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Millipore plasmid dna
Comparison of the polony method to other quantitative PCR methods. ( a ) Comparison of quantification using the polony, droplet digital PCR (ddPCR) and the SYBR Green and Taqman qPCR methods with specific and degenerate primer and probe sets. Two concentrations of genomic <t>DNA</t> from the <t>Syn5</t> phage were used: S1=10 4 copies·ml -1 and S2=10 6 copies·ml -1 ). ( b ) Simulation of differences in viral quantification using degenerate primers in qPCR assays for two phage genotypes that amplified with different efficiencies. The simulation is based on the 25% difference in amplification efficiencies in SYBR Green qPCR assays of 46.6 ± 3.3 and 72.2 ± 2.5% for Syn5 and S-TIP37, respectively (n=5 independent experiments). Differences of 625 fold are found after 40 cycles.
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1) Product Images from "Quantification of diverse virus populations in the environment using the polony method"

Article Title: Quantification of diverse virus populations in the environment using the polony method

Journal: Nature microbiology

doi: 10.1038/s41564-017-0045-y

Comparison of the polony method to other quantitative PCR methods. ( a ) Comparison of quantification using the polony, droplet digital PCR (ddPCR) and the SYBR Green and Taqman qPCR methods with specific and degenerate primer and probe sets. Two concentrations of genomic DNA from the Syn5 phage were used: S1=10 4 copies·ml -1 and S2=10 6 copies·ml -1 ). ( b ) Simulation of differences in viral quantification using degenerate primers in qPCR assays for two phage genotypes that amplified with different efficiencies. The simulation is based on the 25% difference in amplification efficiencies in SYBR Green qPCR assays of 46.6 ± 3.3 and 72.2 ± 2.5% for Syn5 and S-TIP37, respectively (n=5 independent experiments). Differences of 625 fold are found after 40 cycles.
Figure Legend Snippet: Comparison of the polony method to other quantitative PCR methods. ( a ) Comparison of quantification using the polony, droplet digital PCR (ddPCR) and the SYBR Green and Taqman qPCR methods with specific and degenerate primer and probe sets. Two concentrations of genomic DNA from the Syn5 phage were used: S1=10 4 copies·ml -1 and S2=10 6 copies·ml -1 ). ( b ) Simulation of differences in viral quantification using degenerate primers in qPCR assays for two phage genotypes that amplified with different efficiencies. The simulation is based on the 25% difference in amplification efficiencies in SYBR Green qPCR assays of 46.6 ± 3.3 and 72.2 ± 2.5% for Syn5 and S-TIP37, respectively (n=5 independent experiments). Differences of 625 fold are found after 40 cycles.

Techniques Used: Real-time Polymerase Chain Reaction, Digital PCR, SYBR Green Assay, Amplification

Related Articles

Isolation:

Article Title: Deletion of the Gene Encoding p60 in Listeria monocytogenes Leads to Abnormal Cell Division and Loss of Actin-Based Motility
Article Snippet: Restriction enzymes (Amersham Pharmacia Biotech), calf alkaline phosphatase (Amersham Pharmacia Biotech), and T4 DNA ligase (Life Technologies) were utilized according to the manufacturer's instructions. .. Chromosomal DNA from L. monocytogenes was isolated by resuspending bacteria in Tris-EDTA (pH 8.2) containing 2 mg of lysozyme (Sigma) ml−1 . .. After incubation at 37°C for 15 min, bacteria were harvested by centrifugation and further treated with DNAzol (Life Technologies) according to the manufacturer's instructions.

Fluorescence:

Article Title: In vitro culture expansion impairs chondrogenic differentiation and the therapeutic effect of mesenchymal stem cells by regulating the unfolded protein response
Article Snippet: The GAG content was measured using the dimethylmethylene blue dye binding assay and the absorbance was measured at 525 nm using a microplate reader (Thermo, Karlsruhe, Germany). .. The cellularity was measured based on the DNA content using Hoechst 33258 (Sigma) and the fluorescence intensity was measured at 460 nm using a microplate fluorescence reader (FLX800, Bio-tec, Burlington, Vermont, USA). ..

