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Mirus Bio plasmid dna
Overexpression of Cx43 in Sertoli cells rescues PFOS-induced Sertoli cell BTB disruption through proper localization of adhesion proteins at the cell-cell interface. Sertoli cells were cultured at 0.1–0.15 × 10 6 cells/cm 2 for two days. They were transfected with pCI-neo/Cx43 vs. pCI-neo empty vector (control, Ctrl) and then subjected to PFOS treatment, and cells were harvested for IF analysis as noted in the regimen shown in Fig. 3A . In cells overexpressed with Cx43 (pCI-neo/Cx43), the fluorescent intensity for Cx43 at cell-cell interface was moderately stronger than control (pCI-neo empty vector alone) cells. However, PFOS induced considerable down-regulation of Cx43 and claudin 11 at the cell-cell interface (pCI-neo+PFOS), PFOS also perturbed the localization of TJ protein ZO-1, and basal ES proteins N-cadherin and β-catenin. Importantly, overexpression of Cx43 in PFOS treated cells (pCI-neo/Cx43+PFOS) rescued the PFOS-mediated BTB disruption by maintaining the expression of Cx43 and claudin 11. This also induced proper localization of TJ protein ZO-1 and basal ES proteins N-cadherin and ß-catenin at the cell-cell interface. Plasmid <t>DNA</t> (pCI-neo vs. pCI-neo/Cx43) was labeled with Cy3 and appeared as red fluorescence to annotate successful <t>transfection.</t> Scale bar, 30 μm, in the first micrograph, which applies to the remaining micrographs in the same panel. These results are representative micrographs from an experiment, and n = 3 independent experiments were performed using different batches of Sertoli cells and yielded similar results. Each bar graph is a mean ± SD of n = 3 experiments. ** P
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1) Product Images from "Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43"

Article Title: Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

Journal: Scientific Reports

doi: 10.1038/srep29667

Overexpression of Cx43 in Sertoli cells rescues PFOS-induced Sertoli cell BTB disruption through proper localization of adhesion proteins at the cell-cell interface. Sertoli cells were cultured at 0.1–0.15 × 10 6 cells/cm 2 for two days. They were transfected with pCI-neo/Cx43 vs. pCI-neo empty vector (control, Ctrl) and then subjected to PFOS treatment, and cells were harvested for IF analysis as noted in the regimen shown in Fig. 3A . In cells overexpressed with Cx43 (pCI-neo/Cx43), the fluorescent intensity for Cx43 at cell-cell interface was moderately stronger than control (pCI-neo empty vector alone) cells. However, PFOS induced considerable down-regulation of Cx43 and claudin 11 at the cell-cell interface (pCI-neo+PFOS), PFOS also perturbed the localization of TJ protein ZO-1, and basal ES proteins N-cadherin and β-catenin. Importantly, overexpression of Cx43 in PFOS treated cells (pCI-neo/Cx43+PFOS) rescued the PFOS-mediated BTB disruption by maintaining the expression of Cx43 and claudin 11. This also induced proper localization of TJ protein ZO-1 and basal ES proteins N-cadherin and ß-catenin at the cell-cell interface. Plasmid DNA (pCI-neo vs. pCI-neo/Cx43) was labeled with Cy3 and appeared as red fluorescence to annotate successful transfection. Scale bar, 30 μm, in the first micrograph, which applies to the remaining micrographs in the same panel. These results are representative micrographs from an experiment, and n = 3 independent experiments were performed using different batches of Sertoli cells and yielded similar results. Each bar graph is a mean ± SD of n = 3 experiments. ** P
Figure Legend Snippet: Overexpression of Cx43 in Sertoli cells rescues PFOS-induced Sertoli cell BTB disruption through proper localization of adhesion proteins at the cell-cell interface. Sertoli cells were cultured at 0.1–0.15 × 10 6 cells/cm 2 for two days. They were transfected with pCI-neo/Cx43 vs. pCI-neo empty vector (control, Ctrl) and then subjected to PFOS treatment, and cells were harvested for IF analysis as noted in the regimen shown in Fig. 3A . In cells overexpressed with Cx43 (pCI-neo/Cx43), the fluorescent intensity for Cx43 at cell-cell interface was moderately stronger than control (pCI-neo empty vector alone) cells. However, PFOS induced considerable down-regulation of Cx43 and claudin 11 at the cell-cell interface (pCI-neo+PFOS), PFOS also perturbed the localization of TJ protein ZO-1, and basal ES proteins N-cadherin and β-catenin. Importantly, overexpression of Cx43 in PFOS treated cells (pCI-neo/Cx43+PFOS) rescued the PFOS-mediated BTB disruption by maintaining the expression of Cx43 and claudin 11. This also induced proper localization of TJ protein ZO-1 and basal ES proteins N-cadherin and ß-catenin at the cell-cell interface. Plasmid DNA (pCI-neo vs. pCI-neo/Cx43) was labeled with Cy3 and appeared as red fluorescence to annotate successful transfection. Scale bar, 30 μm, in the first micrograph, which applies to the remaining micrographs in the same panel. These results are representative micrographs from an experiment, and n = 3 independent experiments were performed using different batches of Sertoli cells and yielded similar results. Each bar graph is a mean ± SD of n = 3 experiments. ** P

Techniques Used: Over Expression, Cell Culture, Transfection, Plasmid Preparation, Expressing, Labeling, Fluorescence

