Structured Review

Eppendorf AG cell cytokine mrna qrt pcr
Lack of Trif has no impact on myocardial inflammation after I/R. Mice were subjected to I/R (45 min/4 h or 24 h). A, Myocardial <t>cytokine</t> protein expression. Twenty‐four hours after reperfusion, cytokine proteins were measured using Luminex. B, Myocardial cytokine <t>mRNA.</t> Four hours after reperfusion, IFN mRNAs were assayed using <t>qRT‐PCR.</t> C, Myocardial neutrophil infiltration after I/R. Left, total CD45 + /Gr‐1 + cells in the hearts of WT, Trif −/− , or MyD88 −/− mice that were subjected to sham or I/R. n=3 mice/group. Right, representative examples of FACS plots of myocardial cells from mice subjected to I/R. FSC, forward scatter; SSC, side scatter. * P
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1) Product Images from "Role of Extracellular RNA and TLR3-Trif Signaling in Myocardial Ischemia-Reperfusion Injury"

Article Title: Role of Extracellular RNA and TLR3-Trif Signaling in Myocardial Ischemia-Reperfusion Injury

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

doi: 10.1161/JAHA.113.000683

Lack of Trif has no impact on myocardial inflammation after I/R. Mice were subjected to I/R (45 min/4 h or 24 h). A, Myocardial cytokine protein expression. Twenty‐four hours after reperfusion, cytokine proteins were measured using Luminex. B, Myocardial cytokine mRNA. Four hours after reperfusion, IFN mRNAs were assayed using qRT‐PCR. C, Myocardial neutrophil infiltration after I/R. Left, total CD45 + /Gr‐1 + cells in the hearts of WT, Trif −/− , or MyD88 −/− mice that were subjected to sham or I/R. n=3 mice/group. Right, representative examples of FACS plots of myocardial cells from mice subjected to I/R. FSC, forward scatter; SSC, side scatter. * P
Figure Legend Snippet: Lack of Trif has no impact on myocardial inflammation after I/R. Mice were subjected to I/R (45 min/4 h or 24 h). A, Myocardial cytokine protein expression. Twenty‐four hours after reperfusion, cytokine proteins were measured using Luminex. B, Myocardial cytokine mRNA. Four hours after reperfusion, IFN mRNAs were assayed using qRT‐PCR. C, Myocardial neutrophil infiltration after I/R. Left, total CD45 + /Gr‐1 + cells in the hearts of WT, Trif −/− , or MyD88 −/− mice that were subjected to sham or I/R. n=3 mice/group. Right, representative examples of FACS plots of myocardial cells from mice subjected to I/R. FSC, forward scatter; SSC, side scatter. * P

Techniques Used: Mouse Assay, Expressing, Luminex, Quantitative RT-PCR, FACS

Effect of RNase administration on myocardial inflammation, apoptosis, and infarct sizes after I/R. A, Effect of RNase on myocardial cytokine mRNA. Mice were treated with N.S. or RNase and subjected to sham or I/R. Three hours after reperfusion, myocardial cytokines were analyzed by qRT‐PCR. Sham, n=3; I/R, n=5. B, Effect of RNase treatment on myocardial infiltration of CD45 + 1/Gr‐1 + neutrophil, CD3 + T lymphocyte and F4/80 + macrophage after I/R. A representative FACS plot of neutrophils is shown. C, Effect of RNase on myocardial NF‐κB activity. IR. Sham, n=3; I/R, n=5. D, Effect of RNase on myocardial caspase‐3 activity after 4 hours of reperfusion. Sham, n=3; I/R, n=5. E, Effect of RNase on MI after I/R. n=19/group. F, Representative of TTC staining (left) and fluorescent microsphere distribution (right) of myocardial sections from N.S.‐ or RNase‐treated mice. AAR is indicated by the areas devoid of red fluorescent light and the infarct area shown as white. * P
Figure Legend Snippet: Effect of RNase administration on myocardial inflammation, apoptosis, and infarct sizes after I/R. A, Effect of RNase on myocardial cytokine mRNA. Mice were treated with N.S. or RNase and subjected to sham or I/R. Three hours after reperfusion, myocardial cytokines were analyzed by qRT‐PCR. Sham, n=3; I/R, n=5. B, Effect of RNase treatment on myocardial infiltration of CD45 + 1/Gr‐1 + neutrophil, CD3 + T lymphocyte and F4/80 + macrophage after I/R. A representative FACS plot of neutrophils is shown. C, Effect of RNase on myocardial NF‐κB activity. IR. Sham, n=3; I/R, n=5. D, Effect of RNase on myocardial caspase‐3 activity after 4 hours of reperfusion. Sham, n=3; I/R, n=5. E, Effect of RNase on MI after I/R. n=19/group. F, Representative of TTC staining (left) and fluorescent microsphere distribution (right) of myocardial sections from N.S.‐ or RNase‐treated mice. AAR is indicated by the areas devoid of red fluorescent light and the infarct area shown as white. * P