Transfection:

Article Title: Targeting the centriolar replication factor STIL synergizes with DNA damaging agents for treatment of ovarian cancer
Article Snippet: For the HRR/ NHEJ balance, the ratio between green and red cells in each condition was calculated. .. Cell cycle DNA content analysis 48h following siRNA transfection, HeyA8 and IGROV1 cells were collected, fixed in ice-cold EtOH over-night and stained the following day with Propidium Iodide (PI, Sigma P4864). .. Analysis was performed on the Gallios flow cytometer and data were elaborated using Kaluza software (Beckman Coulter).

Staining:

Article Title: Targeting the centriolar replication factor STIL synergizes with DNA damaging agents for treatment of ovarian cancer
Article Snippet: For the HRR/ NHEJ balance, the ratio between green and red cells in each condition was calculated. .. Cell cycle DNA content analysis 48h following siRNA transfection, HeyA8 and IGROV1 cells were collected, fixed in ice-cold EtOH over-night and stained the following day with Propidium Iodide (PI, Sigma P4864). .. Analysis was performed on the Gallios flow cytometer and data were elaborated using Kaluza software (Beckman Coulter).

Article Title: The trans Golgi Network Is Lost from Cells Infected with African Swine Fever Virus
Article Snippet: Primary antibodies were added to samples diluted in blocking buffer and visualized by second antibodies conjugated to Alexa 488 (green) or Alexa 594 (red) purchased from Molecular Probes (Leiden, The Netherlands). .. Viral and cellular DNA was stained with DAPI (4′-6-diamidino-2-phenylindole) purchased from Sigma, St. Louis, Mo. .. Cells were mounted in Fluoromount-G (Southern Biotechnology Associates, Birmingham, Ala.), and in most studies, the cells were viewed at ×60/1.4 NA with a Nikon E800 microscope.

Article Title: Survival of Neural Stem Cells Undergoing DNA Damage-Induced Astrocytic Differentiation in Self-Renewal-Promoting Conditions In Vitro
Article Snippet: For wide-field immunofluorescence microscopy, cells on glass cover slips were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X100. .. After blocking and antibody staining in 0.5% BSA/0.2% gelatin in PBS (secondary antibody AlexaFluor 488-labeled, Invitrogen), nuclear DNA was stained by DAPI (Sigma Aldrich). .. Gene expression analysis Total RNA was extracted from live cells with Trizol reagent (Invitrogen).

Polymerase Chain Reaction:

Article Title: Role of toll-like receptor signalling in Aβ uptake and clearance
Article Snippet: .. Mouse tail DNA was extracted using the Extract-N-Amp tissue PCR kit (Sigma-Aldrich, St Louis, MO) according to the manufacturer’s protocol. .. The PCR was carried out in 25 μl vol using 1 μl of tail DNA extract and 200 nM each of two primers for the TLR4 gene through 35 cycles consisting of 30 s at 94°C, 30 s at 55°C and 1 min at 72°C.

Blocking Assay:

Article Title: Survival of Neural Stem Cells Undergoing DNA Damage-Induced Astrocytic Differentiation in Self-Renewal-Promoting Conditions In Vitro
Article Snippet: For wide-field immunofluorescence microscopy, cells on glass cover slips were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X100. .. After blocking and antibody staining in 0.5% BSA/0.2% gelatin in PBS (secondary antibody AlexaFluor 488-labeled, Invitrogen), nuclear DNA was stained by DAPI (Sigma Aldrich). .. Gene expression analysis Total RNA was extracted from live cells with Trizol reagent (Invitrogen).