Overexpression of Cx43 in Sertoli cells restores actin microfilament organization via proper spatiotemporal expression of actin binding/regulatory proteins Arp3 and Eps8. ( A ) We next examined the mechanism by which Cx43 overexpression rescues the PFOS-induced Sertoli cell TJ-barrier disruption. Exposure of Sertoli cells with an established functional TJ-permeability barrier to PFOS for 24 hr was found to induce mis-orgranization of actin microfilaments wherein they displayed extensive defragmentation across the Sertoli cell cytosol. However, overexpression of Cx43 restored organization of actin microfilaments in Sertoli cells, apparently via proper spatiotemporal expression of actin binding and regulatory proteins Arp3 and Eps8, such that these proteins localized to the Sertoli cell-cell interface, similar to the control cells expressed with the empty pCI-neo vector. Plasmid DNA (pCI-neo vs. pCI-neo/Cx43) was labeled with Cy3 and appeared as red fluorescence to annotate successful transfection. Scale bar, 30 μm, which applies to all other micrographs. ( B ) Relative fluorescent intensity of Arp3 and Eps8 at the Sertoli cell-cell interface was quantified in the PFOS treatment group (pCI-neo+PFOS) vs. control (Ctrl, transfected with pCI-neo vector alone), Cx43 overexpression (pCI-neo/Cx43) and Cx43 overexpression plus PFOS (pCI-neo/Cx43+PFOS) groups. Each bar is a mean ± SD by taking the average fluorescent intensity from two opposite ends between adjacent Sertoli cells (see corresponding colored boxed area for Arp3 vs. Eps8 from different groups) from 50 randomly selected cells in each experiment with n = 3 independent experiments. ** P
Figure Legend Snippet: Overexpression of Cx43 in Sertoli cells restores actin microfilament organization via proper spatiotemporal expression of actin binding/regulatory proteins Arp3 and Eps8. ( A ) We next examined the mechanism by which Cx43 overexpression rescues the PFOS-induced Sertoli cell TJ-barrier disruption. Exposure of Sertoli cells with an established functional TJ-permeability barrier to PFOS for 24 hr was found to induce mis-orgranization of actin microfilaments wherein they displayed extensive defragmentation across the Sertoli cell cytosol. However, overexpression of Cx43 restored organization of actin microfilaments in Sertoli cells, apparently via proper spatiotemporal expression of actin binding and regulatory proteins Arp3 and Eps8, such that these proteins localized to the Sertoli cell-cell interface, similar to the control cells expressed with the empty pCI-neo vector. Plasmid DNA (pCI-neo vs. pCI-neo/Cx43) was labeled with Cy3 and appeared as red fluorescence to annotate successful transfection. Scale bar, 30 μm, which applies to all other micrographs. ( B ) Relative fluorescent intensity of Arp3 and Eps8 at the Sertoli cell-cell interface was quantified in the PFOS treatment group (pCI-neo+PFOS) vs. control (Ctrl, transfected with pCI-neo vector alone), Cx43 overexpression (pCI-neo/Cx43) and Cx43 overexpression plus PFOS (pCI-neo/Cx43+PFOS) groups. Each bar is a mean ± SD by taking the average fluorescent intensity from two opposite ends between adjacent Sertoli cells (see corresponding colored boxed area for Arp3 vs. Eps8 from different groups) from 50 randomly selected cells in each experiment with n = 3 independent experiments. ** P

Techniques Used: Over Expression, Expressing, Binding Assay, Functional Assay, Permeability, Plasmid Preparation, Labeling, Fluorescence, Transfection

2) Product Images from "14-3-3 Proteins Restrain the Exo1 Nuclease to Prevent Overresection *"

Article Title: 14-3-3 Proteins Restrain the Exo1 Nuclease to Prevent Overresection *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.644005

The interaction of Exo1 with 14-3-3s promotes cell survival after DNA damage. A , protein levels of Exo1, mCherry-Difopein, and PCNA in control knockdown or Exo1 knockdown U2OS cells expressing mCherry-Difopein(WT) or mCherry-Difopein(MUT). B , RPA phosphorylation
Figure Legend Snippet: The interaction of Exo1 with 14-3-3s promotes cell survival after DNA damage. A , protein levels of Exo1, mCherry-Difopein, and PCNA in control knockdown or Exo1 knockdown U2OS cells expressing mCherry-Difopein(WT) or mCherry-Difopein(MUT). B , RPA phosphorylation

Techniques Used: Expressing, Recombinase Polymerase Amplification

3) Product Images from "Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA"

Article Title: Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkg801

Binding stability of LNA and PNA ODNs to plasmid in gene gun preparations. ( A ) Agarose gel (without EtBr) of 0.024 µM plasmid pGG2XGFP incubated with 4 µM Alexa Fluor 568 (LNA) or rhodamine-labelled (PNA) ODNs before (b) and after (a) plasmid binding to gold beads. ( B ) As (A) with EtBr. Lane M, 100 bp DNA ladder. ( C ) Agarose gel (without EtBr) of 0.023 µM plasmid gWiz incubated with 4 µM rhodamine-labelled LNA or PNA ODNs after (a) plasmid binding to gold beads, unless stated as before (b). ( D ) As (C) with EtBr. Lane M, 1 kb DNA ladder; Mirus, pGL3CMV rhodamine labelled with Mirus Label IT kit.
Figure Legend Snippet: Binding stability of LNA and PNA ODNs to plasmid in gene gun preparations. ( A ) Agarose gel (without EtBr) of 0.024 µM plasmid pGG2XGFP incubated with 4 µM Alexa Fluor 568 (LNA) or rhodamine-labelled (PNA) ODNs before (b) and after (a) plasmid binding to gold beads. ( B ) As (A) with EtBr. Lane M, 100 bp DNA ladder. ( C ) Agarose gel (without EtBr) of 0.023 µM plasmid gWiz incubated with 4 µM rhodamine-labelled LNA or PNA ODNs after (a) plasmid binding to gold beads, unless stated as before (b). ( D ) As (C) with EtBr. Lane M, 1 kb DNA ladder; Mirus, pGL3CMV rhodamine labelled with Mirus Label IT kit.

