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Gentra Systems post transfection pt
Immunocytochemical detection of Col-I. Specific anti-Col-I primary monoclonal antibodies ( A ) were used to stain the non-transfected human chondrocytes (NTC) and those following <t>transfection</t> with pCMV-SPORT6 IGF-I ( IGF-I ), pCMV-SPORT6 SOX9 ( SOX9 ), or cotransfection with both plasmids ( IGF-I/SOX9 ). Images were taken on days 3, 6, and 9 post-transfection. Once transfected, the cells were incubated at 37°C in a 5% CO 2 atmosphere. Quantified in situ densitometry is shown in the bar graph ( B ). Data are reported as the average and standard deviation of the densitometry value normalized to the number of cells in the evaluated area (expressed in pixels ×10 7 ), which was performed on all chondrocytes observed in 32 images on days 3, 6, and 9 post-transfection. ***P
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1) Product Images from "Cotransfected human chondrocytes: over-expression ofIGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans"

Article Title: Cotransfected human chondrocytes: over-expression ofIGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans

Journal: Brazilian Journal of Medical and Biological Research

doi: 10.1590/1414-431X20154732

Immunocytochemical detection of Col-I. Specific anti-Col-I primary monoclonal antibodies ( A ) were used to stain the non-transfected human chondrocytes (NTC) and those following transfection with pCMV-SPORT6 IGF-I ( IGF-I ), pCMV-SPORT6 SOX9 ( SOX9 ), or cotransfection with both plasmids ( IGF-I/SOX9 ). Images were taken on days 3, 6, and 9 post-transfection. Once transfected, the cells were incubated at 37°C in a 5% CO 2 atmosphere. Quantified in situ densitometry is shown in the bar graph ( B ). Data are reported as the average and standard deviation of the densitometry value normalized to the number of cells in the evaluated area (expressed in pixels ×10 7 ), which was performed on all chondrocytes observed in 32 images on days 3, 6, and 9 post-transfection. ***P
Figure Legend Snippet: Immunocytochemical detection of Col-I. Specific anti-Col-I primary monoclonal antibodies ( A ) were used to stain the non-transfected human chondrocytes (NTC) and those following transfection with pCMV-SPORT6 IGF-I ( IGF-I ), pCMV-SPORT6 SOX9 ( SOX9 ), or cotransfection with both plasmids ( IGF-I/SOX9 ). Images were taken on days 3, 6, and 9 post-transfection. Once transfected, the cells were incubated at 37°C in a 5% CO 2 atmosphere. Quantified in situ densitometry is shown in the bar graph ( B ). Data are reported as the average and standard deviation of the densitometry value normalized to the number of cells in the evaluated area (expressed in pixels ×10 7 ), which was performed on all chondrocytes observed in 32 images on days 3, 6, and 9 post-transfection. ***P

Techniques Used: Staining, Transfection, Cotransfection, Incubation, In Situ, Standard Deviation

Immunocytochemical detection of Col-II. Specific anti-Col-II primary monoclonal antibodies ( A ) were used to stain the non-transfected human chondrocytes (NTC) and those transfected with plasmids pCMV-SPORT6 IGF-I ( IGF-I ), pCMV-SPORT6 SOX9 ( SOX9 ), or following cotransfection with both plasmids ( IGF-I/SOX9 ). Images were taken on days 3, 6, and 9 post-transfection. Once transfected, the cells were incubated at 37°C in a 5% CO 2 atmosphere. Quantified in situ densitometry is shown in the bar graph ( B ). Data are reported as the average and standard deviation of the densitometry value normalized to the number of cells in the evaluated area (expressed in pixels ×10 7 ), which was performed on all chondrocytes observed in 32 images on days 3, 6, and 9 post-transfection. ***P
Figure Legend Snippet: Immunocytochemical detection of Col-II. Specific anti-Col-II primary monoclonal antibodies ( A ) were used to stain the non-transfected human chondrocytes (NTC) and those transfected with plasmids pCMV-SPORT6 IGF-I ( IGF-I ), pCMV-SPORT6 SOX9 ( SOX9 ), or following cotransfection with both plasmids ( IGF-I/SOX9 ). Images were taken on days 3, 6, and 9 post-transfection. Once transfected, the cells were incubated at 37°C in a 5% CO 2 atmosphere. Quantified in situ densitometry is shown in the bar graph ( B ). Data are reported as the average and standard deviation of the densitometry value normalized to the number of cells in the evaluated area (expressed in pixels ×10 7 ), which was performed on all chondrocytes observed in 32 images on days 3, 6, and 9 post-transfection. ***P

Techniques Used: Staining, Transfection, Cotransfection, Incubation, In Situ, Standard Deviation

Glycosaminoglycans (GAG) produced by the chondrocytes. The conditioned media from each culture condition were mixed with dimethyl methylene blue dye, and the amount of GAG was quantified by spectrophotometry. The bars correspond to the non-transfected chondrocytes (NTC) and those transfected with pCMV-SPORT6 IGF-I ( IGF-I ), pCMV-SPORT6 SOX9 ( SOX9 ), or both plasmids ( IGF-I/SOX9 ). Data are reported as the mean and standard deviation of the data normalized to the total protein content based on three experiments on days 3, 6, and 9 post-transfection. ***P
Figure Legend Snippet: Glycosaminoglycans (GAG) produced by the chondrocytes. The conditioned media from each culture condition were mixed with dimethyl methylene blue dye, and the amount of GAG was quantified by spectrophotometry. The bars correspond to the non-transfected chondrocytes (NTC) and those transfected with pCMV-SPORT6 IGF-I ( IGF-I ), pCMV-SPORT6 SOX9 ( SOX9 ), or both plasmids ( IGF-I/SOX9 ). Data are reported as the mean and standard deviation of the data normalized to the total protein content based on three experiments on days 3, 6, and 9 post-transfection. ***P

Techniques Used: Produced, Spectrophotometry, Transfection, Standard Deviation

2) Product Images from "A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes"

Article Title: A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005545

ALT-803 induces HIV transcription from both a primary cell model of latency and from ex vivo patient samples. Primary CD4 + T-cells from healthy donor PBMC were activated, infected with a luciferase reporter HIV virus, and allowed to return to a resting state. This is similar to a post-activation latency model that has been previously described [ 17 ] with additional minor modifications (see Methods ). After 1 week, infected cells were treated with the agents at concentrations indicated for 2 days. A. Shown are luminescence values from luciferase, indicating dose-dependent HIV reactivation by each of the compounds tested. B. Compound associated cytotoxicity was determined in latently-infected cells in parallel with the virus activation assay using Cell Titer Glo (CTG) Dose dependent cytotoxicity was observed with SAHA and romidepsin but not with IL-15 or ALT-803. C and D. PBMC were isolated from HIV-infected subjects who had been suppressed by ART for at least 2 years. Cultures were maintained in the presence of ARVs. Either 1 nM of ALT-803 or 5 ng/ml PMA + 500 ng/ml ionomycin were added for 7 days. HIV activation was measured by quantitating viral RNA in cell-free supernatant using COBAS qPCR. C. The geometric mean of viral copies/ml for each donor are indicated for control (DMSO) and ALT-803 treated wells, following 7 days of treatment. D . The fold HIV activations of all donors are plotted as the ratio of the viral copies/ml for ALT-803 treated or PMA/ionomycin treated to control wells. These results indicate that ALT-803 reactivates virus from the natural patient reservoir, though to a lesser degree than PMA/ionomycin. E-I. Cryopreserved CD4 + T-cells from ARV-treated HIV-infected subjects were thawed, CFSE-labeled, and treated with either 1 nM ALT-803 or 200 ng/ml of PHA for 7 days in the presence of ARVs. E . Activation of CD4 + T-cells within PBMCs was measured at day 7 by flow cytometry, gating on CD3 + CD4 + T-cells and assessing CD69 expression. Each line represents a different subject. P values were calculated by RM one-way ANOVA with Holm-Sidak’s multiple comparison test. F . Proliferation of CD4+ T-cells within PBCs was measured at day 7 by flow, gating on CD3 + CD4 + T-cells and assessing proliferation as %CFSE dim cells. P values were calculated by RM one-way ANOVA with Holm-Sidak’s multiple comparison test. G-I. Cell associated DNA was isolated from purified CD4 + T-cells following 6 days of stimulation. Absolute copies of HIV-Gag and RPP30 were determined by droplet digital PCR and used to calculate copies of HIV per 10 6 CD4 + T-cells. Shown are results from three different subjects. Quantifications were determined in triplicate ( G ) or quadruplicate ( H I ). Means and SEM are shown and the P value was calculated by one-way ANOVA with Holm-Sidak’s multiple comparison test.
Figure Legend Snippet: ALT-803 induces HIV transcription from both a primary cell model of latency and from ex vivo patient samples. Primary CD4 + T-cells from healthy donor PBMC were activated, infected with a luciferase reporter HIV virus, and allowed to return to a resting state. This is similar to a post-activation latency model that has been previously described [ 17 ] with additional minor modifications (see Methods ). After 1 week, infected cells were treated with the agents at concentrations indicated for 2 days. A. Shown are luminescence values from luciferase, indicating dose-dependent HIV reactivation by each of the compounds tested. B. Compound associated cytotoxicity was determined in latently-infected cells in parallel with the virus activation assay using Cell Titer Glo (CTG) Dose dependent cytotoxicity was observed with SAHA and romidepsin but not with IL-15 or ALT-803. C and D. PBMC were isolated from HIV-infected subjects who had been suppressed by ART for at least 2 years. Cultures were maintained in the presence of ARVs. Either 1 nM of ALT-803 or 5 ng/ml PMA + 500 ng/ml ionomycin were added for 7 days. HIV activation was measured by quantitating viral RNA in cell-free supernatant using COBAS qPCR. C. The geometric mean of viral copies/ml for each donor are indicated for control (DMSO) and ALT-803 treated wells, following 7 days of treatment. D . The fold HIV activations of all donors are plotted as the ratio of the viral copies/ml for ALT-803 treated or PMA/ionomycin treated to control wells. These results indicate that ALT-803 reactivates virus from the natural patient reservoir, though to a lesser degree than PMA/ionomycin. E-I. Cryopreserved CD4 + T-cells from ARV-treated HIV-infected subjects were thawed, CFSE-labeled, and treated with either 1 nM ALT-803 or 200 ng/ml of PHA for 7 days in the presence of ARVs. E . Activation of CD4 + T-cells within PBMCs was measured at day 7 by flow cytometry, gating on CD3 + CD4 + T-cells and assessing CD69 expression. Each line represents a different subject. P values were calculated by RM one-way ANOVA with Holm-Sidak’s multiple comparison test. F . Proliferation of CD4+ T-cells within PBCs was measured at day 7 by flow, gating on CD3 + CD4 + T-cells and assessing proliferation as %CFSE dim cells. P values were calculated by RM one-way ANOVA with Holm-Sidak’s multiple comparison test. G-I. Cell associated DNA was isolated from purified CD4 + T-cells following 6 days of stimulation. Absolute copies of HIV-Gag and RPP30 were determined by droplet digital PCR and used to calculate copies of HIV per 10 6 CD4 + T-cells. Shown are results from three different subjects. Quantifications were determined in triplicate ( G ) or quadruplicate ( H I ). Means and SEM are shown and the P value was calculated by one-way ANOVA with Holm-Sidak’s multiple comparison test.

Techniques Used: Ex Vivo, Infection, Luciferase, Activation Assay, CTG Assay, Isolation, Real-time Polymerase Chain Reaction, Labeling, Flow Cytometry, Cytometry, Expressing, Purification, Digital PCR

3) Product Images from "A low-cost open-source SNP genotyping platform for association mapping applications"

Article Title: A low-cost open-source SNP genotyping platform for association mapping applications

Journal: Genome Biology

doi: 10.1186/gb-2005-6-12-r105

Principle of OLA-based SNP genotyping. (a) For each polymorphism, a set of three genotyping oligos are allowed to anneal to denatured PCR product (blue) in the presence of Taq DNA ligase. Ligation of up- and downstream oligos occurs only if there is a perfect match to template. Upstream oligos are color-coded gray (M13 forward amplification primer sequence), red/green (a pair of barcode sequences), and black (assay-specific sequence flanking the query SNP). The downstream oligo is 5'-phosphorylated, and color-coded gray (reverse complemented sequence of the M13 reverse amplification primer), and black (assay-specific flanking sequence). (b) Addition of common M13 primers (gray) allows amplification of all ligated products. (c) After arraying amplified OLA products, membranes are hybridized with probes complementary to the barcode sequences. Probes can be fluorescently labeled with infrared (IR) fluors and both alleles hybridized simultaneously, or radiolabeled and hybridized sequentially.
Figure Legend Snippet: Principle of OLA-based SNP genotyping. (a) For each polymorphism, a set of three genotyping oligos are allowed to anneal to denatured PCR product (blue) in the presence of Taq DNA ligase. Ligation of up- and downstream oligos occurs only if there is a perfect match to template. Upstream oligos are color-coded gray (M13 forward amplification primer sequence), red/green (a pair of barcode sequences), and black (assay-specific sequence flanking the query SNP). The downstream oligo is 5'-phosphorylated, and color-coded gray (reverse complemented sequence of the M13 reverse amplification primer), and black (assay-specific flanking sequence). (b) Addition of common M13 primers (gray) allows amplification of all ligated products. (c) After arraying amplified OLA products, membranes are hybridized with probes complementary to the barcode sequences. Probes can be fluorescently labeled with infrared (IR) fluors and both alleles hybridized simultaneously, or radiolabeled and hybridized sequentially.

Techniques Used: Polymerase Chain Reaction, Ligation, Amplification, Sequencing, Labeling

4) Product Images from "Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies"

Article Title: Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies

Journal: PLoS ONE

doi: 10.1371/journal.pone.0187072

Analysis of HepG2 cells containing the 4CYPs-POR MAC. (A) Flowchart of the MAC transfer from donor CHO cells to recipient HepG2 cells via MMCT method, which comprises the following steps: micronucleation of donor cells by colcemid, enucleation by cytochalasin B, and microcell purification and fusion with recipient HepG2 cells. HepG2 hybrids were selected with 400 μg/mL G418 and picked for clonal expansion. (B) G418-resistant clones are screened by genomic PCR to determine the presence of the 4CYPs-POR transgene. (C) Representative metaphase fluorescence in situ hybridization images of TC-HepG2 cells. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled pPAC-4CYPs-POR (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrows indicate MAC vectors, and the inset shows an enlarged image of the MAC.
Figure Legend Snippet: Analysis of HepG2 cells containing the 4CYPs-POR MAC. (A) Flowchart of the MAC transfer from donor CHO cells to recipient HepG2 cells via MMCT method, which comprises the following steps: micronucleation of donor cells by colcemid, enucleation by cytochalasin B, and microcell purification and fusion with recipient HepG2 cells. HepG2 hybrids were selected with 400 μg/mL G418 and picked for clonal expansion. (B) G418-resistant clones are screened by genomic PCR to determine the presence of the 4CYPs-POR transgene. (C) Representative metaphase fluorescence in situ hybridization images of TC-HepG2 cells. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled pPAC-4CYPs-POR (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrows indicate MAC vectors, and the inset shows an enlarged image of the MAC.

