5). ( A ) Quantitative analyses of siRNA and PTX uptake by flow cytometry. Fluorescence signals of Cy3 and Oregon Green PTX are presented by four-quadrant diagram and histogram (400×). (a) Mock; (b) siRNA; (c) PEI-PLA/siRNA/PEG-PAsp; (d) PTX; (e) PEI-PLA/PTX/PEG-PAsp; (f) PEI-PLA/siRNA/PTX; (g) PEI-PLA/siRNA/PTX/PEG-PAsp. ( B ) Confocal laser scanning microscope images of cells treated with different formulations of NPs: (a) siRNA; (b) PEI-PLA/siRNA/PEG-PAsp; (c) PTX; (d) PEI-PLA/PTX/PEG-PAsp; (e) PEI-PLA/siRNA/PTX/PEG-PAsp. For each column, from left to right: nuclei were stained by DAPI (blue); Oregon Green PTX fluorescence in cells (green); Cy3 signal in cells (red); merged with nucleus, Cy3-siRNA, and Oregon Green PTX. The yellow stains (arrows) in the cytoplasm indicates that the NPs successfully enters into the cell nucleus, and could easily escape from the endosomes via the proton sponge effect of PEI. Abbreviations: PTX, paclitaxel; NPs, nanoparticles; PEI-PLA, polyethyleneimine-block-polylactic acid; PEG-PAsp, poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt).

    Article Snippet: Branched polyethyleneimine (MW=1.8 kDa, bPEI1.8k ) was purchased from Alfa Aesar (Ward Hill, MA, USA).

    Techniques: Incubation, Flow Cytometry, Cytometry, Fluorescence, Proximity Ligation Assay, Laser-Scanning Microscopy, Staining, Blocking Assay

    Characterization of the NPs. ( A ) Particle size (intensity diameter) and zeta potential of PEI-PLA/PTX/siRNA/PEG-PAsp NPs. ( B ) Transmission electron microscopy image of PEI-PLA/PTX/siRNA/PEG-PAsp NPs. The scale bar represents 100 nm. Abbreviations: NPs, nanoparticles; PEI-PLA, polyethyleneimine-block-polylactic acid; PTX, paclitaxel; PEG-PAsp, poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt).

    Journal: International Journal of Nanomedicine

    Article Title: Smart polymeric nanoparticles with pH-responsive and PEG-detachable properties for co-delivering paclitaxel and survivin siRNA to enhance antitumor outcomes

    doi: 10.2147/IJN.S161426

    Figure Lengend Snippet: Characterization of the NPs. ( A ) Particle size (intensity diameter) and zeta potential of PEI-PLA/PTX/siRNA/PEG-PAsp NPs. ( B ) Transmission electron microscopy image of PEI-PLA/PTX/siRNA/PEG-PAsp NPs. The scale bar represents 100 nm. Abbreviations: NPs, nanoparticles; PEI-PLA, polyethyleneimine-block-polylactic acid; PTX, paclitaxel; PEG-PAsp, poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt).

    Article Snippet: Branched polyethyleneimine (MW=1.8 kDa, bPEI1.8k ) was purchased from Alfa Aesar (Ward Hill, MA, USA).

    Techniques: Proximity Ligation Assay, Transmission Assay, Electron Microscopy, Blocking Assay

    ( A ) Synthesis of the PEI-PLA. The PLA-COOH was conjugated to PEI through amide formation in the presence of EDC and NHS, obtaining the cationic amphiphilic copolymer of PEI-PLA. ( B ) 1 H-NMR spectra of PEI and PEI-PLA in D 2 O. The peak of PEI appears at around 2.6 ppm. In PEI-PLA, a new broad peak appears at 2.3–3.4 ppm, which is attributed to the protons of methylene (–CH 2 CH 2 –) in PEI. The signals at δ =1.20 ppm (a) and δ =4.08 ppm (b) correspond to −CH 3 and (–CH) proton in the PLA block of PEI-PLA, respectively. Abbreviations: PEI-PLA, polyethyleneimine-block-polylactic acid; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS, N -hydroxysuccinimide; NMR, nuclear magnetic resonance.