Techniques Used: Binding Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

4) Product Images from "Enhancement of Poly(orthoester) Microspheres for DNA Vaccine Delivery by Blending with Poly(ethylenimine)"

Article Title: Enhancement of Poly(orthoester) Microspheres for DNA Vaccine Delivery by Blending with Poly(ethylenimine)

Journal:

doi: 10.1016/j.biomaterials.2008.03.01

Confocal micrographs of P2 microspheres at 100x optical magnification with (a) FITC-labeled PEI (b) Rhodamine-labeled plasmid DNA and unlabelled PEI (c) Both FITC-PEI and Rhodamine-DNA (d) Cy5-labelled DNA (without PEI). Scale bar indicates 5 um.
Figure Legend Snippet: Confocal micrographs of P2 microspheres at 100x optical magnification with (a) FITC-labeled PEI (b) Rhodamine-labeled plasmid DNA and unlabelled PEI (c) Both FITC-PEI and Rhodamine-DNA (d) Cy5-labelled DNA (without PEI). Scale bar indicates 5 um.

Techniques Used: Labeling, Plasmid Preparation

5) Product Images from "Cellular Uptake of Cationic Polymer-DNA Complexes Via Caveolae Plays a Pivotal Role in Gene Transfection in COS-7 Cells"

Article Title: Cellular Uptake of Cationic Polymer-DNA Complexes Via Caveolae Plays a Pivotal Role in Gene Transfection in COS-7 Cells

Journal: Pharmaceutical Research

doi: 10.1007/s11095-007-9287-3

Intracellular location of pDMAEMA-based polyplexes in COS-7 cells using spectral imaging. Polyplexes with rhodamine labeled DNA co-localized partly with transferrin alexa 488 ( a ) and partly with cholera toxin B Alexa 488 ( b ) after 1-h incubation. Representative pictures are displayed.
Figure Legend Snippet: Intracellular location of pDMAEMA-based polyplexes in COS-7 cells using spectral imaging. Polyplexes with rhodamine labeled DNA co-localized partly with transferrin alexa 488 ( a ) and partly with cholera toxin B Alexa 488 ( b ) after 1-h incubation. Representative pictures are displayed.

Techniques Used: Imaging, Labeling, Incubation

Intracellular location of linear PEI-based polyplexes in COS-7 cells. With transferrin Alexa 488 clear co-localization is observed for rhodamine labeled DNA ( a ) the large PEI-based polyplexes also partly co-localize with cholera toxin B ( b ) after 1-h incubation. Representative pictures are displayed.
Figure Legend Snippet: Intracellular location of linear PEI-based polyplexes in COS-7 cells. With transferrin Alexa 488 clear co-localization is observed for rhodamine labeled DNA ( a ) the large PEI-based polyplexes also partly co-localize with cholera toxin B ( b ) after 1-h incubation. Representative pictures are displayed.

Techniques Used: Labeling, Incubation

6) Product Images from "p-FAK-Tyr397 regulates spermatid adhesion in the rat testis via its effects on F-actin organization at the ectoplasmic specialization"

Article Title: p-FAK-Tyr397 regulates spermatid adhesion in the rat testis via its effects on F-actin organization at the ectoplasmic specialization

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00254.2013

Overexpression of phosphorylated (p) focal adhesion kinase (FAK) p-FAK-Tyr 397 via the use of FAK Y397E phosphomimetic mutant in rat testes in vivo impairs spermiation. Testes were transfected with plasmid DNA as described in materials and methods with
Figure Legend Snippet: Overexpression of phosphorylated (p) focal adhesion kinase (FAK) p-FAK-Tyr 397 via the use of FAK Y397E phosphomimetic mutant in rat testes in vivo impairs spermiation. Testes were transfected with plasmid DNA as described in materials and methods with

Techniques Used: Over Expression, Mutagenesis, In Vivo, Transfection, Plasmid Preparation

7) Product Images from "PhiC31-based Site-Specific Transgenesis System for Production of Transgenic Bovine Embryos by Somatic Cell Nuclear Transfer and Intracytoplasmic Sperm Injection"

Article Title: PhiC31-based Site-Specific Transgenesis System for Production of Transgenic Bovine Embryos by Somatic Cell Nuclear Transfer and Intracytoplasmic Sperm Injection

Journal: Cell Journal (Yakhteh)

doi: 10.22074/cellj.2018.4385.

Head sperm DNA uptake, sperm mediated gene transfer and screening for EGFP expression in SMGT derived embryos. A. Sperm decondensation and exogenous DNA uptake, I. Phase contrast microscopy observation for decondensedsperm, II. Hoechst staining for decondensedsperm, III. The pattern of DNA uptake by decondensedsperm. This pattern depicted by arrowseither over acrosomal ridge or in the post-acrosomal region, B. SMGT derived embryos, I. Blastocysts formation by injection of sperm which incubatedwith 200 ng pDB2 for 30 minutes at RT, II. Blastocysts formation by injectionof sperm which incubated with 1000 ng pDB2 for 30 minutes at RT, and C. RT-PCR for detection of EGFP expressionin SMGT derived embryos. Lane 1: DNA marker, I. SMGT derived embryos obtained by injection of sperm whichincubated by 200 ng pDB2 for 30 minutes at RT, and II. SMGT derived embryosobtained by injection of sperm which incubated by 1000 ng pDB2 for 30 minutes at RT (scale bars: 15 µm). SMGT; Sperm mediated gene transfer and RT-PCR; Real time-polymerase chain reaction.
Figure Legend Snippet: Head sperm DNA uptake, sperm mediated gene transfer and screening for EGFP expression in SMGT derived embryos. A. Sperm decondensation and exogenous DNA uptake, I. Phase contrast microscopy observation for decondensedsperm, II. Hoechst staining for decondensedsperm, III. The pattern of DNA uptake by decondensedsperm. This pattern depicted by arrowseither over acrosomal ridge or in the post-acrosomal region, B. SMGT derived embryos, I. Blastocysts formation by injection of sperm which incubatedwith 200 ng pDB2 for 30 minutes at RT, II. Blastocysts formation by injectionof sperm which incubated with 1000 ng pDB2 for 30 minutes at RT, and C. RT-PCR for detection of EGFP expressionin SMGT derived embryos. Lane 1: DNA marker, I. SMGT derived embryos obtained by injection of sperm whichincubated by 200 ng pDB2 for 30 minutes at RT, and II. SMGT derived embryosobtained by injection of sperm which incubated by 1000 ng pDB2 for 30 minutes at RT (scale bars: 15 µm). SMGT; Sperm mediated gene transfer and RT-PCR; Real time-polymerase chain reaction.