Techniques Used: Purification, Clone Assay, Polymerase Chain Reaction, Fluorescence, In Situ Hybridization, Labeling

Construction and analysis of the 4CYPs-POR MAC vector in CHO cells. (A) Genomic PCR and (B) RT-PCR analysis of CHO cells containing the 4CYPs-POR MAC. N, negative control (parental CHO cells); P, positive control (PAC-4CYPs-POR). (C) FISH analysis of donor CHO cells containing the 4CYPs-POR MAC. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled 4CYPs-POR PAC (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrow indicates MAC vector, and the inset shows enlarged image of the MAC.
Figure Legend Snippet: Construction and analysis of the 4CYPs-POR MAC vector in CHO cells. (A) Genomic PCR and (B) RT-PCR analysis of CHO cells containing the 4CYPs-POR MAC. N, negative control (parental CHO cells); P, positive control (PAC-4CYPs-POR). (C) FISH analysis of donor CHO cells containing the 4CYPs-POR MAC. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled 4CYPs-POR PAC (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrow indicates MAC vector, and the inset shows enlarged image of the MAC.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Fluorescence In Situ Hybridization, Labeling

5) Product Images from "Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini"

Article Title: Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0000398

Genome structure of the cathepsin F gene of Opisthorchis viverrini . (A) Structure of the O. viverrini cathepsin F gene locus, as determined by nucleotide sequence analysis of a ∼3 kb PCR product of genomic DNA of O. viverrini . The top panel shows the size of the cDNA while the bottom panel shows a schematic of positions and relative sizes of the 7 exons and 6 introns (arrowed) that comprise the gene. The 5′-flanking region was generated by inverse PCR. (B) Schematic to compare the structure and exon numbers of the C1 family genes, including the cathepsin F genes of O. viverrini , Schistosoma mansoni , and Homo sapiens , and the cathepsin L gene of S. mansoni . Colored blocks represent exons, thin lines represent introns, and the asterisks identify the exon(s) that encodes the catalytic Cys residue. Database accession numbers are provided for the illustrated sequences. (C) Comparison of the exon/intron structures of O. viverrini cathepsin F and human cathepsin F. The two enzymes were aligned for maximal homology and the amino acid sequences corresponding to an exon for each protein was separated by red (human cathepsin F) or blue ( O. viverrini cathepsin F) arrows. Exon numbering is shown below the blocks of amino acid sequences. Positions of the active site triad of Cys, His and Asn residues are indicated with stars. The position of signal sequence cleavage site is indicated with gray arrows, and the position of FhCL1 trans -processing cleavage site between prosegment and mature enzyme indicated with the yellow arrow.
Figure Legend Snippet: Genome structure of the cathepsin F gene of Opisthorchis viverrini . (A) Structure of the O. viverrini cathepsin F gene locus, as determined by nucleotide sequence analysis of a ∼3 kb PCR product of genomic DNA of O. viverrini . The top panel shows the size of the cDNA while the bottom panel shows a schematic of positions and relative sizes of the 7 exons and 6 introns (arrowed) that comprise the gene. The 5′-flanking region was generated by inverse PCR. (B) Schematic to compare the structure and exon numbers of the C1 family genes, including the cathepsin F genes of O. viverrini , Schistosoma mansoni , and Homo sapiens , and the cathepsin L gene of S. mansoni . Colored blocks represent exons, thin lines represent introns, and the asterisks identify the exon(s) that encodes the catalytic Cys residue. Database accession numbers are provided for the illustrated sequences. (C) Comparison of the exon/intron structures of O. viverrini cathepsin F and human cathepsin F. The two enzymes were aligned for maximal homology and the amino acid sequences corresponding to an exon for each protein was separated by red (human cathepsin F) or blue ( O. viverrini cathepsin F) arrows. Exon numbering is shown below the blocks of amino acid sequences. Positions of the active site triad of Cys, His and Asn residues are indicated with stars. The position of signal sequence cleavage site is indicated with gray arrows, and the position of FhCL1 trans -processing cleavage site between prosegment and mature enzyme indicated with the yellow arrow.

Techniques Used: Sequencing, Polymerase Chain Reaction, Generated, Inverse PCR

6) Product Images from "Identification of a common Wnt-associated genetic signature across multiple cell types in pulmonary arterial hypertension"

Article Title: Identification of a common Wnt-associated genetic signature across multiple cell types in pulmonary arterial hypertension

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00057.2014

Differential regulation of genes by deregulated BMPR2 signaling in iPS-ECL cells is strongly correlated to that in skin fibroblasts from PAH patients. Gene expression arrays were performed using RNA from 21 fibroblast lines derived from HPAH ( n = 10),
Figure Legend Snippet: Differential regulation of genes by deregulated BMPR2 signaling in iPS-ECL cells is strongly correlated to that in skin fibroblasts from PAH patients. Gene expression arrays were performed using RNA from 21 fibroblast lines derived from HPAH ( n = 10),

Techniques Used: Expressing, Derivative Assay

BMPR2 mutation causes persistent gene expression differences across cell types but does not interfere with endothelial differentiation. Expression analysis of WT and BMPR2mut iPS-MSC following 24 h of culture in MSC differentiation medium (Early), differentiated
Figure Legend Snippet: BMPR2 mutation causes persistent gene expression differences across cell types but does not interfere with endothelial differentiation. Expression analysis of WT and BMPR2mut iPS-MSC following 24 h of culture in MSC differentiation medium (Early), differentiated

Techniques Used: Mutagenesis, Expressing

BMPR2 mutation causes increased Wnt pathway gene expression only in differentiated cell types. A : heat map analysis of gene segregation showing high (red) and low (blue) levels of expression. +, Mutant samples. In this set of genes, BMPR2 mutants and
Figure Legend Snippet: BMPR2 mutation causes increased Wnt pathway gene expression only in differentiated cell types. A : heat map analysis of gene segregation showing high (red) and low (blue) levels of expression. +, Mutant samples. In this set of genes, BMPR2 mutants and

Techniques Used: Mutagenesis, Expressing

7) Product Images from "Gene expression and viral prodution in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals"

Article Title: Gene expression and viral prodution in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0437640100

Quantitation of cell-free HIV-1 virions released by latently infected, resting CD4 + T cells from representative viremic ( A and B ) and aviremic ( C and D ) patients. Cell-free supernatants collected from each culture at days 1 and 3 were subjected to Amplicor HIV-1 test (detection limit: 50 copies of HIV-1 RNA per ml) for quantitation of HIV-1 virions.
Figure Legend Snippet: Quantitation of cell-free HIV-1 virions released by latently infected, resting CD4 + T cells from representative viremic ( A and B ) and aviremic ( C and D ) patients. Cell-free supernatants collected from each culture at days 1 and 3 were subjected to Amplicor HIV-1 test (detection limit: 50 copies of HIV-1 RNA per ml) for quantitation of HIV-1 virions.

Techniques Used: Quantitation Assay, Infection

Frequency of latently infected, resting CD4 + T cells carrying HIV-1 proviral DNA ( A ) and cell-associated, unspliced HIV-1 RNA ( B ) from viremic and aviremic patients. Freshly isolated, resting CD4 + T cells were subjected to real-time PCR for detection of HIV-1 proviral DNA and cell-associated HIV-1 RNA. The geometric mean values are shown as black bars.
Figure Legend Snippet: Frequency of latently infected, resting CD4 + T cells carrying HIV-1 proviral DNA ( A ) and cell-associated, unspliced HIV-1 RNA ( B ) from viremic and aviremic patients. Freshly isolated, resting CD4 + T cells were subjected to real-time PCR for detection of HIV-1 proviral DNA and cell-associated HIV-1 RNA. The geometric mean values are shown as black bars.

Techniques Used: Infection, Isolation, Real-time Polymerase Chain Reaction

Levels of cell-free HIV-1 virions released by latently infected, resting CD4 + T cells from viremic and aviremic patients. Cell-free supernatants from each culture harvested at day 1 were subjected to Amplicor HIV-1 test (detection limit: 50 copies of HIV-1 RNA per ml). The geometric mean values are shown as black bars.
Figure Legend Snippet: Levels of cell-free HIV-1 virions released by latently infected, resting CD4 + T cells from viremic and aviremic patients. Cell-free supernatants from each culture harvested at day 1 were subjected to Amplicor HIV-1 test (detection limit: 50 copies of HIV-1 RNA per ml). The geometric mean values are shown as black bars.

Techniques Used: Infection

8) Product Images from "Recurrent Tandem Gene Duplication Gave Rise to Functionally Divergent Genes in Drosophila"

Article Title: Recurrent Tandem Gene Duplication Gave Rise to Functionally Divergent Genes in Drosophila

Journal:

doi: 10.1093/molbev/msn089

Positive Selection of CG32706 Orthologs in D. simulans , D. sechellia , and D. mauritiana
Figure Legend Snippet: Positive Selection of CG32706 Orthologs in D. simulans , D. sechellia , and D. mauritiana

Techniques Used: Selection

Positive Selection of CG32706 Orthologs in D. simulans , D. sechellia , and D. mauritiana
Figure Legend Snippet: Positive Selection of CG32706 Orthologs in D. simulans , D. sechellia , and D. mauritiana

Techniques Used: Selection

Positive Selection of CG32706 Orthologs in D. simulans , D. sechellia , and D. mauritiana
Figure Legend Snippet: Positive Selection of CG32706 Orthologs in D. simulans , D. sechellia , and D. mauritiana

Techniques Used: Selection

9) Product Images from "Integration of HPV6 and Downregulation of AKR1C3 Expression Mark Malignant Transformation in a Patient with Juvenile-Onset Laryngeal Papillomatosis"

Article Title: Integration of HPV6 and Downregulation of AKR1C3 Expression Mark Malignant Transformation in a Patient with Juvenile-Onset Laryngeal Papillomatosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0057207

Genetic analysis of the tumor. ( A ) Sequence analysis of APOT PCR product. Analysis of the chimeric HPV/AKR1C3 mRNA showed that it is spliced from the HPV6 splice donor site at nucleotide 745 to intron one of the AKR1C3 gene. ( B, C ) ArrayCGH analysis. ( B ) Large scale ideogram of chromosome 10. AKR1C3 is marked by an arrow. ( C ) Overwiev of all chromosomes. Arrows indicate 3q loss and AKR1C3 integration site on chromosome 10 and a large deletion on chromosome 3. Green colored regions indicate DNA loss. Algorism z-score, Threshold 2.5.
Figure Legend Snippet: Genetic analysis of the tumor. ( A ) Sequence analysis of APOT PCR product. Analysis of the chimeric HPV/AKR1C3 mRNA showed that it is spliced from the HPV6 splice donor site at nucleotide 745 to intron one of the AKR1C3 gene. ( B, C ) ArrayCGH analysis. ( B ) Large scale ideogram of chromosome 10. AKR1C3 is marked by an arrow. ( C ) Overwiev of all chromosomes. Arrows indicate 3q loss and AKR1C3 integration site on chromosome 10 and a large deletion on chromosome 3. Green colored regions indicate DNA loss. Algorism z-score, Threshold 2.5.

Techniques Used: Sequencing, Polymerase Chain Reaction

10) Product Images from "Intervertebral Disc Degeneration "

Article Title: Intervertebral Disc Degeneration

Journal: The American Journal of Pathology

doi:

Cyclic stretch induces IVD cell apoptosis in vitro. Rabbit AF cells were kept as static controls, subjected to stretch at 15% for 24 hours, or treated with 1 μmol/L staurosporine for 10 hours. A: Genomic DNAs isolated from static, stretched, or staurosporine-treated AF cells were subjected to LM-PCR analysis followed by agarose gel electrophoresis. The data represent results of three independent experiments. B: The nuclear chromatin of AF cells was detected by Hoechst 33258 staining and indicates the cells undergoing apoptosis. Arrows indicate condensed and brightly stained nuclei. A representative section is shown at ×200 magnification. C: Quantitative evaluation of apoptosis was assayed by FACS after PI staining. Results are presented as the percentage of cells below the G 0 /G 1 peak (containing hypodiploid DNA) and are expressed as mean ± SD of three independent experiments. * indicates P
Figure Legend Snippet: Cyclic stretch induces IVD cell apoptosis in vitro. Rabbit AF cells were kept as static controls, subjected to stretch at 15% for 24 hours, or treated with 1 μmol/L staurosporine for 10 hours. A: Genomic DNAs isolated from static, stretched, or staurosporine-treated AF cells were subjected to LM-PCR analysis followed by agarose gel electrophoresis. The data represent results of three independent experiments. B: The nuclear chromatin of AF cells was detected by Hoechst 33258 staining and indicates the cells undergoing apoptosis. Arrows indicate condensed and brightly stained nuclei. A representative section is shown at ×200 magnification. C: Quantitative evaluation of apoptosis was assayed by FACS after PI staining. Results are presented as the percentage of cells below the G 0 /G 1 peak (containing hypodiploid DNA) and are expressed as mean ± SD of three independent experiments. * indicates P

Techniques Used: In Vitro, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, FACS

Cyclic stretch-induced apoptosis is initiated by MDA. Rabbit AF cells were kept as static controls ( A and B ), stretched at 15% for 24 hours, or treated with 1 μmol/L staurosporine or kept as static controls ( C and D ), stretched at 15% for 24 hours, or treated with 100 μmol/L Z-LEHD-FMK. A: The caspase-9 activity in cell lysates was measured by detecting the cleavage of LEHD- p -NA, determined by use of a colorimetric plate reader at 405 nm. Results are expressed as mean ± SD from three independent experiments. B: ΔΨ was assayed by FACS after DiOC 6 staining. The percentage represents the fraction of cells with decreased DiOC 6 fluorescence. C: Quantitative evaluation of apoptosis was assayed by FACS after PI staining. Results are presented as the percentage of cells below the G 0 /G 1 peak (containing hypodiploid DNA) and expressed as mean ± SD from three independent experiments. D: DNA fragmentation was examined after LM-PCR analysis of genomic DNA followed by agarose gel electrophoresis. * indicates P
Figure Legend Snippet: Cyclic stretch-induced apoptosis is initiated by MDA. Rabbit AF cells were kept as static controls ( A and B ), stretched at 15% for 24 hours, or treated with 1 μmol/L staurosporine or kept as static controls ( C and D ), stretched at 15% for 24 hours, or treated with 100 μmol/L Z-LEHD-FMK. A: The caspase-9 activity in cell lysates was measured by detecting the cleavage of LEHD- p -NA, determined by use of a colorimetric plate reader at 405 nm. Results are expressed as mean ± SD from three independent experiments. B: ΔΨ was assayed by FACS after DiOC 6 staining. The percentage represents the fraction of cells with decreased DiOC 6 fluorescence. C: Quantitative evaluation of apoptosis was assayed by FACS after PI staining. Results are presented as the percentage of cells below the G 0 /G 1 peak (containing hypodiploid DNA) and expressed as mean ± SD from three independent experiments. D: DNA fragmentation was examined after LM-PCR analysis of genomic DNA followed by agarose gel electrophoresis. * indicates P

Techniques Used: Multiple Displacement Amplification, Activity Assay, FACS, Staining, Fluorescence, Polymerase Chain Reaction, Agarose Gel Electrophoresis

11) Product Images from "A porcine model of neurofibromatosis type 1 that mimics the human disease"

Article Title: A porcine model of neurofibromatosis type 1 that mimics the human disease

Journal: JCI Insight

doi: 10.1172/jci.insight.120402

Generation of NF1 +/ex42del -targeted miniswine. ( A ) Gene targeting scheme depicting the deletion of exon 42. Exons 41–43 are shown as gray boxes, and the Blast R cassette is shown in red. Dotted vertical lines indicate the gene-targeting construct boundaries. Arrows indicate PCR primers. NF1 Southern blot probe is indicated by a black line and labeled “probe.” Red arrowhead represents remaining loxP site. Figure is not to scale. ( B ) Representative PCR genotyping. Lanes 1–7 represent individual miniswine, lane 8 is a no-template control, lane 9 is an NF1 +/ex42del with Blast R control, and lane 10 is an NF1 +/ex42del Blast R -excised control. PCR primer locations are indicated in A . ( C ) Representative genomic Southern blot with NF1 (upper panel) and Blast R (lower panel) probes. NcoI/EcoNi-digested DNA from WT NF1 miniswine yields a 3.0 kb product, whereas the same digest of NF1 +/ex42del Blast R –excised yields a product of 3.7 kb. NF1 +/ex42del with Blast R yields a product of 4.9 kb. Lanes 1–13 represent 13 individual piglets. Lane 14 is an NF1 +/ex42del with Blast R control. ( D ) Control litter presents with no pigmentation. Characteristic café au lait macules ( E and F , back rump and head regions), auxiliary/inguinal freckling ( G , flank), and cutaneous tumors ( H , neck with cross-sectional insert) are evident on NF1 +/ex42del miniswine.
Figure Legend Snippet: Generation of NF1 +/ex42del -targeted miniswine. ( A ) Gene targeting scheme depicting the deletion of exon 42. Exons 41–43 are shown as gray boxes, and the Blast R cassette is shown in red. Dotted vertical lines indicate the gene-targeting construct boundaries. Arrows indicate PCR primers. NF1 Southern blot probe is indicated by a black line and labeled “probe.” Red arrowhead represents remaining loxP site. Figure is not to scale. ( B ) Representative PCR genotyping. Lanes 1–7 represent individual miniswine, lane 8 is a no-template control, lane 9 is an NF1 +/ex42del with Blast R control, and lane 10 is an NF1 +/ex42del Blast R -excised control. PCR primer locations are indicated in A . ( C ) Representative genomic Southern blot with NF1 (upper panel) and Blast R (lower panel) probes. NcoI/EcoNi-digested DNA from WT NF1 miniswine yields a 3.0 kb product, whereas the same digest of NF1 +/ex42del Blast R –excised yields a product of 3.7 kb. NF1 +/ex42del with Blast R yields a product of 4.9 kb. Lanes 1–13 represent 13 individual piglets. Lane 14 is an NF1 +/ex42del with Blast R control. ( D ) Control litter presents with no pigmentation. Characteristic café au lait macules ( E and F , back rump and head regions), auxiliary/inguinal freckling ( G , flank), and cutaneous tumors ( H , neck with cross-sectional insert) are evident on NF1 +/ex42del miniswine.