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    Millipore non targeting shrna
    HIF-1α stabilisation regulates cell area. (A) Schematic drawing of HIF-1 and its regulation in normoxia and hypoxia. The stability of the HIF-1α subunit is regulated by prolyl-4-hydroxylase domain (PHD) enzymes in an oxygen dependent manner. Following hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL) recognises HIF-1α subunits and targets them for rapid ubiquitination and proteasomal degradation under normoxic conditions. Hypoxia impairs the hydroxylation, which results in HIF-1α stabilisation, nuclear accumulation, heterodimerisation with HIF-1β and subsequent hypoxia-inducible gene expression. (B) Inhibition of PHDs by DMOG causes HIF-1α stabilisation under normoxic conditions. L929 cells were treated with 1 mM DMOG for 48 hrs and total extracts were analysed by immunoblot with the respective antibodies. (C) The cell area of DMOG treated cells increased significantly. The cell area of single cells was measured and was calculated as fold change compared to 20% O 2. (D) Two HIF-1α knock down cell clones (c1, c2) and a non-target control <t>shRNA</t> cell clone <t>(shC)</t> were obtained via stable transduction of specific sh-plasmids. The HIF-1α knock down was verified in western blot experiments of cell lysates from cells incubated at 20% O 2 or 1% O 2 . β-tubulin was used as a loading control. (E) HIF-1α knock down cell clones (c1, c2) do not respond to hypoxia with an increase in cell area. The cell area of single cells was measured and was calculated as fold change compared to the cell clone cultivated at 20% O 2 . (F) Flow cytometry analysis of cell volume after incubation in normoxia and hypoxia. shC, c1 and c2 cells were harvested after 24 hrs of normoxic (20% O 2 ) or hypoxic (1% O 2 ) incubation. Single cell suspension was prepared by enzymatic digestion. Note that hypoxia increases the cell volume independently of the HIF-1α knock down.
    Non Targeting Shrna, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore mission plko
    sh RNA ‐mediated depletion of HDAC 1 in human CMC s. Representative (A) epifluorescence microscopy images (n=3; scale=400 μm) and (B and C) flow cytometric analysis (values are mean± SEM; n=3) of CMC s 72 hours after transduction with the <t>MISSION</t> pLKO <t>.1‐puro‐</t> <t>CMV</t> ‐Turbo GFP Positive Control Vector (1:3 or 1:10 viral titer dilutions). Flow cytometry data were arcsine‐transformed and analyzed by unpaired, 1‐way ANOVA . P values were calculated using the post hoc Bonferroni multiple comparison test. The efficacy of HDAC 1 knockdown in CMC s assessed by (D) q PCR (values are mean± SEM; n=4) and (E) Western blot (representative image; n=4). F, Bar graph denoting densitometric quantification of resulting HDAC 1 immunoblots. Values represent mean protein expression (relative to β‐actin)± SEM (n=4). qPCR and Western blot data were log base 10 (y=log 10 y) transformed and analyzed by unpaired, 1‐way ANOVA . P values were calculated using the post hoc Bonferroni multiple comparison test. CMC s indicates cardiac mesenchymal stromal cells; FSC‐A, forward scatter pulse area; GFP, green fluorescent protein; HDAC 1, histone deacetylase 1; sh HDAC 1, short hairpin RNA ‐histone deacetylase 1; sh NT , short hairpin RNA nontarget; UT , untransduced.
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    HIF-1α stabilisation regulates cell area. (A) Schematic drawing of HIF-1 and its regulation in normoxia and hypoxia. The stability of the HIF-1α subunit is regulated by prolyl-4-hydroxylase domain (PHD) enzymes in an oxygen dependent manner. Following hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL) recognises HIF-1α subunits and targets them for rapid ubiquitination and proteasomal degradation under normoxic conditions. Hypoxia impairs the hydroxylation, which results in HIF-1α stabilisation, nuclear accumulation, heterodimerisation with HIF-1β and subsequent hypoxia-inducible gene expression. (B) Inhibition of PHDs by DMOG causes HIF-1α stabilisation under normoxic conditions. L929 cells were treated with 1 mM DMOG for 48 hrs and total extracts were analysed by immunoblot with the respective antibodies. (C) The cell area of DMOG treated cells increased significantly. The cell area of single cells was measured and was calculated as fold change compared to 20% O 2. (D) Two HIF-1α knock down cell clones (c1, c2) and a non-target control shRNA cell clone (shC) were obtained via stable transduction of specific sh-plasmids. The HIF-1α knock down was verified in western blot experiments of cell lysates from cells incubated at 20% O 2 or 1% O 2 . β-tubulin was used as a loading control. (E) HIF-1α knock down cell clones (c1, c2) do not respond to hypoxia with an increase in cell area. The cell area of single cells was measured and was calculated as fold change compared to the cell clone cultivated at 20% O 2 . (F) Flow cytometry analysis of cell volume after incubation in normoxia and hypoxia. shC, c1 and c2 cells were harvested after 24 hrs of normoxic (20% O 2 ) or hypoxic (1% O 2 ) incubation. Single cell suspension was prepared by enzymatic digestion. Note that hypoxia increases the cell volume independently of the HIF-1α knock down.