Techniques Used: Mouse Assay, Quantitative RT-PCR, FACS, Activity Assay, Staining

2) Product Images from "A New Mutation, hap1-2, Reveals a C Terminal Domain Function in AtMago Protein and Its Biological Effects in Male Gametophyte Development in Arabidopsis thaliana"

Article Title: A New Mutation, hap1-2, Reveals a C Terminal Domain Function in AtMago Protein and Its Biological Effects in Male Gametophyte Development in Arabidopsis thaliana

Journal: PLoS ONE

doi: 10.1371/journal.pone.0148200

Both AtMago and AtMagoΔC are detected in P-bodies. Scale bar = 50 μm. A zoom-in view is shown for each image with a scale bar of 10 μm.
Figure Legend Snippet: Both AtMago and AtMagoΔC are detected in P-bodies. Scale bar = 50 μm. A zoom-in view is shown for each image with a scale bar of 10 μm.

Techniques Used:

CTD of AtMago is required for AtMago-AtY14 interaction, but not for AtMago-AteIF4AIII. Western blots showing results from in vitro pull-down assay for AtMagoΔC-AtY14 (A), AtMagoΔC-AteIF4AIII (B), and various AtMago C terminal truncations-AtY14 (C).
Figure Legend Snippet: CTD of AtMago is required for AtMago-AtY14 interaction, but not for AtMago-AteIF4AIII. Western blots showing results from in vitro pull-down assay for AtMagoΔC-AtY14 (A), AtMagoΔC-AteIF4AIII (B), and various AtMago C terminal truncations-AtY14 (C).

Techniques Used: Western Blot, In Vitro, Pull Down Assay

Characterization and complementation of the HAP1/hap1-2 mutant. (A) HAP1 gene structure. Exons were shown as colored boxes; introns were shown as straight lines; UTRs were shown as gray boxes. The T-DNA insertion in hap1-1 and hap1-2 was indicated by red arrow. (B) rtPCR results showed the expression of a full-length transcript ( AtMago ), a truncated transcript ( AtMagoΔC ) and GAPDH in qrt , qrt;HAP1/hap1-2 , qrt;sr45-1 and qrt;upf3-1 mutant plants. Total RNAs were extracted from the inflorescence tissue. The exon composition of the transcript and the position of used primers were shown next to the corresponding rtPCR results. (C) GUS stained pollen grains from qrt , qrt;HAP1/hap1-1 and qrt;HAP1/hap1-2 showing the identity of mutant pollen grains. Scale bar = 20 μm. (D) Seed number per silique in qrt and qrt;HAP1/hap1-2 plants. Scale bar = 1 mm. The quantification was done with sixteen siliques. Student t-test was used for statistical analysis. Error bars represent standard deviations. (E) HAP1 gene complemented hap1-2 mutant phenotype. The mature plants, GUS-stained pollen grains and seed yield per plant were shown in qrt , qrt;HAP1/hap1-2 , HAP1;qrt; hap1-2 . (F) qPCR results showing that the AtMago gene expression level was fully recovered in the transgenic plant. GAPDH was used as control. Three biological replicates were used in the analysis. Error bar showed standard deviation. Statistical significance compared to qrt was measured by student t-test ( p
Figure Legend Snippet: Characterization and complementation of the HAP1/hap1-2 mutant. (A) HAP1 gene structure. Exons were shown as colored boxes; introns were shown as straight lines; UTRs were shown as gray boxes. The T-DNA insertion in hap1-1 and hap1-2 was indicated by red arrow. (B) rtPCR results showed the expression of a full-length transcript ( AtMago ), a truncated transcript ( AtMagoΔC ) and GAPDH in qrt , qrt;HAP1/hap1-2 , qrt;sr45-1 and qrt;upf3-1 mutant plants. Total RNAs were extracted from the inflorescence tissue. The exon composition of the transcript and the position of used primers were shown next to the corresponding rtPCR results. (C) GUS stained pollen grains from qrt , qrt;HAP1/hap1-1 and qrt;HAP1/hap1-2 showing the identity of mutant pollen grains. Scale bar = 20 μm. (D) Seed number per silique in qrt and qrt;HAP1/hap1-2 plants. Scale bar = 1 mm. The quantification was done with sixteen siliques. Student t-test was used for statistical analysis. Error bars represent standard deviations. (E) HAP1 gene complemented hap1-2 mutant phenotype. The mature plants, GUS-stained pollen grains and seed yield per plant were shown in qrt , qrt;HAP1/hap1-2 , HAP1;qrt; hap1-2 . (F) qPCR results showing that the AtMago gene expression level was fully recovered in the transgenic plant. GAPDH was used as control. Three biological replicates were used in the analysis. Error bar showed standard deviation. Statistical significance compared to qrt was measured by student t-test ( p

Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Real-time Polymerase Chain Reaction, Transgenic Assay, Standard Deviation

3) Product Images from "The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis"

Article Title: The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18030576

GSDMB alternative splicing (AS) pattern. ( a ) Schematic representation of the GSDMB gene. Exons are represented by boxes and are approximately drawn to scale; untranslated regions (UTRs) are depicted by smaller boxes; introns are represented by lines. Constitutive exons are colored in light grey and alternative exons in dark grey. The three reverse-transcription (RT)-PCR assays (A, B, and C) performed to cover the GSDMB coding region are indicated; ( b ) An illustrative agarose gel (2%) shows the amplification products of the three RT-PCR assays. For “A” and “C” assays, the unique amplification product and their length, expressed as base pairs (bp), are schematized on the left. For the “B” assay, multiple specific amplification products were obtained, corresponding to several AS events involving exons 4 to 9, which were further analyzed by both fluorescent RT-PCR and direct sequencing (see further). MW: molecular-weight marker (pUC9- Hae III); ( c ) The left panel represents a demonstrative GeneMapper window showing the fluorescent RT-PCR products of the “B” assay (on RNA extracted from a healthy individual, heterozygous for the rs11078928 polymorphism), using the forward primer labelled with the HEX fluorophore. The filled peaks, shaded in grey, correspond to the RT-PCR products; empty peaks represent the size standard (ROX-500 HD). The vertical axis indicates the length of amplified products in bp, whereas the horizontal axis shows fluorescence units. The schematic representation and the name of the corresponding isoform product are indicated on the right side. The alternative exon 6* is depicted in black. F”: full length, “*” indicates the presence of exon 6* in the isoform, “∆” is followed by the skipped exon, separated by “-” or “,” if the exons are contiguous or not, respectively. The accession number of University of California, Santa Cruz (UCSC) Genome Browser-annotated isoforms is also indicated. The presence/absence in the transcript of a premature termination codon (PTC) is indicated by a “+”/“−”, respectively.
Figure Legend Snippet: GSDMB alternative splicing (AS) pattern. ( a ) Schematic representation of the GSDMB gene. Exons are represented by boxes and are approximately drawn to scale; untranslated regions (UTRs) are depicted by smaller boxes; introns are represented by lines. Constitutive exons are colored in light grey and alternative exons in dark grey. The three reverse-transcription (RT)-PCR assays (A, B, and C) performed to cover the GSDMB coding region are indicated; ( b ) An illustrative agarose gel (2%) shows the amplification products of the three RT-PCR assays. For “A” and “C” assays, the unique amplification product and their length, expressed as base pairs (bp), are schematized on the left. For the “B” assay, multiple specific amplification products were obtained, corresponding to several AS events involving exons 4 to 9, which were further analyzed by both fluorescent RT-PCR and direct sequencing (see further). MW: molecular-weight marker (pUC9- Hae III); ( c ) The left panel represents a demonstrative GeneMapper window showing the fluorescent RT-PCR products of the “B” assay (on RNA extracted from a healthy individual, heterozygous for the rs11078928 polymorphism), using the forward primer labelled with the HEX fluorophore. The filled peaks, shaded in grey, correspond to the RT-PCR products; empty peaks represent the size standard (ROX-500 HD). The vertical axis indicates the length of amplified products in bp, whereas the horizontal axis shows fluorescence units. The schematic representation and the name of the corresponding isoform product are indicated on the right side. The alternative exon 6* is depicted in black. F”: full length, “*” indicates the presence of exon 6* in the isoform, “∆” is followed by the skipped exon, separated by “-” or “,” if the exons are contiguous or not, respectively. The accession number of University of California, Santa Cruz (UCSC) Genome Browser-annotated isoforms is also indicated. The presence/absence in the transcript of a premature termination codon (PTC) is indicated by a “+”/“−”, respectively.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Sequencing, Molecular Weight, Marker, Fluorescence