Techniques Used: Construct, Polymerase Chain Reaction, Southern Blot, Labeling

12) Product Images from "Human Artificial Chromosome with a Conditional Centromere for Gene Delivery and Gene Expression"

Article Title: Human Artificial Chromosome with a Conditional Centromere for Gene Delivery and Gene Expression

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsq020

Insertion of the loxP cassette into the tet-O HAC by homologous recombination in DT40 cells. (A) Morphology of A9BH-3 hybrid cells. (B) Genomic DNA from A9BH-3 hybrid cells was analysed by PCR using HAC-specific primers, HRL-F/R and BACpr-F/R. (C) FISH of DT40/BH-1 cells using the hCot1 probe. The tet-O HAC is indicated by an arrowhead. (D) Insertion of the loxP/HPRT cassette into the tet-O HAC using the targeting vector 5–4–3, which carries the regions of homology (black boxes) to BAC vector sequences in the HAC. (E) FISH analysis of DT40/BHI 1–38 (left) and DT40/BHI 2–2 (right) cells. FISH analysis was performed using a biotin-labelled probe for the loxP cassette (green) and digoxigenin-labelled human hCot1 (red).
Figure Legend Snippet: Insertion of the loxP cassette into the tet-O HAC by homologous recombination in DT40 cells. (A) Morphology of A9BH-3 hybrid cells. (B) Genomic DNA from A9BH-3 hybrid cells was analysed by PCR using HAC-specific primers, HRL-F/R and BACpr-F/R. (C) FISH of DT40/BH-1 cells using the hCot1 probe. The tet-O HAC is indicated by an arrowhead. (D) Insertion of the loxP/HPRT cassette into the tet-O HAC using the targeting vector 5–4–3, which carries the regions of homology (black boxes) to BAC vector sequences in the HAC. (E) FISH analysis of DT40/BHI 1–38 (left) and DT40/BHI 2–2 (right) cells. FISH analysis was performed using a biotin-labelled probe for the loxP cassette (green) and digoxigenin-labelled human hCot1 (red).

Techniques Used: HAC Assay, Homologous Recombination, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Plasmid Preparation, BAC Assay

13) Product Images from "Genetic Variations in HSPA8 Gene Associated with Coronary Heart Disease Risk in a Chinese Population"

Article Title: Genetic Variations in HSPA8 Gene Associated with Coronary Heart Disease Risk in a Chinese Population

Journal: PLoS ONE

doi: 10.1371/journal.pone.0009684

Linkage disequilibrium (D' and r 2 ) between single nucleotide polymorphisms in HSPA8 gene. Based on the data on the resequencing of the HSPA8 gene, a total of 22 single nucleotide polymorphisms were analyzed by JLIN software to analyze linkage disequilibrium (D' and r 2 ) between SNPs.
Figure Legend Snippet: Linkage disequilibrium (D' and r 2 ) between single nucleotide polymorphisms in HSPA8 gene. Based on the data on the resequencing of the HSPA8 gene, a total of 22 single nucleotide polymorphisms were analyzed by JLIN software to analyze linkage disequilibrium (D' and r 2 ) between SNPs.

Techniques Used: Software

Effects of haplotypes in promoter of HSPA8 on luciferase activity. Transient luciferase reporter gene expression assays with constructs containing different haplotypes of HSPA8 promoter in human bronchial epithelial cells (HBE) and human umbilical vein endothelial cells (HUVEC). Upper: schematic representation of different reporter gene constructs. Variants of -703C/T, -357C/T, and -308A/G represent rs2236660, rs2236659, and rs2236658 respectively; Lower: luciferase expression of these constructs. All constructs were cotransfected with pRL-SV40 to standardize the transfection efficiency. Fold increase of luciferase activity was calculated by defining the activity of the empty pGL3-Basic vector as 1. All experiments were performed in triplicates at least in three independent transfection experiments and each value represents mean (standard deviation). *and † indicated that P
Figure Legend Snippet: Effects of haplotypes in promoter of HSPA8 on luciferase activity. Transient luciferase reporter gene expression assays with constructs containing different haplotypes of HSPA8 promoter in human bronchial epithelial cells (HBE) and human umbilical vein endothelial cells (HUVEC). Upper: schematic representation of different reporter gene constructs. Variants of -703C/T, -357C/T, and -308A/G represent rs2236660, rs2236659, and rs2236658 respectively; Lower: luciferase expression of these constructs. All constructs were cotransfected with pRL-SV40 to standardize the transfection efficiency. Fold increase of luciferase activity was calculated by defining the activity of the empty pGL3-Basic vector as 1. All experiments were performed in triplicates at least in three independent transfection experiments and each value represents mean (standard deviation). *and † indicated that P

Techniques Used: Luciferase, Activity Assay, Expressing, Construct, Transfection, Plasmid Preparation, Standard Deviation

Construction of haplotype and tagging SNPs selection in HSPA8 gene. A total of 22 single nucleotide polymorphisms (SNPs) were analyzed by htSNPer1.0 software to select tagSNPs. 1.0 indicated rs2236660 and 22.0, the last SNP rs4802. The results showed that rs2236660, rs2236658, rs10892958 and rs1461496 were selected for tagSNPs.
Figure Legend Snippet: Construction of haplotype and tagging SNPs selection in HSPA8 gene. A total of 22 single nucleotide polymorphisms (SNPs) were analyzed by htSNPer1.0 software to select tagSNPs. 1.0 indicated rs2236660 and 22.0, the last SNP rs4802. The results showed that rs2236660, rs2236658, rs10892958 and rs1461496 were selected for tagSNPs.

Techniques Used: Selection, Software

14) Product Images from "Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies"

Article Title: Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies

Journal: PLoS ONE

doi: 10.1371/journal.pone.0187072

Analysis of HepG2 cells containing the 4CYPs-POR MAC. (A) Flowchart of the MAC transfer from donor CHO cells to recipient HepG2 cells via MMCT method, which comprises the following steps: micronucleation of donor cells by colcemid, enucleation by cytochalasin B, and microcell purification and fusion with recipient HepG2 cells. HepG2 hybrids were selected with 400 μg/mL G418 and picked for clonal expansion. (B) G418-resistant clones are screened by genomic PCR to determine the presence of the 4CYPs-POR transgene. (C) Representative metaphase fluorescence in situ hybridization images of TC-HepG2 cells. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled pPAC-4CYPs-POR (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrows indicate MAC vectors, and the inset shows an enlarged image of the MAC.
Figure Legend Snippet: Analysis of HepG2 cells containing the 4CYPs-POR MAC. (A) Flowchart of the MAC transfer from donor CHO cells to recipient HepG2 cells via MMCT method, which comprises the following steps: micronucleation of donor cells by colcemid, enucleation by cytochalasin B, and microcell purification and fusion with recipient HepG2 cells. HepG2 hybrids were selected with 400 μg/mL G418 and picked for clonal expansion. (B) G418-resistant clones are screened by genomic PCR to determine the presence of the 4CYPs-POR transgene. (C) Representative metaphase fluorescence in situ hybridization images of TC-HepG2 cells. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled pPAC-4CYPs-POR (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrows indicate MAC vectors, and the inset shows an enlarged image of the MAC.

Techniques Used: Purification, Clone Assay, Polymerase Chain Reaction, Fluorescence, In Situ Hybridization, Labeling

Construction and analysis of the 4CYPs-POR MAC vector in CHO cells. (A) Genomic PCR and (B) RT-PCR analysis of CHO cells containing the 4CYPs-POR MAC. N, negative control (parental CHO cells); P, positive control (PAC-4CYPs-POR). (C) FISH analysis of donor CHO cells containing the 4CYPs-POR MAC. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled 4CYPs-POR PAC (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrow indicates MAC vector, and the inset shows enlarged image of the MAC.
Figure Legend Snippet: Construction and analysis of the 4CYPs-POR MAC vector in CHO cells. (A) Genomic PCR and (B) RT-PCR analysis of CHO cells containing the 4CYPs-POR MAC. N, negative control (parental CHO cells); P, positive control (PAC-4CYPs-POR). (C) FISH analysis of donor CHO cells containing the 4CYPs-POR MAC. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled 4CYPs-POR PAC (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrow indicates MAC vector, and the inset shows enlarged image of the MAC.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Fluorescence In Situ Hybridization, Labeling

15) Product Images from "Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor ?1 Expression ▿Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor ?1 Expression ▿ ‡"

Article Title: Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor ?1 Expression ▿Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor ?1 Expression ▿ ‡

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01484-08

GFRα1 expression after the adenovirus-mediated transfer of Ape1/Ref-1 in GM00637 cells. (A) The GM00637 cells were transfected with Ad-LacZ or Ad-Ape1/Ref-1 at a multiplicity of infection of 50, and the cells were harvested 48 h after the infection. The total RNA was extracted and subjected to semiquantitative RT-PCR using the Ape1/Ref-1 -, GFRα -, and GAPDH -specific primers. (B) Protein extracts prepared 48 h after the infection with Ad-LacZ or Ad-Ape1/Ref-1. A 20-μg portion of the total protein was loaded onto an SDS-polyacrylamide gel for Western blot analysis. Antibodies to Ape1/Ref-1 and GFRα1 were used. The detection of α-tubulin was used as the loading control. (C) The GM00637 cells were transfected with control pcDNA3 vector (control), repair Ape1/Ref-1 mutant expression vector (ΔRD), or redox Ape1/Ref-1 mutant expression vector (ΔAPD), and the total RNA was then extracted and subjected to semiquantitative RT-PCR using the Redox Ape1/Ref-1 , Repair Ape1/Ref-1 , GFRα, and GAPDH primers.
Figure Legend Snippet: GFRα1 expression after the adenovirus-mediated transfer of Ape1/Ref-1 in GM00637 cells. (A) The GM00637 cells were transfected with Ad-LacZ or Ad-Ape1/Ref-1 at a multiplicity of infection of 50, and the cells were harvested 48 h after the infection. The total RNA was extracted and subjected to semiquantitative RT-PCR using the Ape1/Ref-1 -, GFRα -, and GAPDH -specific primers. (B) Protein extracts prepared 48 h after the infection with Ad-LacZ or Ad-Ape1/Ref-1. A 20-μg portion of the total protein was loaded onto an SDS-polyacrylamide gel for Western blot analysis. Antibodies to Ape1/Ref-1 and GFRα1 were used. The detection of α-tubulin was used as the loading control. (C) The GM00637 cells were transfected with control pcDNA3 vector (control), repair Ape1/Ref-1 mutant expression vector (ΔRD), or redox Ape1/Ref-1 mutant expression vector (ΔAPD), and the total RNA was then extracted and subjected to semiquantitative RT-PCR using the Redox Ape1/Ref-1 , Repair Ape1/Ref-1 , GFRα, and GAPDH primers.

Techniques Used: Expressing, Transfection, Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Mutagenesis

Ape1/Ref-1 increases c-Src phosphorylation and cellular proliferation in response to GDNF through GFRα1. (A) Ad-Ape1/Ref-1-infected GM00637 cells were transfected with the mock, control siRNA (Cont), or GFRα1 siRNA. At 48 h after transfection, cells were incubated with GDNF (10 ng/ml) for 1 h. Equal amounts (20 μg of proteins) of the cell lysates were separated by SDS-10% PAGE and then transferred onto a nitrocellulose membrane. The membrane was immunoblotted with anti-Ape1/Ref-1, anti-GFRα1, or anti-phospho c-Src (Tyr418) antibodies. To control for equal loading, the membrane was stripped and reprobed against nonphosphorylated c-Src and α-tubulin. (B) Ad-Ape1/Ref-1-infected GM00637 cells were transfected with the mock, control-siRNA (Cont), Ape1/Ref-1-siRNA (Ape1), or GFRα1-siRNA. At 48 h after transfection, cells were then incubated with GDNF (10 ng/ml) for an additional 24 h. The total protein was extracted from the cells, and equal amounts (20 μg of proteins) of the cell lysates were separated by SDS-10% PAGE and then transferred onto a nitrocellulose membrane. The membrane was immunoblotted with anti-Ape1/Ref-1, anti-GFRα1, or anti-β-actin antibodies. (C) Ad-LacZ (LacZ)- or Ad-Ape1/Ref-1 (Ape1)-infected GM00637 cells were transfected with control siRNA or GFRα1 siRNA and then incubated with or without GDNF (10 ng/ml) for up to 72 h. The number of cells was determined by counting the cells every 24 h after GDNF treatment. Each value is the mean ± the SD from three separate experiments. **, P
Figure Legend Snippet: Ape1/Ref-1 increases c-Src phosphorylation and cellular proliferation in response to GDNF through GFRα1. (A) Ad-Ape1/Ref-1-infected GM00637 cells were transfected with the mock, control siRNA (Cont), or GFRα1 siRNA. At 48 h after transfection, cells were incubated with GDNF (10 ng/ml) for 1 h. Equal amounts (20 μg of proteins) of the cell lysates were separated by SDS-10% PAGE and then transferred onto a nitrocellulose membrane. The membrane was immunoblotted with anti-Ape1/Ref-1, anti-GFRα1, or anti-phospho c-Src (Tyr418) antibodies. To control for equal loading, the membrane was stripped and reprobed against nonphosphorylated c-Src and α-tubulin. (B) Ad-Ape1/Ref-1-infected GM00637 cells were transfected with the mock, control-siRNA (Cont), Ape1/Ref-1-siRNA (Ape1), or GFRα1-siRNA. At 48 h after transfection, cells were then incubated with GDNF (10 ng/ml) for an additional 24 h. The total protein was extracted from the cells, and equal amounts (20 μg of proteins) of the cell lysates were separated by SDS-10% PAGE and then transferred onto a nitrocellulose membrane. The membrane was immunoblotted with anti-Ape1/Ref-1, anti-GFRα1, or anti-β-actin antibodies. (C) Ad-LacZ (LacZ)- or Ad-Ape1/Ref-1 (Ape1)-infected GM00637 cells were transfected with control siRNA or GFRα1 siRNA and then incubated with or without GDNF (10 ng/ml) for up to 72 h. The number of cells was determined by counting the cells every 24 h after GDNF treatment. Each value is the mean ± the SD from three separate experiments. **, P

Techniques Used: Infection, Transfection, Incubation, Polyacrylamide Gel Electrophoresis

16) Product Images from "The Catalytic Subunit of DNA-Dependent Protein Kinase Regulates Proliferation, Telomere Length, and Genomic Stability in Human Somatic Cells ▿The Catalytic Subunit of DNA-Dependent Protein Kinase Regulates Proliferation, Telomere Length, and Genomic Stability in Human Somatic Cells ▿ †"

Article Title: The Catalytic Subunit of DNA-Dependent Protein Kinase Regulates Proliferation, Telomere Length, and Genomic Stability in Human Somatic Cells ▿The Catalytic Subunit of DNA-Dependent Protein Kinase Regulates Proliferation, Telomere Length, and Genomic Stability in Human Somatic Cells ▿ †

Journal:

doi: 10.1128/MCB.00355-08

Identification of DNA-PK cs +/− cell lines. Four diagnostic PCRs were carried out: an experimental 5′ PCR to confirm correct targeting events on the 5′ side, using the primers PKcs81-83F1 and LArmR; an experimental 3′
Figure Legend Snippet: Identification of DNA-PK cs +/− cell lines. Four diagnostic PCRs were carried out: an experimental 5′ PCR to confirm correct targeting events on the 5′ side, using the primers PKcs81-83F1 and LArmR; an experimental 3′

Techniques Used: Diagnostic Assay, Polymerase Chain Reaction

17) Product Images from "Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications"

Article Title: Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications

Journal: BMC Research Notes

doi: 10.1186/1756-0500-3-239

Internal control amplification . Mean Ct value of PhHV PCR of the 4 materials (A, B, C and D) for the different extraction methods. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.
Figure Legend Snippet: Internal control amplification . Mean Ct value of PhHV PCR of the 4 materials (A, B, C and D) for the different extraction methods. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.