    Journal: PLoS ONE

    Article Title: Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1? in Cofilin Regulation and Cytoplasmic Actin Distribution

    doi: 10.1371/journal.pone.0069128

    Figure Lengend Snippet: HIF-1α stabilisation regulates cell area. (A) Schematic drawing of HIF-1 and its regulation in normoxia and hypoxia. The stability of the HIF-1α subunit is regulated by prolyl-4-hydroxylase domain (PHD) enzymes in an oxygen dependent manner. Following hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL) recognises HIF-1α subunits and targets them for rapid ubiquitination and proteasomal degradation under normoxic conditions. Hypoxia impairs the hydroxylation, which results in HIF-1α stabilisation, nuclear accumulation, heterodimerisation with HIF-1β and subsequent hypoxia-inducible gene expression. (B) Inhibition of PHDs by DMOG causes HIF-1α stabilisation under normoxic conditions. L929 cells were treated with 1 mM DMOG for 48 hrs and total extracts were analysed by immunoblot with the respective antibodies. (C) The cell area of DMOG treated cells increased significantly. The cell area of single cells was measured and was calculated as fold change compared to 20% O 2. (D) Two HIF-1α knock down cell clones (c1, c2) and a non-target control shRNA cell clone (shC) were obtained via stable transduction of specific sh-plasmids. The HIF-1α knock down was verified in western blot experiments of cell lysates from cells incubated at 20% O 2 or 1% O 2 . β-tubulin was used as a loading control. (E) HIF-1α knock down cell clones (c1, c2) do not respond to hypoxia with an increase in cell area. The cell area of single cells was measured and was calculated as fold change compared to the cell clone cultivated at 20% O 2 . (F) Flow cytometry analysis of cell volume after incubation in normoxia and hypoxia. shC, c1 and c2 cells were harvested after 24 hrs of normoxic (20% O 2 ) or hypoxic (1% O 2 ) incubation. Single cell suspension was prepared by enzymatic digestion. Note that hypoxia increases the cell volume independently of the HIF-1α knock down.

    Article Snippet: For generating the shControl (shC) transfected cells, a pLKO.1-puro vector was used containing a non-targeting shRNA (#SHC002, Sigma-Aldrich).

    Techniques: Expressing, Inhibition, Clone Assay, shRNA, Transduction, Western Blot, Incubation, Flow Cytometry, Cytometry

    Depletion of HIF-1α alters cell motility. (A) Wounding assay of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA (shC) cell clone in normoxia and hypoxia. Cells were grown in normoxia and hypoxia and experimental wounds were caused by scratching cell monolayers with a pipet tip. Images were taken at the indicated time points and the cell free area was determined. Hypoxia delayed wound healing. Note that knocking down HIF-1α slowed wound closure down even more (dashed red line). (B) Single cell migration of the HIF-1α knock down cell clones c1 and c2 and the shC cells. Cells were incubated at 20% O 2 and 1% O 2 for 24 hrs. Images were taken over a time period of 360 min and cell movement was analysed. shC cells showed a reduced migration under hypoxic condition, however this effect was not seen in the HIF-1α knock down clones c1 and c2. Numbers within the bars indicate the number of cells analysed. ** p

    Journal: PLoS ONE

    Article Title: Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1? in Cofilin Regulation and Cytoplasmic Actin Distribution

    doi: 10.1371/journal.pone.0069128

    Figure Lengend Snippet: Depletion of HIF-1α alters cell motility. (A) Wounding assay of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA (shC) cell clone in normoxia and hypoxia. Cells were grown in normoxia and hypoxia and experimental wounds were caused by scratching cell monolayers with a pipet tip. Images were taken at the indicated time points and the cell free area was determined. Hypoxia delayed wound healing. Note that knocking down HIF-1α slowed wound closure down even more (dashed red line). (B) Single cell migration of the HIF-1α knock down cell clones c1 and c2 and the shC cells. Cells were incubated at 20% O 2 and 1% O 2 for 24 hrs. Images were taken over a time period of 360 min and cell movement was analysed. shC cells showed a reduced migration under hypoxic condition, however this effect was not seen in the HIF-1α knock down clones c1 and c2. Numbers within the bars indicate the number of cells analysed. ** p

    Article Snippet: For generating the shControl (shC) transfected cells, a pLKO.1-puro vector was used containing a non-targeting shRNA (#SHC002, Sigma-Aldrich).