    Journal: International Journal of Nanomedicine

    Article Title: Smart polymeric nanoparticles with pH-responsive and PEG-detachable properties for co-delivering paclitaxel and survivin siRNA to enhance antitumor outcomes

    doi: 10.2147/IJN.S161426

    Figure Lengend Snippet: ( A ) Synthesis of the PEI-PLA. The PLA-COOH was conjugated to PEI through amide formation in the presence of EDC and NHS, obtaining the cationic amphiphilic copolymer of PEI-PLA. ( B ) 1 H-NMR spectra of PEI and PEI-PLA in D 2 O. The peak of PEI appears at around 2.6 ppm. In PEI-PLA, a new broad peak appears at 2.3–3.4 ppm, which is attributed to the protons of methylene (–CH 2 CH 2 –) in PEI. The signals at δ =1.20 ppm (a) and δ =4.08 ppm (b) correspond to −CH 3 and (–CH) proton in the PLA block of PEI-PLA, respectively. Abbreviations: PEI-PLA, polyethyleneimine-block-polylactic acid; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS, N -hydroxysuccinimide; NMR, nuclear magnetic resonance.

    Article Snippet: Branched polyethyleneimine (MW=1.8 kDa, bPEI1.8k ) was purchased from Alfa Aesar (Ward Hill, MA, USA).

    Techniques: Proximity Ligation Assay, Nuclear Magnetic Resonance, Blocking Assay

    ( A ) Nuclear morphology, ( B ) cell apoptosis, and ( C ) cell cycle change in A549 cells 48 h after treatment with various formulations. Each row, from top to bottom, presents Hoechst staining of cell nuclei, quantitative detection of apoptotic cells, and cell cycle study. For Hoechst staining, cells were stained with Hoechst 33258 reagent at a concentration of 5 µg/mL for 10 min. For the cell apoptotic assay, cells were stained with Annexin V-FITC/PI for 15 min. For the cell cycle assay, cells were stained with PI (50 µg/mL) for 1 h. Chromatin condensation, nuclear fragmentation, and chromosome abnormalities were found in the PEI-PLA/siRNA/PEG-PAsp, PEI-PLA/PTX/siRNAN.C/PEG-PAsp and PEI-PLA/PTX/siRNA/PEG-PAsp groups (arrows). Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide; PTX, paclitaxel; PEI-PLA, polyethyleneimine-block-polylactic acid; PEG-PAsp, poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt).

    Journal: International Journal of Nanomedicine

    Article Title: Smart polymeric nanoparticles with pH-responsive and PEG-detachable properties for co-delivering paclitaxel and survivin siRNA to enhance antitumor outcomes

    doi: 10.2147/IJN.S161426

    Figure Lengend Snippet: ( A ) Nuclear morphology, ( B ) cell apoptosis, and ( C ) cell cycle change in A549 cells 48 h after treatment with various formulations. Each row, from top to bottom, presents Hoechst staining of cell nuclei, quantitative detection of apoptotic cells, and cell cycle study. For Hoechst staining, cells were stained with Hoechst 33258 reagent at a concentration of 5 µg/mL for 10 min. For the cell apoptotic assay, cells were stained with Annexin V-FITC/PI for 15 min. For the cell cycle assay, cells were stained with PI (50 µg/mL) for 1 h. Chromatin condensation, nuclear fragmentation, and chromosome abnormalities were found in the PEI-PLA/siRNA/PEG-PAsp, PEI-PLA/PTX/siRNAN.C/PEG-PAsp and PEI-PLA/PTX/siRNA/PEG-PAsp groups (arrows). Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide; PTX, paclitaxel; PEI-PLA, polyethyleneimine-block-polylactic acid; PEG-PAsp, poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt).

    Article Snippet: Branched polyethyleneimine (MW=1.8 kDa, bPEI1.8k ) was purchased from Alfa Aesar (Ward Hill, MA, USA).

    Techniques: Staining, Concentration Assay, Cell Cycle Assay, Proximity Ligation Assay, Blocking Assay

    In vitro GAP activity of Git2-short toward ARF1. ARF1 (0.5 μg) preloaded with [α- 32 P]GTP were incubated for 5 min at 30°C with buffer alone (lane 1), with 2.5 μg of Git2-short (lane 2), or the CA mutant (lane 3). The nucleotides were then extracted and separated on a polyethyleneimine-cellulose plate and subjected to autoradiography. Positions of GTP and GDP were identified using authentic nucleotides.