Techniques Used: Expressing, Derivative Assay, Microscopy, Staining, Injection, Incubation, Reverse Transcription Polymerase Chain Reaction, Marker, Real-time Polymerase Chain Reaction

8) Product Images from "Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B"

Article Title: Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B

Journal: Journal of Virology

doi: 10.1128/JVI.00197-18

Generation of a cell line stably expressing the JEV C-prM-E protein and preparation of RRPs. (A) Schematic representation of the generation of a stable cell line expressing the JEV C-prM-E protein and the preparation of RRPs. The BJEV-CME cell line stably expressing the C-prM-E protein was generated by transfecting BHK-21 cells with the pCAG-opti-JEV-CME plasmid, followed by cloning and selection with G418. To produce RRPs, BJEV-CME cells were transfected with a DNA-based WNV replicon under the control of the cytomegalovirus (CMV) promoter. The replicon genome lacks the major coding sequence of the structural protein C-prM-E, and the corresponding sequence was replaced with a GFP-coding sequence following the foot-and-mouth disease virus (FMDV) 2A coding sequence. The replicon plasmid was transcribed by the cytomegalovirus promoter into the replicon RNA expressing GFP and the nonstructural replicase protein. BJEV-CME cells express the C-prM-E polyprotein, which is cleaved into the C, prM, and E proteins by replicon RNA-encoded nonstructural protease and endogenous cellular signal peptidase (SP). The replicon RNA amplifies itself again, and the three structural proteins package the replicon RNA into RRPs, which are secreted into the culture medium. When RRPs infect other JEV-susceptible cells, such as BHK-21 cells, the replicon RNA expresses GFP and nonstructural proteins, which amplifies more RNA. However, no structural proteins or additional RRPs are produced in RRP-infected cells, thereby preventing further replication. When BJEV-CME cells are infected with RRPs, the structural proteins expressed by the cells package the replicon RNA into progeny RRPs, and the infection spreads in rounds similar to those for the wild-type virus. UTR, untranslated region. (B) Cloned and selected stable cell lines were identified by an IFA with JEV E protein-specific MAb 5E7. (C) Production of RRPs from the WNV replicon plasmid by transfection of BJEV-CME cell lines. Green florescence was visualized when replicon plasmid pWNVrepdCME-GFP was transfected into BJEV-CME cells (first infection) and BHK-21 cells (second infection). Transfected-cell supernatants were passaged onto fresh cell cultures, as indicated by the arrows, and green florescence was observed only in BJEV-CME cells. At 3 days postinoculation, supernatants of cells infected first were used to inoculate cells, as indicated by the arrows, for a second infection, and green florescence was analyzed at 72 h postinfection. HRr, hammerhead ribozyme; HDVr, hepatitis delta virus ribozyme.
Figure Legend Snippet: Generation of a cell line stably expressing the JEV C-prM-E protein and preparation of RRPs. (A) Schematic representation of the generation of a stable cell line expressing the JEV C-prM-E protein and the preparation of RRPs. The BJEV-CME cell line stably expressing the C-prM-E protein was generated by transfecting BHK-21 cells with the pCAG-opti-JEV-CME plasmid, followed by cloning and selection with G418. To produce RRPs, BJEV-CME cells were transfected with a DNA-based WNV replicon under the control of the cytomegalovirus (CMV) promoter. The replicon genome lacks the major coding sequence of the structural protein C-prM-E, and the corresponding sequence was replaced with a GFP-coding sequence following the foot-and-mouth disease virus (FMDV) 2A coding sequence. The replicon plasmid was transcribed by the cytomegalovirus promoter into the replicon RNA expressing GFP and the nonstructural replicase protein. BJEV-CME cells express the C-prM-E polyprotein, which is cleaved into the C, prM, and E proteins by replicon RNA-encoded nonstructural protease and endogenous cellular signal peptidase (SP). The replicon RNA amplifies itself again, and the three structural proteins package the replicon RNA into RRPs, which are secreted into the culture medium. When RRPs infect other JEV-susceptible cells, such as BHK-21 cells, the replicon RNA expresses GFP and nonstructural proteins, which amplifies more RNA. However, no structural proteins or additional RRPs are produced in RRP-infected cells, thereby preventing further replication. When BJEV-CME cells are infected with RRPs, the structural proteins expressed by the cells package the replicon RNA into progeny RRPs, and the infection spreads in rounds similar to those for the wild-type virus. UTR, untranslated region. (B) Cloned and selected stable cell lines were identified by an IFA with JEV E protein-specific MAb 5E7. (C) Production of RRPs from the WNV replicon plasmid by transfection of BJEV-CME cell lines. Green florescence was visualized when replicon plasmid pWNVrepdCME-GFP was transfected into BJEV-CME cells (first infection) and BHK-21 cells (second infection). Transfected-cell supernatants were passaged onto fresh cell cultures, as indicated by the arrows, and green florescence was observed only in BJEV-CME cells. At 3 days postinoculation, supernatants of cells infected first were used to inoculate cells, as indicated by the arrows, for a second infection, and green florescence was analyzed at 72 h postinfection. HRr, hammerhead ribozyme; HDVr, hepatitis delta virus ribozyme.