    Techniques: Clone Assay, shRNA, Migration, Incubation

    Depletion of HIF-1α does not affect the number of focal contacts and cell spreading. (A) Two HIF-1α knock down cell clones (c1, c2) and a non-target control shRNA cell clone (shC) were stained for vinculin 24 hrs after normoxic (20% O 2 ) or hypoxic (1% O 2 ) incubation. Counting vinculin positive focal contacts showed a higher number of focal contacts in hypoxia in all three cell lines. (B) Flow cytometry analysis of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA cell clone (shC) after 24 hrs of normoxia or hypoxia stained with integrin β1 antibodies. (C) Wt, shC, and the HIF-1α knock down cell clones c1 and c2 cells were lysed after 24 hrs of normoxia (20% O 2 ) or hypoxia (1% O 2 ). Cell extracts were analysed by Western blots. Note that vinculin and integrin β1 levels are not changed in hypoxia. (D) Cell spreading of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA (shC) cell clone in normoxia and hypoxia. Cells were incubated at 20% O 2 and 1% O 2 , trypsinised and replated for 20 min. Cells were fixed and stained with phalloidin-FITC and divided into three categories (a: round, barely spread; b: in the course of spreading; c: well spread). The percentage of cells in each category was determined. All three cell lines spread faster under hypoxic conditions. Numbers within the bars indicate the number of cells analysed. ** p

    Journal: PLoS ONE

    Article Title: Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1? in Cofilin Regulation and Cytoplasmic Actin Distribution

    doi: 10.1371/journal.pone.0069128

    Figure Lengend Snippet: Depletion of HIF-1α does not affect the number of focal contacts and cell spreading. (A) Two HIF-1α knock down cell clones (c1, c2) and a non-target control shRNA cell clone (shC) were stained for vinculin 24 hrs after normoxic (20% O 2 ) or hypoxic (1% O 2 ) incubation. Counting vinculin positive focal contacts showed a higher number of focal contacts in hypoxia in all three cell lines. (B) Flow cytometry analysis of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA cell clone (shC) after 24 hrs of normoxia or hypoxia stained with integrin β1 antibodies. (C) Wt, shC, and the HIF-1α knock down cell clones c1 and c2 cells were lysed after 24 hrs of normoxia (20% O 2 ) or hypoxia (1% O 2 ). Cell extracts were analysed by Western blots. Note that vinculin and integrin β1 levels are not changed in hypoxia. (D) Cell spreading of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA (shC) cell clone in normoxia and hypoxia. Cells were incubated at 20% O 2 and 1% O 2 , trypsinised and replated for 20 min. Cells were fixed and stained with phalloidin-FITC and divided into three categories (a: round, barely spread; b: in the course of spreading; c: well spread). The percentage of cells in each category was determined. All three cell lines spread faster under hypoxic conditions. Numbers within the bars indicate the number of cells analysed. ** p

    Article Snippet: For generating the shControl (shC) transfected cells, a pLKO.1-puro vector was used containing a non-targeting shRNA (#SHC002, Sigma-Aldrich).

    Techniques: Clone Assay, shRNA, Staining, Incubation, Flow Cytometry, Cytometry, Western Blot

    Presence of hCCX-CKR is associated with suppression of chemotaxis towards CXCL10 in T cells (A) Silencing hCCX-CKR using vectors containing DNA encoding for short hairpin RNA targeting hCCX-CKR resulted in a down-regulation of hCCX-CKR expression by freshly

    Journal: British Journal of Pharmacology

    Article Title: Inhibition of CXCR3-mediated chemotaxis by the human chemokine receptor-like protein CCX-CKR

    doi: 10.1111/bph.12042

    Figure Lengend Snippet: Presence of hCCX-CKR is associated with suppression of chemotaxis towards CXCL10 in T cells (A) Silencing hCCX-CKR using vectors containing DNA encoding for short hairpin RNA targeting hCCX-CKR resulted in a down-regulation of hCCX-CKR expression by freshly

    Article Snippet: Plasmid containing GFP-encoding DNA (pmaxGFP; Lonza, Basel, Switzerland) was used as positive control for transfection and the pLKO.1-puro vector containing DNA encoding for non-targeting shRNA (MISSION® shRNA SHC002; Sigma-Aldrich) was used as negative control.