Analysis of exon 6/6* AS in multiple sclerosis (MS) cases and healthy controls. ( a ) Schematic representation of the GSDMB genomic region comprised between exons 4 and 8. Exons are approximately drawn to scale; alternative exons are depicted in dark grey, as in Figure 1 . The dashed arrows below the scheme indicate the primer couple used in the fluorescent-competitive RT-PCR assay; the forward primer is labelled with the HEX fluorophore; ( b ) The left panel shows three GeneMapper windows, representing an example of the fluorescent products obtained for each of the rs11078928 genotypes. The peaks shaded in grey correspond to the RT-PCR products; those not shaded represent the size standard (ROX-500 HD). The schematic representation of the obtained products for each genotype is shown on the right. Exon 6* is depicted in black; ( c ) Boxplots showing the percentage of the Δ6 isoform with respect to the sum of Δ6, F*, and F isoforms measured on RNA extracted from 30 relapsing remitting (RR)-MS cases and 30 controls, grouped on the basis of the rs11078928 genotype. Boxes define the interquartile range; the thick line refers to the median. The number of subjects belonging to each group is also indicated (N). Significance levels of t -tests is shown above the boxplots (** p
Figure Legend Snippet: Analysis of exon 6/6* AS in multiple sclerosis (MS) cases and healthy controls. ( a ) Schematic representation of the GSDMB genomic region comprised between exons 4 and 8. Exons are approximately drawn to scale; alternative exons are depicted in dark grey, as in Figure 1 . The dashed arrows below the scheme indicate the primer couple used in the fluorescent-competitive RT-PCR assay; the forward primer is labelled with the HEX fluorophore; ( b ) The left panel shows three GeneMapper windows, representing an example of the fluorescent products obtained for each of the rs11078928 genotypes. The peaks shaded in grey correspond to the RT-PCR products; those not shaded represent the size standard (ROX-500 HD). The schematic representation of the obtained products for each genotype is shown on the right. Exon 6* is depicted in black; ( c ) Boxplots showing the percentage of the Δ6 isoform with respect to the sum of Δ6, F*, and F isoforms measured on RNA extracted from 30 relapsing remitting (RR)-MS cases and 30 controls, grouped on the basis of the rs11078928 genotype. Boxes define the interquartile range; the thick line refers to the median. The number of subjects belonging to each group is also indicated (N). Significance levels of t -tests is shown above the boxplots (** p