Techniques Used: Stable Transfection, Expressing, Generated, Plasmid Preparation, Clone Assay, Selection, Transfection, Sequencing, Produced, Infection, Immunofluorescence

Related Articles

Transfection:

Article Title: Modulation of TGFβ-inducible hypermotility by EGF and other factors in human prostate epithelial cells and keratinocytes
Article Snippet: Images were captured on a NIKON E600 Microscope with a SPOT2 digital camera using SPOTcam v.3.5.5 software (Digital Instruments, Tonowanda, NY). .. The amphotropic retroviral packaging cell line Phoenix [ ] was transfected with pBABE/bmi1.puro [ ] or pWzl.Bsd/TERT [ ] plasmid DNA using TransIT-LT1 transfection reagent (Mirus Bio LCC, Madison, WI). ..

Article Title: Multiple cytosolic DNA sensors bind plasmid DNA after transfection
Article Snippet: The collected Z-stacks were then visualized in Imaris software (Bitplane, Zurich, Switzerland). .. For colocalization studies, pDNA was stained before transfection with Label IT Nucleic Acid Labeling Kit, MPF488, (MIR7100, Mirus Bio, Madison, USA) according to manufacturer's instructions. .. Five minutes after the transfection, culture media was added to the wells and slides were incubated for a total of 15 min.

Article Title: Interaction of Hepatitis C Virus Nonstructural Protein 5A with Core Protein Is Critical for the Production of Infectious Virus Particles
Article Snippet: For coexpression of FLAG-tagged core protein and HA-tagged NS5A, cells were seeded onto 35-mm wells of a six-well cell culture plate and cultured overnight. .. Plasmid DNAs (2 μg) were transfected into cells using TransIT-LT1 transfection reagent (Mirus, Madison, WI). .. Cells were harvested at 48 h posttransfection, washed three times with 1 ml of ice-cold phosphate-buffered saline (PBS), and suspended in 0.25 ml lysis buffer (20 mM Tris-HCl [pH 7.4] containing 135 mM NaCl, 1% Triton X-100, 0.05% sodium dodecyl sulfate [SDS], and 10% glycerol) supplemented with 50 mM NaF, 5 mM Na3 VO4 , 1 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF).

Article Title: Multiple cytosolic DNA sensors bind plasmid DNA after transfection
Article Snippet: After 5 min of incubation, DMEM was added and the plates were incubated at 37°C in 5% CO2 for the time designated in each experiment. .. Transfections were also performed using Transit LT1 (Mirus Bio LC, Madison, WI, USA) per manufacturer's instructions. .. Transfection efficiency Transfection efficiency was quantified 24 h after transfection as previously described ( ).

Plasmid Preparation:

Article Title: Modulation of TGFβ-inducible hypermotility by EGF and other factors in human prostate epithelial cells and keratinocytes
Article Snippet: Images were captured on a NIKON E600 Microscope with a SPOT2 digital camera using SPOTcam v.3.5.5 software (Digital Instruments, Tonowanda, NY). .. The amphotropic retroviral packaging cell line Phoenix [ ] was transfected with pBABE/bmi1.puro [ ] or pWzl.Bsd/TERT [ ] plasmid DNA using TransIT-LT1 transfection reagent (Mirus Bio LCC, Madison, WI). ..

Article Title: Interaction of Hepatitis C Virus Nonstructural Protein 5A with Core Protein Is Critical for the Production of Infectious Virus Particles
Article Snippet: For coexpression of FLAG-tagged core protein and HA-tagged NS5A, cells were seeded onto 35-mm wells of a six-well cell culture plate and cultured overnight. .. Plasmid DNAs (2 μg) were transfected into cells using TransIT-LT1 transfection reagent (Mirus, Madison, WI). .. Cells were harvested at 48 h posttransfection, washed three times with 1 ml of ice-cold phosphate-buffered saline (PBS), and suspended in 0.25 ml lysis buffer (20 mM Tris-HCl [pH 7.4] containing 135 mM NaCl, 1% Triton X-100, 0.05% sodium dodecyl sulfate [SDS], and 10% glycerol) supplemented with 50 mM NaF, 5 mM Na3 VO4 , 1 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF).

Article Title: Quantification of Cellular and Nuclear Uptake Rates of Polymeric Gene Delivery Nanoparticles and DNA Plasmids via Flow Cytometry
Article Snippet: A Mirus Label IT® Tracker™ Cy™ 3 Kit was used to conjugate Cy3 to enhanced Green Fluorescent Protein (eGFP-N1; 4733 base pairs (bp)) plasmids. .. In brief, 75 µg plasmid DNA (pDNA; 1 µg/µL), 60 µL of Mirus Buffer A and 415 µL of ultrapure distilled water (DNase and RNase free) was added to 50 µL of the Cy3 dye reagent and incubated for at least 3 hours; at each hour the vials were briefly centrifuged to ensure all reagents were homogeneously reacting. .. Subsequently, 60 µL of 3 M sodium acetate (NaAc) and 1880 µL of ice cold 190 proof ethanol (EtOH) were added and the solution was kept at −20°C for at least 30 additional minutes.

Staining:

Article Title: Multiple cytosolic DNA sensors bind plasmid DNA after transfection
Article Snippet: The collected Z-stacks were then visualized in Imaris software (Bitplane, Zurich, Switzerland). .. For colocalization studies, pDNA was stained before transfection with Label IT Nucleic Acid Labeling Kit, MPF488, (MIR7100, Mirus Bio, Madison, USA) according to manufacturer's instructions. .. Five minutes after the transfection, culture media was added to the wells and slides were incubated for a total of 15 min.

Labeling:

Article Title: Multiple cytosolic DNA sensors bind plasmid DNA after transfection
Article Snippet: The collected Z-stacks were then visualized in Imaris software (Bitplane, Zurich, Switzerland). .. For colocalization studies, pDNA was stained before transfection with Label IT Nucleic Acid Labeling Kit, MPF488, (MIR7100, Mirus Bio, Madison, USA) according to manufacturer's instructions. .. Five minutes after the transfection, culture media was added to the wells and slides were incubated for a total of 15 min.