    Journal: Molecular Biology of the Cell

    Article Title: An ADP-Ribosylation Factor GTPase-activating Protein Git2-short/KIAA0148 Is Involved in Subcellular Localization of Paxillin and Actin Cytoskeletal Organization

    doi:

    Figure Lengend Snippet: In vitro GAP activity of Git2-short toward ARF1. ARF1 (0.5 μg) preloaded with [α- 32 P]GTP were incubated for 5 min at 30°C with buffer alone (lane 1), with 2.5 μg of Git2-short (lane 2), or the CA mutant (lane 3). The nucleotides were then extracted and separated on a polyethyleneimine-cellulose plate and subjected to autoradiography. Positions of GTP and GDP were identified using authentic nucleotides.

    Article Snippet: Bound nucleotides were eluted in 2 M formic acid and were separated by chromatography on polyethyleneimine-cellulose plates (Merck, Rahway, NJ) in 1 M formic acid/1 M LiCl.

    Techniques: In Vitro, Activity Assay, Incubation, Mutagenesis, Autoradiography

    Confocal laser scanning microscopy analysis. Left panels: Human gingival mesenchymal stem cells (hGMSCs) anti-ANA positive nuclei in a 3D-PLA + hGMSCs, b 3D-PLA + extracellular vesicles (EVs) + hGMSCs, and c 3D-PLA + polyethyleneimine (PEI)-EVs + hGMSCs (green fluorescent). Middle panels: Confocal contrast image of 3D-PLA (gray) grafted in mice calvaria after 6 weeks of implantation. Right panels: Merged images of the two abovementioned channels. Scale bars = 10 μm. *, scaffold; C, rat calvaria

    Journal: Stem Cell Research & Therapy

    Article Title: Three-dimensional printed PLA scaffold and human gingival stem cell-derived extracellular vesicles: a new tool for bone defect repair

    doi: 10.1186/s13287-018-0850-0

    Figure Lengend Snippet: Confocal laser scanning microscopy analysis. Left panels: Human gingival mesenchymal stem cells (hGMSCs) anti-ANA positive nuclei in a 3D-PLA + hGMSCs, b 3D-PLA + extracellular vesicles (EVs) + hGMSCs, and c 3D-PLA + polyethyleneimine (PEI)-EVs + hGMSCs (green fluorescent). Middle panels: Confocal contrast image of 3D-PLA (gray) grafted in mice calvaria after 6 weeks of implantation. Right panels: Merged images of the two abovementioned channels. Scale bars = 10 μm. *, scaffold; C, rat calvaria

    Article Snippet: Engineered EVs preparation The EVs pellet (100 μL) was resuspended with 2 mL PBS, and 2 mL of branched polyethyleneimine solution (PEI; MW 25,000; Sigma-Aldrich, Milan, Italy) (0.05 mg/mL in 0.3 M NaCl) was added to the EVs suspension in PBS and the mixture was incubated for 20 min at room temperature.

    Techniques: Confocal Laser Scanning Microscopy, Proximity Ligation Assay, Mouse Assay

    Histological evaluation. Samples harvested at 6 weeks after the calvarial defects were transplanted with a 3D-PLA scaffold or b 3D-PLA + human gingival mesenchymal stem cells (hGMSCs). Left panels (1): The images at low magnification (4×) showed 3D-PLA and 3D-PLA + hGMSCs integrated smoothly with the host tissue. Middle panels (2): High-magnification images (10×) showing the contact area between 3D-PLA and 3D-PLA + hGMSCs with bone calvaria grafted at 6 weeks postsurgery. Right panels (3): Images obtained at 40× objective showing the connective tissue between 3D-PLA and 3D-PLA + hGMSCs and bone host tissue. Samples harvested at 6 weeks after the calvarial defects was transplanted with c 3D-PLA + extracellular vesicles (EVs) scaffold or d 3D-PLA + EVs + hGMSCs. Left panels (1): The images at low magnification (4×) showed 3D-PLA + EVs and 3D-PLA + EVs + hGMSCs integrated smoothly with the host. Middle panels (2): High-magnification images (10×) showing the new bone formation stained with acid fuchsin in both samples grafted at 6 weeks postsurgery. Right panels (3): Images obtained at 40× objective showed a zone with new mineralized matrix inside 3D PLA scaffold for both samples. In particular, in the 3D-PLA + EVs (c3) sample in the contact zone some blood vessels are valuable. Samples harvested at 6 weeks after the calvarial defects was transplanted with e 3D-PLA + polyethyleneimine (PEI)-EVs scaffold or f 3D-PLA + PEI-EVs + hGMSCs. Left panels (1): The images at low magnification (4×) showed 3D-PLA + PEI-EVs and 3D-PLA + PEI-EVs + hGMSCs integrated smoothly with the host. Middle panels (2): High-magnification images (10×) showing the new bone formation stained with acid fuchsin in both samples grafted at 6 weeks postsurgery. Right panels (3): Images obtained at 40× objective showed new bone formation inside the scaffold structure. In particular, in 3D-PLA + PEI-EVs there were numerous blood vessels present around the new bone deposition area. Scale bars = 10 μm. *, scaffold; V, vessels; B, new bone. Acid fuchsin-toluidine blue staining