Techniques Used: Amplification, Polymerase Chain Reaction, DNA Extraction

Assessment of maximum amplicon length: PCR product yield . Mean relative yield (%) of 200, 400 and 600 bp PCR products of 4 materials (A, B, C and D). The prot. K + QIAamp extraction's yield was set at 100%. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.
Figure Legend Snippet: Assessment of maximum amplicon length: PCR product yield . Mean relative yield (%) of 200, 400 and 600 bp PCR products of 4 materials (A, B, C and D). The prot. K + QIAamp extraction's yield was set at 100%. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.

Techniques Used: Amplification, Polymerase Chain Reaction, DNA Extraction

SNP analysis using real time PCR . A . Two representative component plots generated after proteinase K treatment followed by EasyMAG extraction from colon tissue B and real time amplification using SNP assay rs1350138. The light grey line indicates allele 1 (VIC label), the dark grey line indicates allele 2 (FAM label). B . Mean Ct value of SNP rs2043731 and rs1350138 of the 4 materials (A, B, C and D) in JBZ 4× mastermix or ABI 2× mastermix for the different extraction methods. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.
Figure Legend Snippet: SNP analysis using real time PCR . A . Two representative component plots generated after proteinase K treatment followed by EasyMAG extraction from colon tissue B and real time amplification using SNP assay rs1350138. The light grey line indicates allele 1 (VIC label), the dark grey line indicates allele 2 (FAM label). B . Mean Ct value of SNP rs2043731 and rs1350138 of the 4 materials (A, B, C and D) in JBZ 4× mastermix or ABI 2× mastermix for the different extraction methods. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.

Techniques Used: Real-time Polymerase Chain Reaction, Generated, Amplification, DNA Extraction

Assessment of maximum amplicon length: visualisation on gel . Gel image of 200-400-600 bp multiplex PCR-products of representative tissue B. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G. Neg = ultrapure water in PCR, Pos = QIAamp extracted DNA from EDTA-blood.
Figure Legend Snippet: Assessment of maximum amplicon length: visualisation on gel . Gel image of 200-400-600 bp multiplex PCR-products of representative tissue B. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G. Neg = ultrapure water in PCR, Pos = QIAamp extracted DNA from EDTA-blood.

Techniques Used: Amplification, Multiplex Assay, Polymerase Chain Reaction, DNA Extraction

18) Product Images from "Isolation and Mutagenesis of a Capsule-Like Complex (CLC) from Francisella tularensis, and Contribution of the CLC to F. tularensis Virulence in Mice"

Article Title: Isolation and Mutagenesis of a Capsule-Like Complex (CLC) from Francisella tularensis, and Contribution of the CLC to F. tularensis Virulence in Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0019003

RT-PCR of the DNA region FTL_1421 from LVSΔ1423/1422. LVS and LVSΔ1423/1422 were grown on Glc-CDMA for at least 2 days, the RNA isolated, converted to cDNA, and amplified by PCR to determine if a transcript from DNA downstream of the mutation was made. Lanes: M, 1 kb+ DNA molecular size standards; 1, control amplification of FTL_1425-1424 in LVS; 2, amplification of FTL_1425-1424 upstream of the mutation in LVSΔ1423-1422; 3, control amplification of FTL_1425-1424 upstream of the mutation (no bacteria); 4, control amplification of FTL_1421 from LVS; 5, amplification of FTL_1421 immediately downstream of the mutation in LVSΔ1423/1422. The presence of a normal band of about 351 bp from LVSΔ1423/1422 indicated that the mutation had no polar effect on downstream genes.
Figure Legend Snippet: RT-PCR of the DNA region FTL_1421 from LVSΔ1423/1422. LVS and LVSΔ1423/1422 were grown on Glc-CDMA for at least 2 days, the RNA isolated, converted to cDNA, and amplified by PCR to determine if a transcript from DNA downstream of the mutation was made. Lanes: M, 1 kb+ DNA molecular size standards; 1, control amplification of FTL_1425-1424 in LVS; 2, amplification of FTL_1425-1424 upstream of the mutation in LVSΔ1423-1422; 3, control amplification of FTL_1425-1424 upstream of the mutation (no bacteria); 4, control amplification of FTL_1421 from LVS; 5, amplification of FTL_1421 immediately downstream of the mutation in LVSΔ1423/1422. The presence of a normal band of about 351 bp from LVSΔ1423/1422 indicated that the mutation had no polar effect on downstream genes.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Gas Chromatography, Isolation, Amplification, Polymerase Chain Reaction, Mutagenesis

19) Product Images from "Gene targeting in adult rhesus macaque fibroblasts"

Article Title: Gene targeting in adult rhesus macaque fibroblasts

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-8-31

PCR screening strategy . a) A 3.9 kb PCR band occurs in the non-targeted wild type rhesus macaque fibroblast DNA. The reverse screening primer anneals to the deleted part of exon 6. If a targeting event has occurred, there will be no 3.9 kb PCR band since that part if exon 6 has been deleted. b) M = 1 kb Marker, HTF = HPRT targeted fibroblasts, WT = wild type rhesus fibroblasts, ATM = ataxia telangiectasia mutated (positive control).
Figure Legend Snippet: PCR screening strategy . a) A 3.9 kb PCR band occurs in the non-targeted wild type rhesus macaque fibroblast DNA. The reverse screening primer anneals to the deleted part of exon 6. If a targeting event has occurred, there will be no 3.9 kb PCR band since that part if exon 6 has been deleted. b) M = 1 kb Marker, HTF = HPRT targeted fibroblasts, WT = wild type rhesus fibroblasts, ATM = ataxia telangiectasia mutated (positive control).

Techniques Used: Polymerase Chain Reaction, Marker, Positive Control

hTERT genomic PCR assay . a) pCI-neo-hTERT construct. Primers (arrows) amplify a 2960 bp product that includes the 3' end of the hTERT cassette and the 5' end of the NEO r cassette. b) M = 1 kb Marker, WT = wild type rhesus fibroblasts, HTF = TERT transfected fibroblasts (Experiment #8, HPRT null clone).
Figure Legend Snippet: hTERT genomic PCR assay . a) pCI-neo-hTERT construct. Primers (arrows) amplify a 2960 bp product that includes the 3' end of the hTERT cassette and the 5' end of the NEO r cassette. b) M = 1 kb Marker, WT = wild type rhesus fibroblasts, HTF = TERT transfected fibroblasts (Experiment #8, HPRT null clone).

Techniques Used: Polymerase Chain Reaction, Construct, Marker, Transfection

RT-PCR for TERT expression in rhesus fibroblast cells . a) CDS = coding region, T2A = hTERT-T2A, T2B = hTERT-T2B, ENF3 = MK TERT ENF3, ENR3 = MK TERT ENR3, blue line = portions of TERT included in pCI-neo-hTERT construct. b) M = 100 bp marker; WT = wild type rhesus fibroblasts; ES = rhesus embryonic stem cells; TTF = TERT transfected fibroblasts (experiment #8, HPRT null clone) T3' = Amplicons of 3' UTR TERT found in endogenous TERT mRNA but not present in construct hTERT mRNA; TC = Amplicons of coding TERT found in both endogenous and construct hTERT mRNA.
Figure Legend Snippet: RT-PCR for TERT expression in rhesus fibroblast cells . a) CDS = coding region, T2A = hTERT-T2A, T2B = hTERT-T2B, ENF3 = MK TERT ENF3, ENR3 = MK TERT ENR3, blue line = portions of TERT included in pCI-neo-hTERT construct. b) M = 100 bp marker; WT = wild type rhesus fibroblasts; ES = rhesus embryonic stem cells; TTF = TERT transfected fibroblasts (experiment #8, HPRT null clone) T3' = Amplicons of 3' UTR TERT found in endogenous TERT mRNA but not present in construct hTERT mRNA; TC = Amplicons of coding TERT found in both endogenous and construct hTERT mRNA.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Construct, Marker, Transfection

20) Product Images from "A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells"

Article Title: A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky332

High-efficiency editing by human A3A and A3Bctd editosomes. ( A ) Representative fluorescence microscopy images of ACE-activated, mCherry-positive 293T cells catalyzed by human A3A, human A3Bctd, or rat APOBEC1/BE3 editosomes (mCherry codon 59-directed gRNA versus NS-gRNA; inset white bar = 30 μm). ( B ) Quantification of the experiment in panel ‘A’ together with 2 independent parallel experiments ( n = 3; average ± SD). The corresponding immunoblots of expressed APOBEC–Cas9n-UGI constructs are shown below (low and high exposures to help visualize BE3) with HSP90 as a loading control. ( C ) Time course of ACE activation in 293T cells catalyzed by human A3A, human A3Bctd, or rat APOBEC1/BE3 editosomes (mCherry codon 59-directed gRNA versus NS-gRNA; n = 3; mean ± SD; error bars smaller than symbols are not shown). ( D ) Titration data for 293T cells co-transfected with the ACE reporter, mCherry codon 59-directed gRNA, and different amounts of the indicated editosome constructs (100–600 ng; n = 3; mean ± SD). BE3i has an intron in the rat APOBEC1 portion of the construct, identical to the intron required for propagation of A3A and A3Bctd constructs in E. coli and for expression in mammalian cells.
Figure Legend Snippet: High-efficiency editing by human A3A and A3Bctd editosomes. ( A ) Representative fluorescence microscopy images of ACE-activated, mCherry-positive 293T cells catalyzed by human A3A, human A3Bctd, or rat APOBEC1/BE3 editosomes (mCherry codon 59-directed gRNA versus NS-gRNA; inset white bar = 30 μm). ( B ) Quantification of the experiment in panel ‘A’ together with 2 independent parallel experiments ( n = 3; average ± SD). The corresponding immunoblots of expressed APOBEC–Cas9n-UGI constructs are shown below (low and high exposures to help visualize BE3) with HSP90 as a loading control. ( C ) Time course of ACE activation in 293T cells catalyzed by human A3A, human A3Bctd, or rat APOBEC1/BE3 editosomes (mCherry codon 59-directed gRNA versus NS-gRNA; n = 3; mean ± SD; error bars smaller than symbols are not shown). ( D ) Titration data for 293T cells co-transfected with the ACE reporter, mCherry codon 59-directed gRNA, and different amounts of the indicated editosome constructs (100–600 ng; n = 3; mean ± SD). BE3i has an intron in the rat APOBEC1 portion of the construct, identical to the intron required for propagation of A3A and A3Bctd constructs in E. coli and for expression in mammalian cells.

Techniques Used: Fluorescence, Microscopy, Western Blot, Construct, Activation Assay, Titration, Transfection, Expressing

ACE enriches for base-editing events at heterologous genomic loci. ( A ) Schematic of a co-transfection experiment resulting in ACE reporter activation (yellow shading represents mCherry and eGFP double-positive cells). FANCF and the Pst I restriction assay used to quantify chromosomal base editing of this locus. Base editing events destroy the Pst I cleavage site and block cleavage of the 452 bp amplicon into 260 and 192 bp products. ( B ) Representative agarose gels images showing the results of FANCF base editing by A3A and A3Bctd editsomes in 293T cells. The percentage of base editing was calculated by dividing the percentage of substrate band by the total of substrate and product bands following Pst I cleavage for both unsorted and mCherry-positive cell populations. ( C ) Sanger sequencing results for the gRNA-binding region of the FANCF gene, which was recovered by high-fidelity PCR using genomic DNA from mCherry-positive 293T. Mutated nucleotides are highlighted in red and deleted nucleotides are indicated by hyphens. The number of times each sequence was recovered is indicated to the right.
Figure Legend Snippet: ACE enriches for base-editing events at heterologous genomic loci. ( A ) Schematic of a co-transfection experiment resulting in ACE reporter activation (yellow shading represents mCherry and eGFP double-positive cells). FANCF and the Pst I restriction assay used to quantify chromosomal base editing of this locus. Base editing events destroy the Pst I cleavage site and block cleavage of the 452 bp amplicon into 260 and 192 bp products. ( B ) Representative agarose gels images showing the results of FANCF base editing by A3A and A3Bctd editsomes in 293T cells. The percentage of base editing was calculated by dividing the percentage of substrate band by the total of substrate and product bands following Pst I cleavage for both unsorted and mCherry-positive cell populations. ( C ) Sanger sequencing results for the gRNA-binding region of the FANCF gene, which was recovered by high-fidelity PCR using genomic DNA from mCherry-positive 293T. Mutated nucleotides are highlighted in red and deleted nucleotides are indicated by hyphens. The number of times each sequence was recovered is indicated to the right.

Techniques Used: Cotransfection, Activation Assay, Restriction Assay, Blocking Assay, Amplification, Sequencing, Binding Assay, Polymerase Chain Reaction

A real-time fluorescent reporter for APOBEC- and Cas9-mediated editing. ( A ) Schematic of the APOBEC- and Cas9-mediated editing (ACE) reporter in the context of a lentiviral construct with a CMV promoter that drives expression of a bicistronic message encoding mutant mCherry and wild-type eGFP. The sequence of the gRNA displaced DNA strand is shown below with flanking APOBEC 5′-TCA deamination hotspots (red), PAM sites, and 43 bp insertion labeled. See text for a description of editing, cleavage, and processing events required for reversion to mCherry-positive. Ribbon schematics of defective mCherry (gray) and functionally restored mCherry (red) with the flexible loop position of residue 59 shown (model based on pdb 2H5Q). ( B ) Schematic of an APOBEC–Cas9n/gRNA editosome engaging a DNA target. C-to-U editing occurs in the ssDNA loop displaced by gRNA annealing to target DNA. ( C ) Representative images of mCherry-positive 293T cells catalyzed by BE3 and mCherry codon 59-directed gRNA (#59-gRNA) but not with NS-gRNA (NS, non-specific; inset white bar = 30 μm). ( D ) Quantification of the base editing experiment in panel C ( n = 3; average ± SD).
Figure Legend Snippet: A real-time fluorescent reporter for APOBEC- and Cas9-mediated editing. ( A ) Schematic of the APOBEC- and Cas9-mediated editing (ACE) reporter in the context of a lentiviral construct with a CMV promoter that drives expression of a bicistronic message encoding mutant mCherry and wild-type eGFP. The sequence of the gRNA displaced DNA strand is shown below with flanking APOBEC 5′-TCA deamination hotspots (red), PAM sites, and 43 bp insertion labeled. See text for a description of editing, cleavage, and processing events required for reversion to mCherry-positive. Ribbon schematics of defective mCherry (gray) and functionally restored mCherry (red) with the flexible loop position of residue 59 shown (model based on pdb 2H5Q). ( B ) Schematic of an APOBEC–Cas9n/gRNA editosome engaging a DNA target. C-to-U editing occurs in the ssDNA loop displaced by gRNA annealing to target DNA. ( C ) Representative images of mCherry-positive 293T cells catalyzed by BE3 and mCherry codon 59-directed gRNA (#59-gRNA) but not with NS-gRNA (NS, non-specific; inset white bar = 30 μm). ( D ) Quantification of the base editing experiment in panel C ( n = 3; average ± SD).