    Techniques: Chemotaxis Assay, shRNA, Expressing

    NME1 suppresses motile and invasive potential of WM1158 melanoma cells by upregulating ITGβ3 expression. (a) Expression of NME1 and ITGβ3 proteins was measured by immunoblot analysis in cells receiving the indicated combinations of forced NME1 expression and shRNAs directed to ITGβ3. shRNA treatments consisted a non-targeting control shRNA (−) or one of two shRNAs specific for ITGβ3 (“a” or “b”). (b) Representative images of wound/scratch assays were acquired 24 h after wound induction in cells receiving the indicated combinations of forced NME1 expression and shRNA-mediated silencing of ITGβ3. Dotted boxes depict borders of original wounds. (c) Graph provides a quantitative analysis of individual cells migrating into wounds. Closed circles and error bars represent means (+/− SEM) derived from 3– 4 independent experiments. Asterisks denote means that are significantly different (*p≤0.05 by ANOVA with Holm-Sidak pairwise testing). (d) Expression of ITGβ3 mRNA was measured in cells used for measurement of single cell invasion activity in 3-dimensional sphere assays after the indicated combination of forced NME1 expression and shRNA sequences (-, non-targeting control; ITGβ3-directed shRNAs “c” and “a”). The shRNA sequence “c” was employed instead of sequence “b” used in panel a, as sequence “b” strongly disrupted spheroid formation. Cultures were co-infected with a lentiviral vector for expression of eGFP for enhanced imaging of invading cells. *, comparison between vector- and NME1-treated; †, denotes significant silencing of ITGβ3 RNA in absence of forced NME1 expression (black bars); ‡, denotes significant silencing of ITGβ3 RNA in presence of forced NME1 expression (white bars). p

    Journal: Experimental cell research

    Article Title: The metastasis suppressor NME1 inhibits melanoma cell motility via direct transcriptional induction of the integrin beta-3 gene

    doi: 10.1016/j.yexcr.2018.11.010

    Figure Lengend Snippet: NME1 suppresses motile and invasive potential of WM1158 melanoma cells by upregulating ITGβ3 expression. (a) Expression of NME1 and ITGβ3 proteins was measured by immunoblot analysis in cells receiving the indicated combinations of forced NME1 expression and shRNAs directed to ITGβ3. shRNA treatments consisted a non-targeting control shRNA (−) or one of two shRNAs specific for ITGβ3 (“a” or “b”). (b) Representative images of wound/scratch assays were acquired 24 h after wound induction in cells receiving the indicated combinations of forced NME1 expression and shRNA-mediated silencing of ITGβ3. Dotted boxes depict borders of original wounds. (c) Graph provides a quantitative analysis of individual cells migrating into wounds. Closed circles and error bars represent means (+/− SEM) derived from 3– 4 independent experiments. Asterisks denote means that are significantly different (*p≤0.05 by ANOVA with Holm-Sidak pairwise testing). (d) Expression of ITGβ3 mRNA was measured in cells used for measurement of single cell invasion activity in 3-dimensional sphere assays after the indicated combination of forced NME1 expression and shRNA sequences (-, non-targeting control; ITGβ3-directed shRNAs “c” and “a”). The shRNA sequence “c” was employed instead of sequence “b” used in panel a, as sequence “b” strongly disrupted spheroid formation. Cultures were co-infected with a lentiviral vector for expression of eGFP for enhanced imaging of invading cells. *, comparison between vector- and NME1-treated; †, denotes significant silencing of ITGβ3 RNA in absence of forced NME1 expression (black bars); ‡, denotes significant silencing of ITGβ3 RNA in presence of forced NME1 expression (white bars). p

    Article Snippet: RNA knockdown was achieved by lentiviral delivery of Mission shRNA (Sigma-Aldrich) against ITGβ3 (TRCN0000003236, ITGβ3 shRNA-a; TRCN0000318546, ITGβ3 shRNA-b; TRCN0000003235, ITGβ3 shRNA-c) or a non-targeted shRNA (SHC002, Sigma-Aldrich) as a negative control.