Incubation:

Article Title: Quantification of Cellular and Nuclear Uptake Rates of Polymeric Gene Delivery Nanoparticles and DNA Plasmids via Flow Cytometry
Article Snippet: A Mirus Label IT® Tracker™ Cy™ 3 Kit was used to conjugate Cy3 to enhanced Green Fluorescent Protein (eGFP-N1; 4733 base pairs (bp)) plasmids. .. In brief, 75 µg plasmid DNA (pDNA; 1 µg/µL), 60 µL of Mirus Buffer A and 415 µL of ultrapure distilled water (DNase and RNase free) was added to 50 µL of the Cy3 dye reagent and incubated for at least 3 hours; at each hour the vials were briefly centrifuged to ensure all reagents were homogeneously reacting. .. Subsequently, 60 µL of 3 M sodium acetate (NaAc) and 1880 µL of ice cold 190 proof ethanol (EtOH) were added and the solution was kept at −20°C for at least 30 additional minutes.

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    Mirus Bio fitc labeled plasmid dna
    Characterization of NCs’ encapsulation efficiency and cargo release profile. (a) EE for <t>FITC-labeled</t> plasmid <t>DNA,</t> water-soluble quantum dots, doxorubicin, and pyrene. The EE of hydrophobic pyrene are generally high in all the formulations, where
    Fitc Labeled Plasmid Dna, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mirus Bio plasmid dna
    <t>DNA</t> nanocarriers choreograph robust and persistent CAR production by lymphocytes in vitro a , Flow cytometry of nanoparticles binding to T cells. Splenocytes from naive C57BL/6 mice were mixed with CD3-targeted nanoparticles carrying <t>Cy5-labelled</t> plasmid DNA. After a 20-min incubation, cells were washed to remove unbound particles, and analysed by flow cytometry. The profiles shown here are representative of eight independent experiments. b , Confocal microscopy establishes that nanoparticles loaded with Cy5-labelled DNA (magenta) are rapidly (within 120 min) internalized from the cell surface. To provide contrast, T cells were labelled with CellTracker Green prior to nanoparticle exposure. The images are representative of 20 randomly chosen fields. Scale bars, 2 μm. c , Flow cytometry of T cells 30 h after incubation with nanoparticles bearing 194-1BBz_2A_GFP genes. The graph displays CD3 + -gated lymphocyte populations. d , Comparison of T-cell transfection efficiencies achieved with DNA nanocarriers that contain microtubule-associated and nuclear localization signalling peptide sequences with those that do not, based on 10 independent experiments. e , Flow cytometry of T cells 30 h after transduction with a lentiviral vector encoding the same 194-1BBz_2A_GFP construct. The numbers within the graphs in c and e show the percentage of CAR + T cells (using GFP as surrogate marker). f , In vitro assay comparing cytotoxicity of nanoparticle-transfected versus lentivirus-transfected T cells against Eμ-ALL01 leukaemia cells, or the B16F10 cell line as a control. To ensure equal CAR expression levels, transfected T cells were sorted using FACS to GFP mean fluorescence intensities (as a surrogate reporter for CAR expression) between 10 3 and 10 4 before using them in the functional assays. Each point represents the mean ± s.e.m. pooled from independent experiments conducted in triplicate. g , ELISA measurements of IL-2 (at 24 h), and IFN-γ and TNF-α (at 48 h) secretion by transfected cells following co-culture with Eμ-ALL01 leukaemia cells. Unstimulated, lentivirus-transduced 194-1BBz CAR T-cells were analysed for comparison. h , Co-delivery of plasmids encoding the hyperactive piggyBac transposase iPB7 promotes persistent CAR gene expression. After transfection and sorting, CAR-positive T cells were cultured and the persistence of their 194-1BBz_2A_GFP expression was measured with flow cytometry. Data are representative of two independent experiments.
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    Characterization of NCs’ encapsulation efficiency and cargo release profile. (a) EE for FITC-labeled plasmid DNA, water-soluble quantum dots, doxorubicin, and pyrene. The EE of hydrophobic pyrene are generally high in all the formulations, where

    Journal: ACS Nano

    Article Title: Multifunctional Nanocapsules for Simultaneous Encapsulation of Hydrophilic and Hydrophobic Compounds and On-Demand Release

    doi: 10.1021/nn205023w

    Figure Lengend Snippet: Characterization of NCs’ encapsulation efficiency and cargo release profile. (a) EE for FITC-labeled plasmid DNA, water-soluble quantum dots, doxorubicin, and pyrene. The EE of hydrophobic pyrene are generally high in all the formulations, where

    Article Snippet: FITC-labeled plasmid DNA (Label IT® Plasmid, 2.7 kb) was purchased from Mirus (Madison, WI).