Techniques Used: Construct, Expressing, Mutagenesis, Sequencing, Labeling

Chromosomal editing by A3A and A3Bctd editosomes. ( A ) Editing of single copy genomic ACE reporter by the indicated editosomes in 293T and HeLa cells ( n = 3, average ± SD). ( B ) Immunoblots corresponding to a representative experiment in panel A showing APOBEC–Cas9n-UGI expression levels and HSP90 as a loading control. ( C ) Sanger sequencing results for the gRNA-binding region of the ACE reporter recovered by high-fidelity PCR of mCherry-positive 293T. Mutated nucleotides are depicted in red and deleted nucleotides by hyphens. The number of times each sequence was recovered is indicated to the right.
Figure Legend Snippet: Chromosomal editing by A3A and A3Bctd editosomes. ( A ) Editing of single copy genomic ACE reporter by the indicated editosomes in 293T and HeLa cells ( n = 3, average ± SD). ( B ) Immunoblots corresponding to a representative experiment in panel A showing APOBEC–Cas9n-UGI expression levels and HSP90 as a loading control. ( C ) Sanger sequencing results for the gRNA-binding region of the ACE reporter recovered by high-fidelity PCR of mCherry-positive 293T. Mutated nucleotides are depicted in red and deleted nucleotides by hyphens. The number of times each sequence was recovered is indicated to the right.

Techniques Used: Western Blot, Expressing, Sequencing, Binding Assay, Polymerase Chain Reaction

ACE reporter activation through Cas9 nucleolytic cleavage. ( A ) Quantification of ACE reporter activation in 293T cells 72 h after co-transfection of ACE reporter, mCherry codon 59 targeting gRNA or NS-gRNA, and A3A-Cas9n-UGI, A3Bctd-Cas9n-UGI, rat APOBEC1-Cas9n-UGI/BE3, Cas9, or Cas9n expression constructs ( n = 3; mean ± SD). ( B ) Anti-Cas9 and anti-HSP90 immunoblots from a representative experiment reported in panel A.
Figure Legend Snippet: ACE reporter activation through Cas9 nucleolytic cleavage. ( A ) Quantification of ACE reporter activation in 293T cells 72 h after co-transfection of ACE reporter, mCherry codon 59 targeting gRNA or NS-gRNA, and A3A-Cas9n-UGI, A3Bctd-Cas9n-UGI, rat APOBEC1-Cas9n-UGI/BE3, Cas9, or Cas9n expression constructs ( n = 3; mean ± SD). ( B ) Anti-Cas9 and anti-HSP90 immunoblots from a representative experiment reported in panel A.

Techniques Used: Activation Assay, Cotransfection, Expressing, Construct, Western Blot

21) Product Images from "Defining a Clade by Morphological, Molecular and Toxinological Criteria: Distinctive Forms related to Conus praecellens A. Adams, 1854 †"

Article Title: Defining a Clade by Morphological, Molecular and Toxinological Criteria: Distinctive Forms related to Conus praecellens A. Adams, 1854 †

Journal: The Nautilus

doi:

Toxinological analysis. Predicted mature toxin sequences from two distinct branches of the O-superfamily of conopeptides for four members of Turriconus: Conus acutangulus, Conus mitratus, Conus praecellensand Conus stupa. An independent comparison of the toxin sequences from the two branches, the hydrophilic ω and the hydrophobic δ, each demonstrate the close relationship between the different species of the Turriconus clade analyzed.
Figure Legend Snippet: Toxinological analysis. Predicted mature toxin sequences from two distinct branches of the O-superfamily of conopeptides for four members of Turriconus: Conus acutangulus, Conus mitratus, Conus praecellensand Conus stupa. An independent comparison of the toxin sequences from the two branches, the hydrophilic ω and the hydrophobic δ, each demonstrate the close relationship between the different species of the Turriconus clade analyzed.

Techniques Used:

An illustration of the “miniexcelsus -like complex”. Shown are five specimens (left five) and close ups of their respective protoconchs (right five). Left, top and bottom: Conus miniexcelsus , middle: Conus excelsus , and right top and bottom: Conus acutangulus. Although the shells are different in pattern and size at maturity, note the similar purplish brown translucent protoconchs, followed by the ivory white early teleoconch whorls before the regular shell pattern is initiated. Typical Conus acutangulus(top right) and the “deep-water form” (bottom right) are both distinctly more nodulose while there is a striking similarity between Conus excelsusand Conus miniexcelsusin their protoconchs and early teleoconch whorls.
Figure Legend Snippet: An illustration of the “miniexcelsus -like complex”. Shown are five specimens (left five) and close ups of their respective protoconchs (right five). Left, top and bottom: Conus miniexcelsus , middle: Conus excelsus , and right top and bottom: Conus acutangulus. Although the shells are different in pattern and size at maturity, note the similar purplish brown translucent protoconchs, followed by the ivory white early teleoconch whorls before the regular shell pattern is initiated. Typical Conus acutangulus(top right) and the “deep-water form” (bottom right) are both distinctly more nodulose while there is a striking similarity between Conus excelsusand Conus miniexcelsusin their protoconchs and early teleoconch whorls.

Techniques Used:

Distinctive forms proposed to belong to the Turriconusclade. Top row (showing the shell and in the inset, a close-up of the corresponding protoconch), from left to right: Conus excelsus; miniexcelsus; acutangulus , “typical form”; acutangulus , “deep-water form”; andremenezi; praecellens , “sowerbii form”; rizali; praecellens , “Aliguay form”. Lower row, from left to right: Conus stupa; stupella; mitratus; cylindraceus. The protoconch of Conus stupais not shown; it is extremely eroded in the figured specimen.
Figure Legend Snippet: Distinctive forms proposed to belong to the Turriconusclade. Top row (showing the shell and in the inset, a close-up of the corresponding protoconch), from left to right: Conus excelsus; miniexcelsus; acutangulus , “typical form”; acutangulus , “deep-water form”; andremenezi; praecellens , “sowerbii form”; rizali; praecellens , “Aliguay form”. Lower row, from left to right: Conus stupa; stupella; mitratus; cylindraceus. The protoconch of Conus stupais not shown; it is extremely eroded in the figured specimen.

Techniques Used:

22) Product Images from "Integration of HPV6 and Downregulation of AKR1C3 Expression Mark Malignant Transformation in a Patient with Juvenile-Onset Laryngeal Papillomatosis"

Article Title: Integration of HPV6 and Downregulation of AKR1C3 Expression Mark Malignant Transformation in a Patient with Juvenile-Onset Laryngeal Papillomatosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0057207

Genetic analysis of the tumor. ( A ) Sequence analysis of APOT PCR product. Analysis of the chimeric HPV/AKR1C3 mRNA showed that it is spliced from the HPV6 splice donor site at nucleotide 745 to intron one of the AKR1C3 gene. ( B, C ) ArrayCGH analysis. ( B ) Large scale ideogram of chromosome 10. AKR1C3 is marked by an arrow. ( C ) Overwiev of all chromosomes. Arrows indicate 3q loss and AKR1C3 integration site on chromosome 10 and a large deletion on chromosome 3. Green colored regions indicate DNA loss. Algorism z-score, Threshold 2.5.
Figure Legend Snippet: Genetic analysis of the tumor. ( A ) Sequence analysis of APOT PCR product. Analysis of the chimeric HPV/AKR1C3 mRNA showed that it is spliced from the HPV6 splice donor site at nucleotide 745 to intron one of the AKR1C3 gene. ( B, C ) ArrayCGH analysis. ( B ) Large scale ideogram of chromosome 10. AKR1C3 is marked by an arrow. ( C ) Overwiev of all chromosomes. Arrows indicate 3q loss and AKR1C3 integration site on chromosome 10 and a large deletion on chromosome 3. Green colored regions indicate DNA loss. Algorism z-score, Threshold 2.5.

Techniques Used: Sequencing, Polymerase Chain Reaction

23) Product Images from "Two BMP responsive elements, STAT, and bZIP/HNF4/COUP motifs of the hepcidin promoter are critical for BMP, SMAD1, and HJV responsiveness"

Article Title: Two BMP responsive elements, STAT, and bZIP/HNF4/COUP motifs of the hepcidin promoter are critical for BMP, SMAD1, and HJV responsiveness

Journal:

doi: 10.1182/blood-2008-05-160184

Finer mapping of the SMAD1, hemojuvelin, BMP2, and BMP9 response in the critical BMP-RE2/bZIP/HNF-4/COUP region . Constructs containing mutations in critical distal BMP-RE2/bZIP/COUP/HNF4 region and also the double mutant BMP-RE1/2 were introduced into
Figure Legend Snippet: Finer mapping of the SMAD1, hemojuvelin, BMP2, and BMP9 response in the critical BMP-RE2/bZIP/HNF-4/COUP region . Constructs containing mutations in critical distal BMP-RE2/bZIP/COUP/HNF4 region and also the double mutant BMP-RE1/2 were introduced into

Techniques Used: Construct, Mutagenesis

24) Product Images from "Refined human artificial chromosome vectors for gene therapy and animal transgenesis"

Article Title: Refined human artificial chromosome vectors for gene therapy and animal transgenesis

Journal: Gene Therapy

doi: 10.1038/gt.2010.147

Analysis of a HAC containing desired genes. ( a – f ) FISH analyses for DT40 (21HAC2) ( a ), DT40 (21HAC3) ( b ), CHO (21HAC1) ( c ), CHO (GFP-21HAC1) ( d ), CHO (21HAC4) ( e ) and CHO (HPRT-21HAC4) ( f ). Digoxigenin-labeled human COT-1 DNA (red) was used to detect the HAC. Biotin-labeled CAG–EGFP, CMV-DsRed and RP6-127C8 (green) were used to detect the marker gene on the HAC in ( a and d ), ( b ) and ( f ), respectively. Chromosomal DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI). The inset shows an enlarged image of the HAC (arrow). ( g ) Expression of the fluorescent gene on the HAC in DT40 cells. Phase-contrast (top panel), GFP-fluorescence (middle panel) and DsRed-fluorescence (bottom panel) micrographs are shown. ( h ) Expression of the fluorescent gene on the 21HAC1 in CHO cells. ( i ) Representative genomic PCR and reverse transcriptase (RT)-PCR data for detecting HPRT-21HAC4 in CHO cells. HT1080 and CHO (21HAC4) cells were used as positive and negative controls, respectively. β-actin was used as an internal control.
Figure Legend Snippet: Analysis of a HAC containing desired genes. ( a – f ) FISH analyses for DT40 (21HAC2) ( a ), DT40 (21HAC3) ( b ), CHO (21HAC1) ( c ), CHO (GFP-21HAC1) ( d ), CHO (21HAC4) ( e ) and CHO (HPRT-21HAC4) ( f ). Digoxigenin-labeled human COT-1 DNA (red) was used to detect the HAC. Biotin-labeled CAG–EGFP, CMV-DsRed and RP6-127C8 (green) were used to detect the marker gene on the HAC in ( a and d ), ( b ) and ( f ), respectively. Chromosomal DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI). The inset shows an enlarged image of the HAC (arrow). ( g ) Expression of the fluorescent gene on the HAC in DT40 cells. Phase-contrast (top panel), GFP-fluorescence (middle panel) and DsRed-fluorescence (bottom panel) micrographs are shown. ( h ) Expression of the fluorescent gene on the 21HAC1 in CHO cells. ( i ) Representative genomic PCR and reverse transcriptase (RT)-PCR data for detecting HPRT-21HAC4 in CHO cells. HT1080 and CHO (21HAC4) cells were used as positive and negative controls, respectively. β-actin was used as an internal control.

Techniques Used: HAC Assay, Fluorescence In Situ Hybridization, Labeling, Marker, Expressing, Fluorescence, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

25) Product Images from "Transcriptional Regulation of N-Acetylglutamate Synthase"

Article Title: Transcriptional Regulation of N-Acetylglutamate Synthase

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029527

DNA-protein avidin-agarose pull-down assay results confirm transcription factor binding. Two probes for the promoter (A) and one probe for the enhancer (B) encompass the highly conserved transcription factor binding motifs of NAGS . The motif colors reflect the colors used in figures 4A and B . Assays followed by immunoblot confirmed binding of Sp1 and CREB, but not C/EBP within the promoter (C) and HNF-1 and NF-Y, but not SMAD3 or AP-2 within the enhancer regions (D). Lanes 1–4 represent precipitated proteins from mouse liver nuclear extract bound to biotinylated probes of the regions of interest (Lane 1), non-biotinylated probes of the regions of interest (Lane 2), biotinylated probes of non-specific regions (Lane 3), and no probe (Lane 4). Lanes 5–8 represent supernatant fluid from overnight incubation of biotinylated probes of the region of interest (Lane 5), non-biotinylated probes of the region of interest (Lane 6), biotinylated probes of the non-specific regions (Lane 7), or no probe (Lane 8). Immunoblots are representative of at least three replicate experiments.
Figure Legend Snippet: DNA-protein avidin-agarose pull-down assay results confirm transcription factor binding. Two probes for the promoter (A) and one probe for the enhancer (B) encompass the highly conserved transcription factor binding motifs of NAGS . The motif colors reflect the colors used in figures 4A and B . Assays followed by immunoblot confirmed binding of Sp1 and CREB, but not C/EBP within the promoter (C) and HNF-1 and NF-Y, but not SMAD3 or AP-2 within the enhancer regions (D). Lanes 1–4 represent precipitated proteins from mouse liver nuclear extract bound to biotinylated probes of the regions of interest (Lane 1), non-biotinylated probes of the regions of interest (Lane 2), biotinylated probes of non-specific regions (Lane 3), and no probe (Lane 4). Lanes 5–8 represent supernatant fluid from overnight incubation of biotinylated probes of the region of interest (Lane 5), non-biotinylated probes of the region of interest (Lane 6), biotinylated probes of the non-specific regions (Lane 7), or no probe (Lane 8). Immunoblots are representative of at least three replicate experiments.

Techniques Used: Avidin-Biotin Assay, Pull Down Assay, Binding Assay, Incubation, Western Blot

26) Product Images from "Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: implications for eradication"

Article Title: Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: implications for eradication

Journal: AIDS (London, England)

doi: 10.1097/QAD.0b013e328340a239

Frequencies of HIV proviral DNA (a) and infectious virus (b) in CD4 + T cells of HIV-infected individuals receiving effective ART for prolonged periods of time
Figure Legend Snippet: Frequencies of HIV proviral DNA (a) and infectious virus (b) in CD4 + T cells of HIV-infected individuals receiving effective ART for prolonged periods of time

Techniques Used: Infection

27) Product Images from "Genotyping on a Thermal Gradient DNA Chip"

Article Title: Genotyping on a Thermal Gradient DNA Chip

Journal: Genome Research

doi: 10.1101/gr.790603

Genotype analyses for two SNPs in the factor VII gene by using a thermal gradient chip. Fluorescence units ( y -axis) as a function of chip island temperature ( x -axis) are shown for hybridization of Cy 5-labeled, denatured PCR, and solution-phase targets to chip-bound normal (N-ASO, red) or mutant (M-ASO, green) oligonucleotide probes. Genomic DNA from individuals either heterozygous (N/M, middle panels), homozygous wild type (N/N, left panels), or homozygous mutant (M/M, right panels) encompassing either the G353A (upper three panels) or C-122T (lower three panels) SNPs in the factor VII gene were amplified by PCR. Samples then were heat denatured and annealed to chips containing the five different thermally isolated islands. Chips were washed and scanned as described in Methods. Numbers in brackets refer to the ratio of intensities of annealing of labeled targets to normal (red)/mutant (green) probe spots on the array.
Figure Legend Snippet: Genotype analyses for two SNPs in the factor VII gene by using a thermal gradient chip. Fluorescence units ( y -axis) as a function of chip island temperature ( x -axis) are shown for hybridization of Cy 5-labeled, denatured PCR, and solution-phase targets to chip-bound normal (N-ASO, red) or mutant (M-ASO, green) oligonucleotide probes. Genomic DNA from individuals either heterozygous (N/M, middle panels), homozygous wild type (N/N, left panels), or homozygous mutant (M/M, right panels) encompassing either the G353A (upper three panels) or C-122T (lower three panels) SNPs in the factor VII gene were amplified by PCR. Samples then were heat denatured and annealed to chips containing the five different thermally isolated islands. Chips were washed and scanned as described in Methods. Numbers in brackets refer to the ratio of intensities of annealing of labeled targets to normal (red)/mutant (green) probe spots on the array.