    Techniques: Expressing, shRNA, Derivative Assay, Activity Assay, Sequencing, Infection, Plasmid Preparation, Imaging

    sh RNA ‐mediated depletion of HDAC 1 in human CMC s. Representative (A) epifluorescence microscopy images (n=3; scale=400 μm) and (B and C) flow cytometric analysis (values are mean± SEM; n=3) of CMC s 72 hours after transduction with the MISSION pLKO .1‐puro‐ CMV ‐Turbo GFP Positive Control Vector (1:3 or 1:10 viral titer dilutions). Flow cytometry data were arcsine‐transformed and analyzed by unpaired, 1‐way ANOVA . P values were calculated using the post hoc Bonferroni multiple comparison test. The efficacy of HDAC 1 knockdown in CMC s assessed by (D) q PCR (values are mean± SEM; n=4) and (E) Western blot (representative image; n=4). F, Bar graph denoting densitometric quantification of resulting HDAC 1 immunoblots. Values represent mean protein expression (relative to β‐actin)± SEM (n=4). qPCR and Western blot data were log base 10 (y=log 10 y) transformed and analyzed by unpaired, 1‐way ANOVA . P values were calculated using the post hoc Bonferroni multiple comparison test. CMC s indicates cardiac mesenchymal stromal cells; FSC‐A, forward scatter pulse area; GFP, green fluorescent protein; HDAC 1, histone deacetylase 1; sh HDAC 1, short hairpin RNA ‐histone deacetylase 1; sh NT , short hairpin RNA nontarget; UT , untransduced.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Histone Deacetylase 1 Depletion Activates Human Cardiac Mesenchymal Stromal Cell Proangiogenic Paracrine Signaling Through a Mechanism Requiring Enhanced Basic Fibroblast Growth Factor Synthesis and Secretion

    doi: 10.1161/JAHA.117.006183

    Figure Lengend Snippet: sh RNA ‐mediated depletion of HDAC 1 in human CMC s. Representative (A) epifluorescence microscopy images (n=3; scale=400 μm) and (B and C) flow cytometric analysis (values are mean± SEM; n=3) of CMC s 72 hours after transduction with the MISSION pLKO .1‐puro‐ CMV ‐Turbo GFP Positive Control Vector (1:3 or 1:10 viral titer dilutions). Flow cytometry data were arcsine‐transformed and analyzed by unpaired, 1‐way ANOVA . P values were calculated using the post hoc Bonferroni multiple comparison test. The efficacy of HDAC 1 knockdown in CMC s assessed by (D) q PCR (values are mean± SEM; n=4) and (E) Western blot (representative image; n=4). F, Bar graph denoting densitometric quantification of resulting HDAC 1 immunoblots. Values represent mean protein expression (relative to β‐actin)± SEM (n=4). qPCR and Western blot data were log base 10 (y=log 10 y) transformed and analyzed by unpaired, 1‐way ANOVA . P values were calculated using the post hoc Bonferroni multiple comparison test. CMC s indicates cardiac mesenchymal stromal cells; FSC‐A, forward scatter pulse area; GFP, green fluorescent protein; HDAC 1, histone deacetylase 1; sh HDAC 1, short hairpin RNA ‐histone deacetylase 1; sh NT , short hairpin RNA nontarget; UT , untransduced.

    Article Snippet: Seventy‐two hours after initial viral particle exposure, cell transduction efficiency was assessed by fluorescent‐mediated detection of green fluorescent protein (GFP) in cells transduced with MISSION pLKO.1‐puro‐CMV‐TurboGFP Positive Control Vector (SHC003; Sigma‐Aldrich).

    Techniques: Epifluorescence Microscopy, Flow Cytometry, Transduction, Positive Control, Plasmid Preparation, Cytometry, Transformation Assay, Polymerase Chain Reaction, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Histone Deacetylase Assay, shRNA