    Journal: Stem Cell Research & Therapy

    Article Title: Three-dimensional printed PLA scaffold and human gingival stem cell-derived extracellular vesicles: a new tool for bone defect repair

    doi: 10.1186/s13287-018-0850-0

    Figure Lengend Snippet: Histological evaluation. Samples harvested at 6 weeks after the calvarial defects were transplanted with a 3D-PLA scaffold or b 3D-PLA + human gingival mesenchymal stem cells (hGMSCs). Left panels (1): The images at low magnification (4×) showed 3D-PLA and 3D-PLA + hGMSCs integrated smoothly with the host tissue. Middle panels (2): High-magnification images (10×) showing the contact area between 3D-PLA and 3D-PLA + hGMSCs with bone calvaria grafted at 6 weeks postsurgery. Right panels (3): Images obtained at 40× objective showing the connective tissue between 3D-PLA and 3D-PLA + hGMSCs and bone host tissue. Samples harvested at 6 weeks after the calvarial defects was transplanted with c 3D-PLA + extracellular vesicles (EVs) scaffold or d 3D-PLA + EVs + hGMSCs. Left panels (1): The images at low magnification (4×) showed 3D-PLA + EVs and 3D-PLA + EVs + hGMSCs integrated smoothly with the host. Middle panels (2): High-magnification images (10×) showing the new bone formation stained with acid fuchsin in both samples grafted at 6 weeks postsurgery. Right panels (3): Images obtained at 40× objective showed a zone with new mineralized matrix inside 3D PLA scaffold for both samples. In particular, in the 3D-PLA + EVs (c3) sample in the contact zone some blood vessels are valuable. Samples harvested at 6 weeks after the calvarial defects was transplanted with e 3D-PLA + polyethyleneimine (PEI)-EVs scaffold or f 3D-PLA + PEI-EVs + hGMSCs. Left panels (1): The images at low magnification (4×) showed 3D-PLA + PEI-EVs and 3D-PLA + PEI-EVs + hGMSCs integrated smoothly with the host. Middle panels (2): High-magnification images (10×) showing the new bone formation stained with acid fuchsin in both samples grafted at 6 weeks postsurgery. Right panels (3): Images obtained at 40× objective showed new bone formation inside the scaffold structure. In particular, in 3D-PLA + PEI-EVs there were numerous blood vessels present around the new bone deposition area. Scale bars = 10 μm. *, scaffold; V, vessels; B, new bone. Acid fuchsin-toluidine blue staining

    Article Snippet: Engineered EVs preparation The EVs pellet (100 μL) was resuspended with 2 mL PBS, and 2 mL of branched polyethyleneimine solution (PEI; MW 25,000; Sigma-Aldrich, Milan, Italy) (0.05 mg/mL in 0.3 M NaCl) was added to the EVs suspension in PBS and the mixture was incubated for 20 min at room temperature.