    Techniques: Labeling, Plasmid Preparation

    DNA nanocarriers choreograph robust and persistent CAR production by lymphocytes in vitro a , Flow cytometry of nanoparticles binding to T cells. Splenocytes from naive C57BL/6 mice were mixed with CD3-targeted nanoparticles carrying Cy5-labelled plasmid DNA. After a 20-min incubation, cells were washed to remove unbound particles, and analysed by flow cytometry. The profiles shown here are representative of eight independent experiments. b , Confocal microscopy establishes that nanoparticles loaded with Cy5-labelled DNA (magenta) are rapidly (within 120 min) internalized from the cell surface. To provide contrast, T cells were labelled with CellTracker Green prior to nanoparticle exposure. The images are representative of 20 randomly chosen fields. Scale bars, 2 μm. c , Flow cytometry of T cells 30 h after incubation with nanoparticles bearing 194-1BBz_2A_GFP genes. The graph displays CD3 + -gated lymphocyte populations. d , Comparison of T-cell transfection efficiencies achieved with DNA nanocarriers that contain microtubule-associated and nuclear localization signalling peptide sequences with those that do not, based on 10 independent experiments. e , Flow cytometry of T cells 30 h after transduction with a lentiviral vector encoding the same 194-1BBz_2A_GFP construct. The numbers within the graphs in c and e show the percentage of CAR + T cells (using GFP as surrogate marker). f , In vitro assay comparing cytotoxicity of nanoparticle-transfected versus lentivirus-transfected T cells against Eμ-ALL01 leukaemia cells, or the B16F10 cell line as a control. To ensure equal CAR expression levels, transfected T cells were sorted using FACS to GFP mean fluorescence intensities (as a surrogate reporter for CAR expression) between 10 3 and 10 4 before using them in the functional assays. Each point represents the mean ± s.e.m. pooled from independent experiments conducted in triplicate. g , ELISA measurements of IL-2 (at 24 h), and IFN-γ and TNF-α (at 48 h) secretion by transfected cells following co-culture with Eμ-ALL01 leukaemia cells. Unstimulated, lentivirus-transduced 194-1BBz CAR T-cells were analysed for comparison. h , Co-delivery of plasmids encoding the hyperactive piggyBac transposase iPB7 promotes persistent CAR gene expression. After transfection and sorting, CAR-positive T cells were cultured and the persistence of their 194-1BBz_2A_GFP expression was measured with flow cytometry. Data are representative of two independent experiments.

    Journal: Nature nanotechnology

    Article Title: In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers

    doi: 10.1038/nnano.2017.57

    Figure Lengend Snippet: DNA nanocarriers choreograph robust and persistent CAR production by lymphocytes in vitro a , Flow cytometry of nanoparticles binding to T cells. Splenocytes from naive C57BL/6 mice were mixed with CD3-targeted nanoparticles carrying Cy5-labelled plasmid DNA. After a 20-min incubation, cells were washed to remove unbound particles, and analysed by flow cytometry. The profiles shown here are representative of eight independent experiments. b , Confocal microscopy establishes that nanoparticles loaded with Cy5-labelled DNA (magenta) are rapidly (within 120 min) internalized from the cell surface. To provide contrast, T cells were labelled with CellTracker Green prior to nanoparticle exposure. The images are representative of 20 randomly chosen fields. Scale bars, 2 μm. c , Flow cytometry of T cells 30 h after incubation with nanoparticles bearing 194-1BBz_2A_GFP genes. The graph displays CD3 + -gated lymphocyte populations. d , Comparison of T-cell transfection efficiencies achieved with DNA nanocarriers that contain microtubule-associated and nuclear localization signalling peptide sequences with those that do not, based on 10 independent experiments. e , Flow cytometry of T cells 30 h after transduction with a lentiviral vector encoding the same 194-1BBz_2A_GFP construct. The numbers within the graphs in c and e show the percentage of CAR + T cells (using GFP as surrogate marker). f , In vitro assay comparing cytotoxicity of nanoparticle-transfected versus lentivirus-transfected T cells against Eμ-ALL01 leukaemia cells, or the B16F10 cell line as a control. To ensure equal CAR expression levels, transfected T cells were sorted using FACS to GFP mean fluorescence intensities (as a surrogate reporter for CAR expression) between 10 3 and 10 4 before using them in the functional assays. Each point represents the mean ± s.e.m. pooled from independent experiments conducted in triplicate. g , ELISA measurements of IL-2 (at 24 h), and IFN-γ and TNF-α (at 48 h) secretion by transfected cells following co-culture with Eμ-ALL01 leukaemia cells. Unstimulated, lentivirus-transduced 194-1BBz CAR T-cells were analysed for comparison. h , Co-delivery of plasmids encoding the hyperactive piggyBac transposase iPB7 promotes persistent CAR gene expression. After transfection and sorting, CAR-positive T cells were cultured and the persistence of their 194-1BBz_2A_GFP expression was measured with flow cytometry. Data are representative of two independent experiments.

    Article Snippet: Plasmid DNA was labelled with Cy5 using the Mirus Label IT Nucleic Acid Labeling Kit with some modifications to the manufacturer’s protocol.

    Techniques: In Vitro, Flow Cytometry, Cytometry, Binding Assay, Mouse Assay, Plasmid Preparation, Incubation, Confocal Microscopy, Transfection, Transduction, Construct, Marker, Expressing, FACS, Fluorescence, Functional Assay, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Cell Culture

    Pin1 enhances DE gene expression during KSHV reactivation. (A) Vero-rKSHV.219 cells were transfected with plasmids expressing indicated proteins (amounts indicated). Total DNA was normalized to 2.5 μg in each transfection by the addition of empty expression vector. The percentage of cells expressing RFP under the control of the DE nut-1 /PAN promoter was counted by FACS 48 h posttransfection. The n -fold RFP-positive (RFP+) cells were graphed by normalizing cells cotransfected with Rta and Pin1 expression vectors to cells transfected with Pin1 expression vector and then comparing each value to cells transfected with Rta expression vector alone, which was set at 1. Values that are significantly different ( P

    Journal: Journal of Virology

    Article Title: The Cellular Peptidyl-Prolyl cis/trans Isomerase Pin1 Regulates Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency

    doi: 10.1128/JVI.02877-13

    Figure Lengend Snippet: Pin1 enhances DE gene expression during KSHV reactivation. (A) Vero-rKSHV.219 cells were transfected with plasmids expressing indicated proteins (amounts indicated). Total DNA was normalized to 2.5 μg in each transfection by the addition of empty expression vector. The percentage of cells expressing RFP under the control of the DE nut-1 /PAN promoter was counted by FACS 48 h posttransfection. The n -fold RFP-positive (RFP+) cells were graphed by normalizing cells cotransfected with Rta and Pin1 expression vectors to cells transfected with Pin1 expression vector and then comparing each value to cells transfected with Rta expression vector alone, which was set at 1. Values that are significantly different ( P

    Article Snippet: Vero-rKSHV.294 cells were seeded at a density of 1 × 105 cells/well in a 6-well plate and transfected with up to 5 μg plasmid DNA using TransIT LT1 reagent (Mirus Bio).