Techniques Used: Chromatin Immunoprecipitation, Fluorescence, Hybridization, Labeling, Polymerase Chain Reaction, Allele-specific Oligonucleotide, Mutagenesis, Amplification, Isolation

28) Product Images from "Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways"

Article Title: Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways

Journal: Journal of Clinical Investigation

doi:

Diagram of the NKX2.5 ). The region enclosed by boxes indicates exon 1 (left) and exon 2 (right); the horizontal line indicates the intron. Shaded boxes indicate the 5′ and 3′ untranslated regions (UTR), respectively. The white box denotes the coding region, and the black boxes indicate the conserved TN domain, homeodomain (HD), and NK domain, respectively, within the coding region. Nonsense mutations are indicated above and missense mutations below the gene diagram.
Figure Legend Snippet: Diagram of the NKX2.5 ). The region enclosed by boxes indicates exon 1 (left) and exon 2 (right); the horizontal line indicates the intron. Shaded boxes indicate the 5′ and 3′ untranslated regions (UTR), respectively. The white box denotes the coding region, and the black boxes indicate the conserved TN domain, homeodomain (HD), and NK domain, respectively, within the coding region. Nonsense mutations are indicated above and missense mutations below the gene diagram.

Techniques Used:

29) Product Images from "A modest but significant effect of CGB5 gene promoter polymorphisms in modulating the risk of recurrent miscarriage"

Article Title: A modest but significant effect of CGB5 gene promoter polymorphisms in modulating the risk of recurrent miscarriage

Journal: Fertility and Sterility

doi: 10.1016/j.fertnstert.2013.02.019

Genomic content of the studied polymorphisms in the CGB5 and CGB8 genes. ( A ) Design of the RFLP experiment for the genotyping of the Danish RM patients and fertile controls. The exons are depicted with gray boxes . A bold arrow shows the direction of gene transcription. The positions of the PCR primers (1F to 4F, 1R to 4R; Supplemental Table 1 ) for the amplification of the CGB5 and CGB8 genic regions are depicted with short arrows . The flanking regions of the genotyped SNPs (c5-155, c5-142, c5+1038, c8+1045) have been zoomed in and aligned between the two duplicate genes. Dots indicate identical nucleotides in the corresponding positions of CGB5 and CGB8 . The SNP code corresponds to the gene name (c5 = CGB5 ) and location relative to mRNA start site. The LD between the four polymorphisms in the CGB5 promoter region is expressed using the r 2 -statistic. ( B ) The SNPs identified in Danes within the resequenced region of CGB8 spanning the upstream region (−350 bp from mRNA start site) and the first exon ( gray box ; up to +400 bp). The proximal promoter of the hCGbeta coding CGB genes necessary for full basal expression has been demonstrated to be located between nucleotide positions −362 and +104 relative to mRNA start site (31) . The direction of gene transcription is shown with a bold arrow . Singleton SNPs are marked with “S,” rare SNPs (MAF,
Figure Legend Snippet: Genomic content of the studied polymorphisms in the CGB5 and CGB8 genes. ( A ) Design of the RFLP experiment for the genotyping of the Danish RM patients and fertile controls. The exons are depicted with gray boxes . A bold arrow shows the direction of gene transcription. The positions of the PCR primers (1F to 4F, 1R to 4R; Supplemental Table 1 ) for the amplification of the CGB5 and CGB8 genic regions are depicted with short arrows . The flanking regions of the genotyped SNPs (c5-155, c5-142, c5+1038, c8+1045) have been zoomed in and aligned between the two duplicate genes. Dots indicate identical nucleotides in the corresponding positions of CGB5 and CGB8 . The SNP code corresponds to the gene name (c5 = CGB5 ) and location relative to mRNA start site. The LD between the four polymorphisms in the CGB5 promoter region is expressed using the r 2 -statistic. ( B ) The SNPs identified in Danes within the resequenced region of CGB8 spanning the upstream region (−350 bp from mRNA start site) and the first exon ( gray box ; up to +400 bp). The proximal promoter of the hCGbeta coding CGB genes necessary for full basal expression has been demonstrated to be located between nucleotide positions −362 and +104 relative to mRNA start site (31) . The direction of gene transcription is shown with a bold arrow . Singleton SNPs are marked with “S,” rare SNPs (MAF,

Techniques Used: Polymerase Chain Reaction, Amplification, Expressing

30) Product Images from "Two BMP responsive elements, STAT, and bZIP/HNF4/COUP motifs of the hepcidin promoter are critical for BMP, SMAD1, and HJV responsiveness"

Article Title: Two BMP responsive elements, STAT, and bZIP/HNF4/COUP motifs of the hepcidin promoter are critical for BMP, SMAD1, and HJV responsiveness

Journal:

doi: 10.1182/blood-2008-05-160184

Finer mapping of the SMAD1, hemojuvelin, BMP2, and BMP9 response in the critical BMP-RE2/bZIP/HNF-4/COUP region . Constructs containing mutations in critical distal BMP-RE2/bZIP/COUP/HNF4 region and also the double mutant BMP-RE1/2 were introduced into
Figure Legend Snippet: Finer mapping of the SMAD1, hemojuvelin, BMP2, and BMP9 response in the critical BMP-RE2/bZIP/HNF-4/COUP region . Constructs containing mutations in critical distal BMP-RE2/bZIP/COUP/HNF4 region and also the double mutant BMP-RE1/2 were introduced into

Techniques Used: Construct, Mutagenesis

A hypothetical scheme of activation of the murine Hamp1 promoter . Fully active promoter complex requires the presence of both the SMAD1/4 complexes as well as STAT3. Elements depicted as critical reduce/impair the ability of the promoter to respond to
Figure Legend Snippet: A hypothetical scheme of activation of the murine Hamp1 promoter . Fully active promoter complex requires the presence of both the SMAD1/4 complexes as well as STAT3. Elements depicted as critical reduce/impair the ability of the promoter to respond to

Techniques Used: Activation Assay

Mapping of SMAD1, HJV, BMP4, and BMP9 responsive region of Hamp1 . Constructs containing various fragments of the murine Hamp1 promoter linked to a firefly reporter were transfected into HepG2 cells and either cotransfected with SMAD1 (A) or hemojuvelin
Figure Legend Snippet: Mapping of SMAD1, HJV, BMP4, and BMP9 responsive region of Hamp1 . Constructs containing various fragments of the murine Hamp1 promoter linked to a firefly reporter were transfected into HepG2 cells and either cotransfected with SMAD1 (A) or hemojuvelin

Techniques Used: Construct, Transfection

The effect of various Hamp1 promoter deletions on the SMAD1, HJV, BMP2, and BMP9 induced response . Firefly reporter constructs driven by the murine Hamp1 promoter were used, 2.5 kb Hamp1 WT was the positive full-length promoter control, 260 bp Hamp1 represented
Figure Legend Snippet: The effect of various Hamp1 promoter deletions on the SMAD1, HJV, BMP2, and BMP9 induced response . Firefly reporter constructs driven by the murine Hamp1 promoter were used, 2.5 kb Hamp1 WT was the positive full-length promoter control, 260 bp Hamp1 represented

Techniques Used: Construct

The effect of SMAD1, SMAD4, and HJV on endogenous HAMP (A) or Hamp1 promoter-driven reporter expression (B) in HepG2 cells . Overexpression of SMAD1 andHJV induced expression of endogenous HAMP mRNA as assessed by Q-RT-PCR (A) as well as expression of
Figure Legend Snippet: The effect of SMAD1, SMAD4, and HJV on endogenous HAMP (A) or Hamp1 promoter-driven reporter expression (B) in HepG2 cells . Overexpression of SMAD1 andHJV induced expression of endogenous HAMP mRNA as assessed by Q-RT-PCR (A) as well as expression of

Techniques Used: Expressing, Over Expression, Reverse Transcription Polymerase Chain Reaction

31) Product Images from "Recurrent Tandem Gene Duplication Gave Rise to Functionally Divergent Genes in Drosophila"

Article Title: Recurrent Tandem Gene Duplication Gave Rise to Functionally Divergent Genes in Drosophila

Journal:

doi: 10.1093/molbev/msn089

Positive Selection of CG32706 Orthologs in D. simulans , D. sechellia , and D. mauritiana
Figure Legend Snippet: Positive Selection of CG32706 Orthologs in D. simulans , D. sechellia , and D. mauritiana

Techniques Used: Selection

Positive Selection of CG32706 Orthologs in D. simulans , D. sechellia , and D. mauritiana
Figure Legend Snippet: Positive Selection of CG32706 Orthologs in D. simulans , D. sechellia , and D. mauritiana

Techniques Used: Selection

Positive Selection of CG32706 Orthologs in D. simulans , D. sechellia , and D. mauritiana
Figure Legend Snippet: Positive Selection of CG32706 Orthologs in D. simulans , D. sechellia , and D. mauritiana

Techniques Used: Selection

32) Product Images from "Identification and Comparison of Eleven Rhesus Macaque Chemokine Receptors"

Article Title: Identification and Comparison of Eleven Rhesus Macaque Chemokine Receptors

Journal: AIDS research and human retroviruses

doi: 10.1089/088922201750290104

Alignment of extracellular amino-terminal domains of ( A ) CCR3, ( B ) CCR5, and ( C ) STRL33. The rhesus macaque ( M. mul. ) counterpart is aligned above the human ( H. sap. ) protein in each alignment. Insertions are represented by spaces; invariant residues between each pair are drawn as hyphens; all other amino acid differences are shown in lower case. Sites of potential tyrosine sulfation are indicated by asterisks. Amino acids are numbered from 1, the amino-terminal methionine, to the last residue at the predicted carboxyl end of the amino-terminal extracellular domain (Pro-39 in CCR3; Pro-35 in CCR5; Lys-32 in STRL33).
Figure Legend Snippet: Alignment of extracellular amino-terminal domains of ( A ) CCR3, ( B ) CCR5, and ( C ) STRL33. The rhesus macaque ( M. mul. ) counterpart is aligned above the human ( H. sap. ) protein in each alignment. Insertions are represented by spaces; invariant residues between each pair are drawn as hyphens; all other amino acid differences are shown in lower case. Sites of potential tyrosine sulfation are indicated by asterisks. Amino acids are numbered from 1, the amino-terminal methionine, to the last residue at the predicted carboxyl end of the amino-terminal extracellular domain (Pro-39 in CCR3; Pro-35 in CCR5; Lys-32 in STRL33).

Techniques Used:

33) Product Images from "Gene expression and viral prodution in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals"

Article Title: Gene expression and viral prodution in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0437640100

Quantitation of cell-free HIV-1 virions released by latently infected, resting CD4 +  T cells from representative viremic ( A  and  B ) and aviremic ( C  and  D ) patients. Cell-free supernatants collected from each culture at days 1 and 3 were subjected to Amplicor HIV-1 test (detection limit: 50 copies of HIV-1 RNA per ml) for quantitation of HIV-1 virions.
Figure Legend Snippet: Quantitation of cell-free HIV-1 virions released by latently infected, resting CD4 + T cells from representative viremic ( A and B ) and aviremic ( C and D ) patients. Cell-free supernatants collected from each culture at days 1 and 3 were subjected to Amplicor HIV-1 test (detection limit: 50 copies of HIV-1 RNA per ml) for quantitation of HIV-1 virions.

Techniques Used: Quantitation Assay, Infection

Frequency of latently infected, resting CD4 +  T cells carrying HIV-1 proviral DNA ( A ) and cell-associated, unspliced HIV-1 RNA ( B ) from viremic and aviremic patients. Freshly isolated, resting CD4 +  T cells were subjected to real-time PCR for detection of HIV-1 proviral DNA and cell-associated HIV-1 RNA. The geometric mean values are shown as black bars.
Figure Legend Snippet: Frequency of latently infected, resting CD4 + T cells carrying HIV-1 proviral DNA ( A ) and cell-associated, unspliced HIV-1 RNA ( B ) from viremic and aviremic patients. Freshly isolated, resting CD4 + T cells were subjected to real-time PCR for detection of HIV-1 proviral DNA and cell-associated HIV-1 RNA. The geometric mean values are shown as black bars.

Techniques Used: Infection, Isolation, Real-time Polymerase Chain Reaction

Clustering of differentially expressed genes in resting CD4 +  T cells of viremic patients, aviremic patients, and healthy HIV-negative donors. Transcription profiles of resting cells from five viremic patients, five aviremic patients, and four HIV-negative donors were examined by using high-density microarrays. Levels of gene expression were assayed on Affymetrix U95A chips, and 535 differentially expressed genes were identified. Genes were grouped by using K-means clustering, and samples were grouped by hierarchical clustering. Clusters 4 and 5 indicate a set of 370 genes that were up-regulated in viremic patients relative to both aviremic and healthy individuals. Statistical analysis (Fisher exact test) of the genes in clusters 4 and 5 determined that the top three functional categories, transcription regulators, RNA processing/modification, and protein trafficking/vesicle, were overrepresented in this list of genes relative to all annotated genes on the chip. Differences in relative levels of gene expression ( Z -score) are indicated in color, where red indicates up-regulation and green indicates down-regulation. Some genes were listed twice because certain probes recognize multiple regions of a single gene. Levels of expression of housekeeping genes are also shown.
Figure Legend Snippet: Clustering of differentially expressed genes in resting CD4 + T cells of viremic patients, aviremic patients, and healthy HIV-negative donors. Transcription profiles of resting cells from five viremic patients, five aviremic patients, and four HIV-negative donors were examined by using high-density microarrays. Levels of gene expression were assayed on Affymetrix U95A chips, and 535 differentially expressed genes were identified. Genes were grouped by using K-means clustering, and samples were grouped by hierarchical clustering. Clusters 4 and 5 indicate a set of 370 genes that were up-regulated in viremic patients relative to both aviremic and healthy individuals. Statistical analysis (Fisher exact test) of the genes in clusters 4 and 5 determined that the top three functional categories, transcription regulators, RNA processing/modification, and protein trafficking/vesicle, were overrepresented in this list of genes relative to all annotated genes on the chip. Differences in relative levels of gene expression ( Z -score) are indicated in color, where red indicates up-regulation and green indicates down-regulation. Some genes were listed twice because certain probes recognize multiple regions of a single gene. Levels of expression of housekeeping genes are also shown.

Techniques Used: Expressing, Functional Assay, Modification, Chromatin Immunoprecipitation

Levels of cell-free HIV-1 virions released by latently infected, resting CD4 +  T cells from viremic and aviremic patients. Cell-free supernatants from each culture harvested at day 1 were subjected to Amplicor HIV-1 test (detection limit: 50 copies of HIV-1 RNA per ml). The geometric mean values are shown as black bars.
Figure Legend Snippet: Levels of cell-free HIV-1 virions released by latently infected, resting CD4 + T cells from viremic and aviremic patients. Cell-free supernatants from each culture harvested at day 1 were subjected to Amplicor HIV-1 test (detection limit: 50 copies of HIV-1 RNA per ml). The geometric mean values are shown as black bars.