    Techniques: Proximity Ligation Assay, Staining

    CT analysis showing bone damage was still present in the 3D-PLA, 3D-PLA + human gingival mesenchymal stem cells (hGMSCs), and 3D-PLA + extracellular vesicles (EVs) groups. On the contrary, in the 3D-PLA + EVs + hGMSCs, 3D-PLA + polyethyleneimine (PEI)-EVs, and 3D-PLA + PEI-EVs + hGMSCs groups the complete repair of the calvarial defect was shown. Arrows indicated bone defects

    Journal: Stem Cell Research & Therapy

    Article Title: Three-dimensional printed PLA scaffold and human gingival stem cell-derived extracellular vesicles: a new tool for bone defect repair

    doi: 10.1186/s13287-018-0850-0

    Figure Lengend Snippet: CT analysis showing bone damage was still present in the 3D-PLA, 3D-PLA + human gingival mesenchymal stem cells (hGMSCs), and 3D-PLA + extracellular vesicles (EVs) groups. On the contrary, in the 3D-PLA + EVs + hGMSCs, 3D-PLA + polyethyleneimine (PEI)-EVs, and 3D-PLA + PEI-EVs + hGMSCs groups the complete repair of the calvarial defect was shown. Arrows indicated bone defects

    Article Snippet: Engineered EVs preparation The EVs pellet (100 μL) was resuspended with 2 mL PBS, and 2 mL of branched polyethyleneimine solution (PEI; MW 25,000; Sigma-Aldrich, Milan, Italy) (0.05 mg/mL in 0.3 M NaCl) was added to the EVs suspension in PBS and the mixture was incubated for 20 min at room temperature.

    Techniques: Proximity Ligation Assay

    Extracellular vesicles (EVs) and polyethyleneimine (PEI)-engineered EVs (PEI-EVs) characterization. a Tapping mode topographic AFM image showing round morphology of EVs and PEI-EVs. b Average size and ζ-potential of EVs and PEI-EVs. High-magnification images (40×) of hGMSCs cured with c EVs and d PEI-EVs panel 1: hGMSCs cultured in the presence of WGA-stained EVs and PEI-EVs observed with confocal laser scanning microscopy after 24 h; panel 2: bright field of hGMSCs/WGA-stained EVs and PEI-EVs. e DLS size distribution histograms of EVs and PEI-EVs. f Western blot showed the positivity for CD9, CD63, CD81, and TSG101. Scale bars in c,d = 10 μm

    Journal: Stem Cell Research & Therapy

    Article Title: Three-dimensional printed PLA scaffold and human gingival stem cell-derived extracellular vesicles: a new tool for bone defect repair

    doi: 10.1186/s13287-018-0850-0

    Figure Lengend Snippet: Extracellular vesicles (EVs) and polyethyleneimine (PEI)-engineered EVs (PEI-EVs) characterization. a Tapping mode topographic AFM image showing round morphology of EVs and PEI-EVs. b Average size and ζ-potential of EVs and PEI-EVs. High-magnification images (40×) of hGMSCs cured with c EVs and d PEI-EVs panel 1: hGMSCs cultured in the presence of WGA-stained EVs and PEI-EVs observed with confocal laser scanning microscopy after 24 h; panel 2: bright field of hGMSCs/WGA-stained EVs and PEI-EVs. e DLS size distribution histograms of EVs and PEI-EVs. f Western blot showed the positivity for CD9, CD63, CD81, and TSG101. Scale bars in c,d = 10 μm

    Article Snippet: Engineered EVs preparation The EVs pellet (100 μL) was resuspended with 2 mL PBS, and 2 mL of branched polyethyleneimine solution (PEI; MW 25,000; Sigma-Aldrich, Milan, Italy) (0.05 mg/mL in 0.3 M NaCl) was added to the EVs suspension in PBS and the mixture was incubated for 20 min at room temperature.

    Techniques: Cell Culture, Whole Genome Amplification, Staining, Confocal Laser Scanning Microscopy, Western Blot

    Sc Sls1p promotes the ATPase activity of Sc Kar2p along with GST-63Jp. Sc Kar2p (1 μM) was incubated with cold ATP (200 μM) and [α- 32 P]ATP (0.1 μCi, 3,000 Ci/mmol). Where indicated, the following proteins were present in the assay: GST-63Jp (2 μM), Sc Sls1p (4 μM), Sc Sls1.5p (4 μM), α-Lact (4 μM), and RCMLA (4 μM). After 10 min of incubation at room temperature, 1 μl of each reaction mixture was spotted in triplicate onto polyethyleneimine TLC plates, and the conversion of [α- 32 P]ATP (black arrowhead) to [α- 32 P]ADP (white arrowhead) was analyzed by using a PhosphorImager (Molecular Dynamics).