    Techniques: Expressing, Transfection, Plasmid Preparation, FACS

    Pin1 does not transactivate the CMV promoter. Pin1 knockout MEFs were transfected with the promoter-reporter plasmid CMV-Luc, together with 10, 30, or 100 ng of Pin1 expression vector (amount of vector indicated by the height of the black triangle) or empty expression vector (0). Total DNA was normalized by the addition of empty expression vector. Luciferase activity was measured 2 days posttransfection, and fold activation was calculated by comparison to CMV-Luc cotransfected with the empty vector alone.

    Journal: Journal of Virology

    Article Title: The Cellular Peptidyl-Prolyl cis/trans Isomerase Pin1 Regulates Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency

    doi: 10.1128/JVI.02877-13

    Figure Lengend Snippet: Pin1 does not transactivate the CMV promoter. Pin1 knockout MEFs were transfected with the promoter-reporter plasmid CMV-Luc, together with 10, 30, or 100 ng of Pin1 expression vector (amount of vector indicated by the height of the black triangle) or empty expression vector (0). Total DNA was normalized by the addition of empty expression vector. Luciferase activity was measured 2 days posttransfection, and fold activation was calculated by comparison to CMV-Luc cotransfected with the empty vector alone.

    Article Snippet: Vero-rKSHV.294 cells were seeded at a density of 1 × 105 cells/well in a 6-well plate and transfected with up to 5 μg plasmid DNA using TransIT LT1 reagent (Mirus Bio).

    Techniques: Knock-Out, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Activation Assay

    Pin1 enhances Rta transactivation of the nut-1 /PAN promoter in uninfected B cells. BL-41 B lymphocytes were electroporated with 5 μg of the promoter-reporter plasmid pnut-1/GL3, together with empty expression vector, indicated amounts of Rta expression vector, or indicated amounts of Pin1 expression vector. Total DNA was normalized to 20 μg in each transfection by the addition of empty expression vector. Luciferase activity was measured from total protein extracts 2 days after transfection. Values were normalized to β-galactosidase expressed from a cotransfected control plasmid, and fold activation was calculated by comparison to empty vector. Symbols: −, vector not transfected; +, 1 μg plasmid; ++, 3 μg plasmid; +++, 9 μg plasmid.

    Journal: Journal of Virology

    Article Title: The Cellular Peptidyl-Prolyl cis/trans Isomerase Pin1 Regulates Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency

    doi: 10.1128/JVI.02877-13

    Figure Lengend Snippet: Pin1 enhances Rta transactivation of the nut-1 /PAN promoter in uninfected B cells. BL-41 B lymphocytes were electroporated with 5 μg of the promoter-reporter plasmid pnut-1/GL3, together with empty expression vector, indicated amounts of Rta expression vector, or indicated amounts of Pin1 expression vector. Total DNA was normalized to 20 μg in each transfection by the addition of empty expression vector. Luciferase activity was measured from total protein extracts 2 days after transfection. Values were normalized to β-galactosidase expressed from a cotransfected control plasmid, and fold activation was calculated by comparison to empty vector. Symbols: −, vector not transfected; +, 1 μg plasmid; ++, 3 μg plasmid; +++, 9 μg plasmid.

    Article Snippet: Vero-rKSHV.294 cells were seeded at a density of 1 × 105 cells/well in a 6-well plate and transfected with up to 5 μg plasmid DNA using TransIT LT1 reagent (Mirus Bio).

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

    Amot S176A but not Amot S176E is required for formation of the nuclear Yap-Tead complex. HEK293-shAmot cells were co-transfected with Amot-WT or Amot-p130 S176A or Amot-p130 S176E . Total lysates (Input) and Pan-Tead or YAP IPs were subjected to immunoblot analysis with anti-Pan-Tead or anti-YAP antibodies, as indicated. The blots shown are representative of three independent biological replicates. ( B ) ChIP analysis of HEK293T-shAmot cells transfected with Amot-WT, Amot-p130 S176A or an empty vector control. Real-time quantitative PCR was performed in eluted DNA using primers targeting the promoter regions of ApoE and Areg . See Table 2 . The data show the means ±s.e.m. from three independent biological replicates (n = 3). Individual pairwise comparisons were assessed by Student's t-test, **p

    Journal: eLife

    Article Title: Regulation of localization and function of the transcriptional co-activator YAP by angiomotin

    doi: 10.7554/eLife.23966

    Figure Lengend Snippet: Amot S176A but not Amot S176E is required for formation of the nuclear Yap-Tead complex. HEK293-shAmot cells were co-transfected with Amot-WT or Amot-p130 S176A or Amot-p130 S176E . Total lysates (Input) and Pan-Tead or YAP IPs were subjected to immunoblot analysis with anti-Pan-Tead or anti-YAP antibodies, as indicated. The blots shown are representative of three independent biological replicates. ( B ) ChIP analysis of HEK293T-shAmot cells transfected with Amot-WT, Amot-p130 S176A or an empty vector control. Real-time quantitative PCR was performed in eluted DNA using primers targeting the promoter regions of ApoE and Areg . See Table 2 . The data show the means ±s.e.m. from three independent biological replicates (n = 3). Individual pairwise comparisons were assessed by Student's t-test, **p

    Article Snippet: On the following day cells were co-transfected with 40 nM of siRNAs (targeting either YAP or a non-targeting siRNA duplex (control)), and 2 μg of plasmid DNA (empty vector control/ wild type Amot-p130/AmotS176A /AmotS176E ) using TransIT-LT1 and TransIT-TKO (Mirus, Fisher Scientific, Illinois) according to manufacturer’s instructions.

    Techniques: Transfection, Chromatin Immunoprecipitation, Plasmid Preparation, Real-time Polymerase Chain Reaction