Techniques Used: Infection

34) Product Images from "A porcine model of neurofibromatosis type 1 that mimics the human disease"

Article Title: A porcine model of neurofibromatosis type 1 that mimics the human disease

Journal: JCI Insight

doi: 10.1172/jci.insight.120402

Generation of NF1 +/ex42del -targeted miniswine. ( A ) Gene targeting scheme depicting the deletion of exon 42. Exons 41–43 are shown as gray boxes, and the Blast R cassette is shown in red. Dotted vertical lines indicate the gene-targeting construct boundaries. Arrows indicate PCR primers. NF1 Southern blot probe is indicated by a black line and labeled “probe.” Red arrowhead represents remaining loxP site. Figure is not to scale. ( B ) Representative PCR genotyping. Lanes 1–7 represent individual miniswine, lane 8 is a no-template control, lane 9 is an NF1 +/ex42del with Blast R control, and lane 10 is an NF1 +/ex42del Blast R -excised control. PCR primer locations are indicated in A . ( C ) Representative genomic Southern blot with NF1 (upper panel) and Blast R (lower panel) probes. NcoI/EcoNi-digested DNA from WT NF1 miniswine yields a 3.0 kb product, whereas the same digest of NF1 +/ex42del Blast R –excised yields a product of 3.7 kb. NF1 +/ex42del with Blast R yields a product of 4.9 kb. Lanes 1–13 represent 13 individual piglets. Lane 14 is an NF1 +/ex42del with Blast R control. ( D ) Control litter presents with no pigmentation. Characteristic café au lait macules ( E and F , back rump and head regions), auxiliary/inguinal freckling ( G , flank), and cutaneous tumors ( H , neck with cross-sectional insert) are evident on NF1 +/ex42del miniswine.
Figure Legend Snippet: Generation of NF1 +/ex42del -targeted miniswine. ( A ) Gene targeting scheme depicting the deletion of exon 42. Exons 41–43 are shown as gray boxes, and the Blast R cassette is shown in red. Dotted vertical lines indicate the gene-targeting construct boundaries. Arrows indicate PCR primers. NF1 Southern blot probe is indicated by a black line and labeled “probe.” Red arrowhead represents remaining loxP site. Figure is not to scale. ( B ) Representative PCR genotyping. Lanes 1–7 represent individual miniswine, lane 8 is a no-template control, lane 9 is an NF1 +/ex42del with Blast R control, and lane 10 is an NF1 +/ex42del Blast R -excised control. PCR primer locations are indicated in A . ( C ) Representative genomic Southern blot with NF1 (upper panel) and Blast R (lower panel) probes. NcoI/EcoNi-digested DNA from WT NF1 miniswine yields a 3.0 kb product, whereas the same digest of NF1 +/ex42del Blast R –excised yields a product of 3.7 kb. NF1 +/ex42del with Blast R yields a product of 4.9 kb. Lanes 1–13 represent 13 individual piglets. Lane 14 is an NF1 +/ex42del with Blast R control. ( D ) Control litter presents with no pigmentation. Characteristic café au lait macules ( E and F , back rump and head regions), auxiliary/inguinal freckling ( G , flank), and cutaneous tumors ( H , neck with cross-sectional insert) are evident on NF1 +/ex42del miniswine.

Techniques Used: Construct, Polymerase Chain Reaction, Southern Blot, Labeling

35) Product Images from "Functional roles of 3?-terminal structures of template RNA during in vivo retrotransposition of non-LTR retrotransposon, R1Bm"

Article Title: Functional roles of 3?-terminal structures of template RNA during in vivo retrotransposition of non-LTR retrotransposon, R1Bm

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki347

In vivo retrotransposition assay for R1 elements using recombinant baculoviruses. ( A ) Diagram of the PCR assay for retrotransposition. Shown at the top of the figure is a diagram of the R1 element expressed from AcNPV. The 5′UTR sequence derived from the polyhedrin promoter and ORF1/ORF2 of R1 are shaded in black and gray, respectively. Nucleotide position is numbered with the transcription initiation site (A of T A AG) defined as +1. EN and RT denote the endonuclease and reverse transcriptase domains, respectively. The amino acid position of each mis-sense mutant is also shown; the 2H209A mutant represents the substitution of the 209 th histidine ( H ) in the ORF 2 for alanine ( A ), and the 2D680A mutant represents the substitution of the 680 th aspartic acid ( D ) in the ORF 2 for alanine ( A ). Shown at the bottom is a diagram of an rDNA unit showing the location of R1 insertion; 14 bp of the target site duplication (TSD) created upon R1 insertion is boxed. The putative cleavage sites by R1-EN are indicated by black arrowheads. Arrows represent the primer set to amplify the 5′ and 3′ junctions between the R1 sequence and the 28S sequence. The thick bar above the GST region which is fused to the R1 ORF1 indicates the probe used for Southern hybridization in Figure 4 . ( B ) Expression of R1 proteins with a baculovirus-based expression system. The total proteins were extracted from Sf9 cells infected with recombinant viruses shown above and run on SDS–PAGE; the predicted molecular weight for R1 GST/(His) 6 /ORF1 was 78 170. Lane M; size markers.
Figure Legend Snippet: In vivo retrotransposition assay for R1 elements using recombinant baculoviruses. ( A ) Diagram of the PCR assay for retrotransposition. Shown at the top of the figure is a diagram of the R1 element expressed from AcNPV. The 5′UTR sequence derived from the polyhedrin promoter and ORF1/ORF2 of R1 are shaded in black and gray, respectively. Nucleotide position is numbered with the transcription initiation site (A of T A AG) defined as +1. EN and RT denote the endonuclease and reverse transcriptase domains, respectively. The amino acid position of each mis-sense mutant is also shown; the 2H209A mutant represents the substitution of the 209 th histidine ( H ) in the ORF 2 for alanine ( A ), and the 2D680A mutant represents the substitution of the 680 th aspartic acid ( D ) in the ORF 2 for alanine ( A ). Shown at the bottom is a diagram of an rDNA unit showing the location of R1 insertion; 14 bp of the target site duplication (TSD) created upon R1 insertion is boxed. The putative cleavage sites by R1-EN are indicated by black arrowheads. Arrows represent the primer set to amplify the 5′ and 3′ junctions between the R1 sequence and the 28S sequence. The thick bar above the GST region which is fused to the R1 ORF1 indicates the probe used for Southern hybridization in Figure 4 . ( B ) Expression of R1 proteins with a baculovirus-based expression system. The total proteins were extracted from Sf9 cells infected with recombinant viruses shown above and run on SDS–PAGE; the predicted molecular weight for R1 GST/(His) 6 /ORF1 was 78 170. Lane M; size markers.

Techniques Used: In Vivo, Recombinant, Polymerase Chain Reaction, Sequencing, Derivative Assay, Mutagenesis, Hybridization, Expressing, Infection, SDS Page, Molecular Weight

3′-junction analysis for retrotransposed R1 elements. ( A ) PCR amplification of the 3′ boundaries between the transposed R1 and the 28S rDNA gene. Sf9 genomic DNAs were extracted 12, 24, 48, and 72 h post-infection (h.p.i.) with AcNPV expressing wild-type R1, 2H209A (EN-deficient mutant) and 2D680A (RT-deficient mutant). The purified DNA was used as template for PCR amplification with a pair of primers, +4941 and 28S(+109) ( Figure 2A ). The PCR products were subjected to 2.5% agarose electrophoresis and stained with ethidium bromide. The molecular size marker is loaded alongside and each size in base pair (bp) was shown on the left of the picture. ( B and C ) 72 h.p.i. 3′ junction clones obtained with wild-type R1 (B) and endonuclease-deficient R1 (2H209A) (C). Shown at the top of each figure is a diagram of the 3′ end structure of the construct. Sequences derived from the R1Bm and the pAcGHLTB vectors are indicated by shaded and open boxes, respectively. The initiation sites for reverse transcription (left of the dotted vertical lines) are indicated by nucleotide numbers. The target DNA regions are shown on the right of the dotted vertical lines. Extra nucleotides at the junction that are not derived from either the 28S gene or the R1 construct are given between the two vertical lines (non-templated). Boxes to the right of the vertical lines represent the 14 bp of TSD. The number of clones containing each insertion type is indicated in the right-most column and the most major type is indicated by an asterisk. The TGT or TG sequences on the 3′ end of the R1 template that can base-pair with the target DNA are also indicated ( Figure 6 ). +, insertions into the site 180 bp upstream of TSD observed for wild-type and endonuclease-defi cient R1.
Figure Legend Snippet: 3′-junction analysis for retrotransposed R1 elements. ( A ) PCR amplification of the 3′ boundaries between the transposed R1 and the 28S rDNA gene. Sf9 genomic DNAs were extracted 12, 24, 48, and 72 h post-infection (h.p.i.) with AcNPV expressing wild-type R1, 2H209A (EN-deficient mutant) and 2D680A (RT-deficient mutant). The purified DNA was used as template for PCR amplification with a pair of primers, +4941 and 28S(+109) ( Figure 2A ). The PCR products were subjected to 2.5% agarose electrophoresis and stained with ethidium bromide. The molecular size marker is loaded alongside and each size in base pair (bp) was shown on the left of the picture. ( B and C ) 72 h.p.i. 3′ junction clones obtained with wild-type R1 (B) and endonuclease-deficient R1 (2H209A) (C). Shown at the top of each figure is a diagram of the 3′ end structure of the construct. Sequences derived from the R1Bm and the pAcGHLTB vectors are indicated by shaded and open boxes, respectively. The initiation sites for reverse transcription (left of the dotted vertical lines) are indicated by nucleotide numbers. The target DNA regions are shown on the right of the dotted vertical lines. Extra nucleotides at the junction that are not derived from either the 28S gene or the R1 construct are given between the two vertical lines (non-templated). Boxes to the right of the vertical lines represent the 14 bp of TSD. The number of clones containing each insertion type is indicated in the right-most column and the most major type is indicated by an asterisk. The TGT or TG sequences on the 3′ end of the R1 template that can base-pair with the target DNA are also indicated ( Figure 6 ). +, insertions into the site 180 bp upstream of TSD observed for wild-type and endonuclease-defi cient R1.

Techniques Used: Polymerase Chain Reaction, Amplification, Infection, Expressing, Mutagenesis, Purification, Electrophoresis, Staining, Marker, Clone Assay, Construct, Derivative Assay

36) Product Images from "Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: implications for eradication"

Article Title: Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: implications for eradication

Journal: AIDS (London, England)

doi: 10.1097/QAD.0b013e328340a239

Frequencies of HIV proviral DNA (a) and infectious virus (b) in CD4 +  T cells of HIV-infected individuals receiving effective ART for prolonged periods of time
Figure Legend Snippet: Frequencies of HIV proviral DNA (a) and infectious virus (b) in CD4 + T cells of HIV-infected individuals receiving effective ART for prolonged periods of time

Techniques Used: Infection

37) Product Images from "Two BMP responsive elements, STAT, and bZIP/HNF4/COUP motifs of the hepcidin promoter are critical for BMP, SMAD1, and HJV responsiveness"

Article Title: Two BMP responsive elements, STAT, and bZIP/HNF4/COUP motifs of the hepcidin promoter are critical for BMP, SMAD1, and HJV responsiveness

Journal:

doi: 10.1182/blood-2008-05-160184

Mapping of SMAD1, HJV, BMP4, and BMP9 responsive region of Hamp1 . Constructs containing various fragments of the murine Hamp1 promoter linked to a firefly reporter were transfected into HepG2 cells and either cotransfected with SMAD1 (A) or hemojuvelin
Figure Legend Snippet: Mapping of SMAD1, HJV, BMP4, and BMP9 responsive region of Hamp1 . Constructs containing various fragments of the murine Hamp1 promoter linked to a firefly reporter were transfected into HepG2 cells and either cotransfected with SMAD1 (A) or hemojuvelin

Techniques Used: Construct, Transfection

38) Product Images from "Conantokin-P, an Unusual Conantokin with a Long Disulfide Loop"

Article Title: Conantokin-P, an Unusual Conantokin with a Long Disulfide Loop

Journal:

doi: 10.1016/j.toxicon.2008.04.178

3.1. Analysis of clones encoding putative conantokins from Conus purpurascens and Conus ermineus
Figure Legend Snippet: 3.1. Analysis of clones encoding putative conantokins from Conus purpurascens and Conus ermineus

Techniques Used: Clone Assay

39) Product Images from "Endothelial progenitor cells display clonal restriction in multiple myeloma"

Article Title: Endothelial progenitor cells display clonal restriction in multiple myeloma

Journal: BMC Cancer

doi: 10.1186/1471-2407-6-161

X-chromosome inactivation patterns in EPCs from MM patients . (A) X-chromosome inactivation status in a MM patient (Case 5, Table 1). XCI patterns were determined at least twice for each sample by incorporating α-[ 33 P]-dCTP into the PCR reaction. For each sample, DNA was either undigested (-) or digested (+) with the methylation-sensitive restriction enzyme Hpa II. The PCR products were separated on 8% sequencing gels and subjected to autoradiography. Intensity of the specific bands corresponding to the AR gene demonstrates a skewed pattern of XCI (predominance of a single band) in EPCs, and a random pattern of XCI (presence of two bands of similar intensity) in hair root cells. A clonal population was defined as a cell population with greater than 77% expression of either one of the X-linked alleles. (B) Distribution of XCI patterns in MM patients and healthy controls. XCI patterns were determined in DNA from EPCs in MM patients (filled triangles), and PBMCs in healthy controls (open triangles) (*p = .05). For clarity, the y-axis begins at 40%.
Figure Legend Snippet: X-chromosome inactivation patterns in EPCs from MM patients . (A) X-chromosome inactivation status in a MM patient (Case 5, Table 1). XCI patterns were determined at least twice for each sample by incorporating α-[ 33 P]-dCTP into the PCR reaction. For each sample, DNA was either undigested (-) or digested (+) with the methylation-sensitive restriction enzyme Hpa II. The PCR products were separated on 8% sequencing gels and subjected to autoradiography. Intensity of the specific bands corresponding to the AR gene demonstrates a skewed pattern of XCI (predominance of a single band) in EPCs, and a random pattern of XCI (presence of two bands of similar intensity) in hair root cells. A clonal population was defined as a cell population with greater than 77% expression of either one of the X-linked alleles. (B) Distribution of XCI patterns in MM patients and healthy controls. XCI patterns were determined in DNA from EPCs in MM patients (filled triangles), and PBMCs in healthy controls (open triangles) (*p = .05). For clarity, the y-axis begins at 40%.