    Journal: Molecular and Cellular Biology

    Article Title: Sls1p Stimulates Sec63p-Mediated Activation of Kar2p in a Conformation-Dependent Manner in the Yeast Endoplasmic Reticulum

    doi:

    Figure Lengend Snippet: Sc Sls1p promotes the ATPase activity of Sc Kar2p along with GST-63Jp. Sc Kar2p (1 μM) was incubated with cold ATP (200 μM) and [α- 32 P]ATP (0.1 μCi, 3,000 Ci/mmol). Where indicated, the following proteins were present in the assay: GST-63Jp (2 μM), Sc Sls1p (4 μM), Sc Sls1.5p (4 μM), α-Lact (4 μM), and RCMLA (4 μM). After 10 min of incubation at room temperature, 1 μl of each reaction mixture was spotted in triplicate onto polyethyleneimine TLC plates, and the conversion of [α- 32 P]ATP (black arrowhead) to [α- 32 P]ADP (white arrowhead) was analyzed by using a PhosphorImager (Molecular Dynamics).

    Article Snippet: Reactions were stopped on ice, and 1 μl was spotted in triplicate onto polyethyleneimine cellulose thin-layer chromatography (TLC) plates (Sigma).

    Techniques: Activity Assay, Incubation, Thin Layer Chromatography

    Single-turnover ATPase assays. (A) A [α- 32 P] Sc Kar2 complex was formed after incubation of Sc Kar2p with 100 μCi of [α- 32 P]ATP for 10 min at room temperature and removal of free nucleotide by rapid gel filtration on microspin G-50 columns. [α- 32 P] Sc Kar2 (1 μM) was further incubated with or without GST-63Jp (1 μM) and/or Sc Sls1p (4 μM) in the presence of cold ATP (100 μM). Aliquots were obtained at 1, 2, 5, and 10 min and then separated from free nucleotide by gel filtration on microspin G-50 columns. Then, 3 μl from each reaction was spotted in triplicate onto polyethyleneimine TLC plates, and the conversion of [α- 32 P]ATP (black arrowhead) to [α- 32 P]ADP (white arrowhead) was analyzed using a PhosphorImager (Molecular Dynamics). K, J, and S represent Sc Kar2p, GST-63Jp, and Sc Sls1p, respectively. Quantification of ATP (B) and ADP (C) was performed with the ImageQuant software, and results were averaged from three independent experiments. Values were plotted as a function of time (K, diamonds; KS, triangles; KJ, squares; KJS, circles).

    Journal: Molecular and Cellular Biology

    Article Title: Sls1p Stimulates Sec63p-Mediated Activation of Kar2p in a Conformation-Dependent Manner in the Yeast Endoplasmic Reticulum

    doi:

    Figure Lengend Snippet: Single-turnover ATPase assays. (A) A [α- 32 P] Sc Kar2 complex was formed after incubation of Sc Kar2p with 100 μCi of [α- 32 P]ATP for 10 min at room temperature and removal of free nucleotide by rapid gel filtration on microspin G-50 columns. [α- 32 P] Sc Kar2 (1 μM) was further incubated with or without GST-63Jp (1 μM) and/or Sc Sls1p (4 μM) in the presence of cold ATP (100 μM). Aliquots were obtained at 1, 2, 5, and 10 min and then separated from free nucleotide by gel filtration on microspin G-50 columns. Then, 3 μl from each reaction was spotted in triplicate onto polyethyleneimine TLC plates, and the conversion of [α- 32 P]ATP (black arrowhead) to [α- 32 P]ADP (white arrowhead) was analyzed using a PhosphorImager (Molecular Dynamics). K, J, and S represent Sc Kar2p, GST-63Jp, and Sc Sls1p, respectively. Quantification of ATP (B) and ADP (C) was performed with the ImageQuant software, and results were averaged from three independent experiments. Values were plotted as a function of time (K, diamonds; KS, triangles; KJ, squares; KJS, circles).

    Article Snippet: Reactions were stopped on ice, and 1 μl was spotted in triplicate onto polyethyleneimine cellulose thin-layer chromatography (TLC) plates (Sigma).

    Techniques: Incubation, Filtration, Thin Layer Chromatography, Software