Techniques Used: Polymerase Chain Reaction, Methylation, Sequencing, Autoradiography, Expressing

PCR analysis and sequencing of immunoglobulin V H gene rearrangement in EPCs in MM patients . A series of 7 V H family-specific primers (V H 1–6) common to the most described members of the corresponding V H family, including two separate primers specific for the V H 4 family (V H 4A and -4B), were used as forward primers. A consensus J H primer was used as reverse primer to amplify rearranged DNA, producing an expected 350-bp PCR product as described by Deane and Norton [22]. (A) Agarose gel analysis of V H 4A family-specific IGH gene rearrangement detected in EPCs and BM cells from a MM patient. Briefly, 1 μg of genomic DNA was amplified with each of the V H family primers and the constant J H primer, which were added at a concentration of 1 μM each. Products were run on 1% agarose gels and visualized with ethidium bromide staining. DNA from MM cell line U266, which contains a V H 1-J H rearrangement, was amplified simultaneously as a positive control for rearrangement, shown as a 350-bp PCR product. Primers specific for apolipoprotein B served as a control to monitor the efficacy of PCR. M indicates marker (1 Kb Plus DNA Ladder [Invitrogen, Carlsbad, CA]). No products were detected from this patient's DNA with the V H 5 or V H 6 primers (not shown). (B) Sequence analysis of IGH rearrangement found in EPCs and BM cells from a MM patient. Sequences obtained from PCR amplification of IGH DNA in EPCs and BM cells shown from Case 14 were compared using FASTA. The 5' V H 4A and J H regions identified by BLAST are shown above the sequences along with numbers indicating the length of the sequences analyzed. The V H 4A-J H rearrangement in EPCs and BM cells from this patient has a 100% sequence overlap.
Figure Legend Snippet: PCR analysis and sequencing of immunoglobulin V H gene rearrangement in EPCs in MM patients . A series of 7 V H family-specific primers (V H 1–6) common to the most described members of the corresponding V H family, including two separate primers specific for the V H 4 family (V H 4A and -4B), were used as forward primers. A consensus J H primer was used as reverse primer to amplify rearranged DNA, producing an expected 350-bp PCR product as described by Deane and Norton [22]. (A) Agarose gel analysis of V H 4A family-specific IGH gene rearrangement detected in EPCs and BM cells from a MM patient. Briefly, 1 μg of genomic DNA was amplified with each of the V H family primers and the constant J H primer, which were added at a concentration of 1 μM each. Products were run on 1% agarose gels and visualized with ethidium bromide staining. DNA from MM cell line U266, which contains a V H 1-J H rearrangement, was amplified simultaneously as a positive control for rearrangement, shown as a 350-bp PCR product. Primers specific for apolipoprotein B served as a control to monitor the efficacy of PCR. M indicates marker (1 Kb Plus DNA Ladder [Invitrogen, Carlsbad, CA]). No products were detected from this patient's DNA with the V H 5 or V H 6 primers (not shown). (B) Sequence analysis of IGH rearrangement found in EPCs and BM cells from a MM patient. Sequences obtained from PCR amplification of IGH DNA in EPCs and BM cells shown from Case 14 were compared using FASTA. The 5' V H 4A and J H regions identified by BLAST are shown above the sequences along with numbers indicating the length of the sequences analyzed. The V H 4A-J H rearrangement in EPCs and BM cells from this patient has a 100% sequence overlap.

Techniques Used: Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Amplification, Concentration Assay, Staining, Positive Control, Marker

40) Product Images from "The CpG Island in the Murine Foxl2 Proximal Promoter Is Differentially Methylated in Primary and Immortalized Cells"

Article Title: The CpG Island in the Murine Foxl2 Proximal Promoter Is Differentially Methylated in Primary and Immortalized Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0076642

The Foxl2 promoter is hypomethylated in homologous cells and tissues. A) Analysis of percent methylation of two regions of the Foxl2 promoter in genomic DNA from LβT2 and NIH3T3 cells as assessed by qAMP (white bars) and pyrosequencing (black bars). Data are from a representative of a 2 replicate experiments, which yielded comparable results. B) Percent methylation of -81/+43 of the Foxl2 promoter in genomic DNA from the indicated murine cell lines. Data are from a single experiment. C) Percent methylation of -81/+43 of the Foxl2 promoter in genomic DNA from the indicated murine tissues Data are from a representative of a 2 replicate experiments, which yielded comparable results.
Figure Legend Snippet: The Foxl2 promoter is hypomethylated in homologous cells and tissues. A) Analysis of percent methylation of two regions of the Foxl2 promoter in genomic DNA from LβT2 and NIH3T3 cells as assessed by qAMP (white bars) and pyrosequencing (black bars). Data are from a representative of a 2 replicate experiments, which yielded comparable results. B) Percent methylation of -81/+43 of the Foxl2 promoter in genomic DNA from the indicated murine cell lines. Data are from a single experiment. C) Percent methylation of -81/+43 of the Foxl2 promoter in genomic DNA from the indicated murine tissues Data are from a representative of a 2 replicate experiments, which yielded comparable results.

Techniques Used: Methylation

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Mass Spectrometry:

Article Title: Mice with Alopecia, Osteoporosis, and Systemic Amyloidosis Due to Mutation in Zdhhc13, a Gene Coding for Palmitoyl Acyltransferase
Article Snippet: .. SNP genotyping using genomic DNA isolated from mouse tails (Puregene DNA purification kit, Gentra Systems, Minneapolis, MN, USA) was performed using high-throughput MALDI-TOF mass spectrometry , . .. Primers and probes flanking the SNPs were designed in multiplex format using SpectroDESIGNER software (Sequenom, San Diego, CA, USA).

Synthesized:

Article Title: Cotransfected human chondrocytes: over-expression ofIGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans
Article Snippet: .. Total RNA extraction and RT-PCR On days 3, 6, and 9 post-transfection (PT), the total RNA was isolated from transfected, CTC and NTC using an RNA cell and tissue purification kit (GENTRA Systems, USA). cDNA was synthesized from each RNA preparation using M-MLV reverse transcriptase (Invitrogen), and the total RNA (500 ng) was treated with 0.5 U of deoxyribonuclease I (DNase I; Invitrogen) to digest genomic DNA. .. The genesSOX9 , IGF-I , and the constitutive gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) were amplified by polymerase chain reaction (PCR) using specific primers ( ).

Isolation:

Article Title: Mice with Alopecia, Osteoporosis, and Systemic Amyloidosis Due to Mutation in Zdhhc13, a Gene Coding for Palmitoyl Acyltransferase
Article Snippet: .. SNP genotyping using genomic DNA isolated from mouse tails (Puregene DNA purification kit, Gentra Systems, Minneapolis, MN, USA) was performed using high-throughput MALDI-TOF mass spectrometry , . .. Primers and probes flanking the SNPs were designed in multiplex format using SpectroDESIGNER software (Sequenom, San Diego, CA, USA).

Article Title: Cotransfected human chondrocytes: over-expression ofIGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans
Article Snippet: .. Total RNA extraction and RT-PCR On days 3, 6, and 9 post-transfection (PT), the total RNA was isolated from transfected, CTC and NTC using an RNA cell and tissue purification kit (GENTRA Systems, USA). cDNA was synthesized from each RNA preparation using M-MLV reverse transcriptase (Invitrogen), and the total RNA (500 ng) was treated with 0.5 U of deoxyribonuclease I (DNase I; Invitrogen) to digest genomic DNA. .. The genesSOX9 , IGF-I , and the constitutive gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) were amplified by polymerase chain reaction (PCR) using specific primers ( ).

Article Title: Human T-Cell Leukemia Virus Type 1 Expressing Nonoverlapping Tax and Rex Genes Replicates and Immortalizes Primary Human T Lymphocytes but Fails To Replicate and Persist In Vivo
Article Snippet: .. Genomic DNA was isolated from permanently transfected cell clones or from immortalized PBMCs using the PUREGENE DNA purification system (Gentra, Minneapolis, MN). ..

Transfection:

Article Title: Cotransfected human chondrocytes: over-expression ofIGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans
Article Snippet: .. Total RNA extraction and RT-PCR On days 3, 6, and 9 post-transfection (PT), the total RNA was isolated from transfected, CTC and NTC using an RNA cell and tissue purification kit (GENTRA Systems, USA). cDNA was synthesized from each RNA preparation using M-MLV reverse transcriptase (Invitrogen), and the total RNA (500 ng) was treated with 0.5 U of deoxyribonuclease I (DNase I; Invitrogen) to digest genomic DNA. .. The genesSOX9 , IGF-I , and the constitutive gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) were amplified by polymerase chain reaction (PCR) using specific primers ( ).

Article Title: Human T-Cell Leukemia Virus Type 1 Expressing Nonoverlapping Tax and Rex Genes Replicates and Immortalizes Primary Human T Lymphocytes but Fails To Replicate and Persist In Vivo
Article Snippet: .. Genomic DNA was isolated from permanently transfected cell clones or from immortalized PBMCs using the PUREGENE DNA purification system (Gentra, Minneapolis, MN). ..

Purification:

Article Title: Cotransfected human chondrocytes: over-expression ofIGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans
Article Snippet: .. Total RNA extraction and RT-PCR On days 3, 6, and 9 post-transfection (PT), the total RNA was isolated from transfected, CTC and NTC using an RNA cell and tissue purification kit (GENTRA Systems, USA). cDNA was synthesized from each RNA preparation using M-MLV reverse transcriptase (Invitrogen), and the total RNA (500 ng) was treated with 0.5 U of deoxyribonuclease I (DNase I; Invitrogen) to digest genomic DNA. .. The genesSOX9 , IGF-I , and the constitutive gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) were amplified by polymerase chain reaction (PCR) using specific primers ( ).

Digital PCR:

Article Title: A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes
Article Snippet: .. Droplet digital PCR Genomic DNA was extracted using the Gentra Puregene kit (Gentra) following the manufacturer’s instructions. .. For each sample, 5 μg DNA was digested with the BsaJI enzyme (NEB) in 1x NEB smartcut buffer.

Polymerase Chain Reaction:

Article Title: A low-cost open-source SNP genotyping platform for association mapping applications
Article Snippet: .. Genomic DNA and PCR amplification Over 2,000 Drosophila melanogaster flies were collected from a single outbred population, and genomic DNA extracted from each using the PureGene cell and tissue DNA isolation kit (Gentra Systems Inc. Minneapolis, MN, USA). ..

Article Title: Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies
Article Snippet: .. Genomic polymerase chain reaction (PCR) Genomic DNA was extracted from cell lines using a genomic DNA extraction kit with DNase-free RNase (Gentra Systems, Minneapolis, USA). ..

High Throughput Screening Assay:

Article Title: Mice with Alopecia, Osteoporosis, and Systemic Amyloidosis Due to Mutation in Zdhhc13, a Gene Coding for Palmitoyl Acyltransferase
Article Snippet: .. SNP genotyping using genomic DNA isolated from mouse tails (Puregene DNA purification kit, Gentra Systems, Minneapolis, MN, USA) was performed using high-throughput MALDI-TOF mass spectrometry , . .. Primers and probes flanking the SNPs were designed in multiplex format using SpectroDESIGNER software (Sequenom, San Diego, CA, USA).

DNA Purification:

Article Title: Mice with Alopecia, Osteoporosis, and Systemic Amyloidosis Due to Mutation in Zdhhc13, a Gene Coding for Palmitoyl Acyltransferase
Article Snippet: .. SNP genotyping using genomic DNA isolated from mouse tails (Puregene DNA purification kit, Gentra Systems, Minneapolis, MN, USA) was performed using high-throughput MALDI-TOF mass spectrometry , . .. Primers and probes flanking the SNPs were designed in multiplex format using SpectroDESIGNER software (Sequenom, San Diego, CA, USA).

Article Title: Human T-Cell Leukemia Virus Type 1 Expressing Nonoverlapping Tax and Rex Genes Replicates and Immortalizes Primary Human T Lymphocytes but Fails To Replicate and Persist In Vivo
Article Snippet: .. Genomic DNA was isolated from permanently transfected cell clones or from immortalized PBMCs using the PUREGENE DNA purification system (Gentra, Minneapolis, MN). ..

Reverse Transcription Polymerase Chain Reaction:

Article Title: Cotransfected human chondrocytes: over-expression ofIGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans
Article Snippet: .. Total RNA extraction and RT-PCR On days 3, 6, and 9 post-transfection (PT), the total RNA was isolated from transfected, CTC and NTC using an RNA cell and tissue purification kit (GENTRA Systems, USA). cDNA was synthesized from each RNA preparation using M-MLV reverse transcriptase (Invitrogen), and the total RNA (500 ng) was treated with 0.5 U of deoxyribonuclease I (DNase I; Invitrogen) to digest genomic DNA. .. The genesSOX9 , IGF-I , and the constitutive gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) were amplified by polymerase chain reaction (PCR) using specific primers ( ).

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    Gentra Systems genomic polymerase chain reaction pcr genomic dna
    Analysis of HepG2 cells containing the 4CYPs-POR MAC. (A) Flowchart of the MAC transfer from donor CHO cells to recipient HepG2 cells via MMCT method, which comprises the following steps: micronucleation of donor cells by colcemid, enucleation by cytochalasin B, and microcell purification and fusion with recipient HepG2 cells. HepG2 hybrids were selected with 400 μg/mL G418 and picked for clonal expansion. (B) G418-resistant clones are screened by genomic <t>PCR</t> to determine the presence of the 4CYPs-POR transgene. (C) Representative metaphase fluorescence in situ hybridization images of TC-HepG2 cells. Digoxigenin-labeled mouse cot-1 <t>DNA</t> (red) was used to detect the MAC. Biotin-labeled pPAC-4CYPs-POR (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrows indicate MAC vectors, and the inset shows an enlarged image of the MAC.
    Genomic Polymerase Chain Reaction Pcr Genomic Dna, supplied by Gentra Systems, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Analysis of HepG2 cells containing the 4CYPs-POR MAC. (A) Flowchart of the MAC transfer from donor CHO cells to recipient HepG2 cells via MMCT method, which comprises the following steps: micronucleation of donor cells by colcemid, enucleation by cytochalasin B, and microcell purification and fusion with recipient HepG2 cells. HepG2 hybrids were selected with 400 μg/mL G418 and picked for clonal expansion. (B) G418-resistant clones are screened by genomic PCR to determine the presence of the 4CYPs-POR transgene. (C) Representative metaphase fluorescence in situ hybridization images of TC-HepG2 cells. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled pPAC-4CYPs-POR (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrows indicate MAC vectors, and the inset shows an enlarged image of the MAC.

    Journal: PLoS ONE

    Article Title: Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies

    doi: 10.1371/journal.pone.0187072

    Figure Lengend Snippet: Analysis of HepG2 cells containing the 4CYPs-POR MAC. (A) Flowchart of the MAC transfer from donor CHO cells to recipient HepG2 cells via MMCT method, which comprises the following steps: micronucleation of donor cells by colcemid, enucleation by cytochalasin B, and microcell purification and fusion with recipient HepG2 cells. HepG2 hybrids were selected with 400 μg/mL G418 and picked for clonal expansion. (B) G418-resistant clones are screened by genomic PCR to determine the presence of the 4CYPs-POR transgene. (C) Representative metaphase fluorescence in situ hybridization images of TC-HepG2 cells. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled pPAC-4CYPs-POR (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrows indicate MAC vectors, and the inset shows an enlarged image of the MAC.

    Article Snippet: Genomic polymerase chain reaction (PCR) Genomic DNA was extracted from cell lines using a genomic DNA extraction kit with DNase-free RNase (Gentra Systems, Minneapolis, USA).

    Techniques: Purification, Clone Assay, Polymerase Chain Reaction, Fluorescence, In Situ Hybridization, Labeling

    Construction and analysis of the 4CYPs-POR MAC vector in CHO cells. (A) Genomic PCR and (B) RT-PCR analysis of CHO cells containing the 4CYPs-POR MAC. N, negative control (parental CHO cells); P, positive control (PAC-4CYPs-POR). (C) FISH analysis of donor CHO cells containing the 4CYPs-POR MAC. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled 4CYPs-POR PAC (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrow indicates MAC vector, and the inset shows enlarged image of the MAC.

    Journal: PLoS ONE

    Article Title: Establishment of a novel hepatocyte model that expresses four cytochrome P450 genes stably via mammalian-derived artificial chromosome for pharmacokinetics and toxicity studies

    doi: 10.1371/journal.pone.0187072

    Figure Lengend Snippet: Construction and analysis of the 4CYPs-POR MAC vector in CHO cells. (A) Genomic PCR and (B) RT-PCR analysis of CHO cells containing the 4CYPs-POR MAC. N, negative control (parental CHO cells); P, positive control (PAC-4CYPs-POR). (C) FISH analysis of donor CHO cells containing the 4CYPs-POR MAC. Digoxigenin-labeled mouse cot-1 DNA (red) was used to detect the MAC. Biotin-labeled 4CYPs-POR PAC (green) was used to detect the 4CYPs-POR cassette in the MAC. Chromosomal DNA was counterstained with DAPI. White arrow indicates MAC vector, and the inset shows enlarged image of the MAC.

    Article Snippet: Genomic polymerase chain reaction (PCR) Genomic DNA was extracted from cell lines using a genomic DNA extraction kit with DNase-free RNase (Gentra Systems, Minneapolis, USA).

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Fluorescence In Situ Hybridization, Labeling