Techniques Used: Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction

Characterization of the GSDMB exonic circular RNA (ecircRNA). ( a ) Schematic representation of the formation of the GSDMB ecircRNA through a backsplicing event between exons 4 and 5. Exons are approximately drawn to scale; exon 4 is depicted in grey, exon 5 in black. The curved arrow joins the 5′ splice site of exon 5 to 3′ splice site of exon 4. Arrows below exon 5 indicate the divergent primer couple used to detect the putative circular RNA (circRNA). On the right, a schematic representation of the circRNA is depicted, with the circular black line representing the product amplified by the primer couple. Below the scheme, direct-sequencing electropherograms show the head-to-tail splice junction, indicated by an arrow, located between GSDMB exons 5 and 4; ( b ) Schematic representation of the linear splicing involving exons 4–5. Arrows below exons 4–5 indicate the primer couple used in RT-PCR assays. Electropherograms showing the junction between exons 4 and 5 is also reported; ( c ) Agarose gel (2%) with the results of the RNase R treatment. The expression of the GSDMB ecircRNA was evaluated by RT-PCR in untreated (−) or RNase R-treated (+) RNA of a healthy control, using the divergent primer couple within exon 5. Expression of total GSDMB linear mRNA was also evaluated, using the RT-PCR assay shown in Figure 2 (linear product; exons 9–11), the assay reported in ( b ) (linear product; exons 4 and 5), as well as an assay using a divergent primer couple on exon 10 (negative control; no circRNA detected). MW: molecular-weight marker (pUC9- Hae III); ( d ) Boxplots show expression levels of the GSDMB ecircRNA measured by semi-quantitative real-time RT-PCR in PBMCs of 30 MS cases and 30 healthy controls. Boxes define the interquartile range; the thick line refers to the median. Results were normalized to expression levels of the HMBS housekeeping gene. The number of subjects belonging to each group is also indicated (N). The significance level of t -test analysis is shown. ** p
Figure Legend Snippet: Characterization of the GSDMB exonic circular RNA (ecircRNA). ( a ) Schematic representation of the formation of the GSDMB ecircRNA through a backsplicing event between exons 4 and 5. Exons are approximately drawn to scale; exon 4 is depicted in grey, exon 5 in black. The curved arrow joins the 5′ splice site of exon 5 to 3′ splice site of exon 4. Arrows below exon 5 indicate the divergent primer couple used to detect the putative circular RNA (circRNA). On the right, a schematic representation of the circRNA is depicted, with the circular black line representing the product amplified by the primer couple. Below the scheme, direct-sequencing electropherograms show the head-to-tail splice junction, indicated by an arrow, located between GSDMB exons 5 and 4; ( b ) Schematic representation of the linear splicing involving exons 4–5. Arrows below exons 4–5 indicate the primer couple used in RT-PCR assays. Electropherograms showing the junction between exons 4 and 5 is also reported; ( c ) Agarose gel (2%) with the results of the RNase R treatment. The expression of the GSDMB ecircRNA was evaluated by RT-PCR in untreated (−) or RNase R-treated (+) RNA of a healthy control, using the divergent primer couple within exon 5. Expression of total GSDMB linear mRNA was also evaluated, using the RT-PCR assay shown in Figure 2 (linear product; exons 9–11), the assay reported in ( b ) (linear product; exons 4 and 5), as well as an assay using a divergent primer couple on exon 10 (negative control; no circRNA detected). MW: molecular-weight marker (pUC9- Hae III); ( d ) Boxplots show expression levels of the GSDMB ecircRNA measured by semi-quantitative real-time RT-PCR in PBMCs of 30 MS cases and 30 healthy controls. Boxes define the interquartile range; the thick line refers to the median. Results were normalized to expression levels of the HMBS housekeeping gene. The number of subjects belonging to each group is also indicated (N). The significance level of t -test analysis is shown. ** p

Techniques Used: Amplification, Sequencing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Negative Control, Molecular Weight, Marker, Quantitative RT-PCR, Mass Spectrometry

Evaluation of GSDMB susceptibility to nonsense-mediated mRNA decay (NMD). The upper panel shows a partial scheme of the GSDMB gene and the position of the primer couple used in the semi-quantitative real-time RT-PCR assay. The lower panel represents GSDMB total expression levels in HEK293 and HepG2 cells lines untreated (NT) or treated for 8 h with cycloheximide. Expression levels of two PRKCA transcripts, sensitive and insensitive to NMD, are also shown. The expression level of the untreated sample was set to 1. Bars represent means + SEM (standard error of the mean) of three independent experiments, each performed in triplicate. Significance levels of t -tests are shown. ** p
Figure Legend Snippet: Evaluation of GSDMB susceptibility to nonsense-mediated mRNA decay (NMD). The upper panel shows a partial scheme of the GSDMB gene and the position of the primer couple used in the semi-quantitative real-time RT-PCR assay. The lower panel represents GSDMB total expression levels in HEK293 and HepG2 cells lines untreated (NT) or treated for 8 h with cycloheximide. Expression levels of two PRKCA transcripts, sensitive and insensitive to NMD, are also shown. The expression level of the untreated sample was set to 1. Bars represent means + SEM (standard error of the mean) of three independent experiments, each performed in triplicate. Significance levels of t -tests are shown. ** p

Techniques Used: Quantitative RT-PCR, Expressing

4) Product Images from "Mesenchymal Stem Cells Attenuate Cisplatin-Induced Nephrotoxicity in iNOS-Dependent Manner"

Article Title: Mesenchymal Stem Cells Attenuate Cisplatin-Induced Nephrotoxicity in iNOS-Dependent Manner

Journal: Stem Cells International

doi: 10.1155/2017/1315378

MSC-CM reduces influx of inflammatory DCs and CTLs in cisplatin-induced acute kidney injury and alters their cytokine profile. Total number of (a) IL-10-producing CD45+CD11c+ DCs, CD4+CD25+FoxP3+ T regulatory cells, (b) TNF- α +CD45+CD11c+ DCs, (c) IFN- γ - and IL-17-producing CD8+ CTL cells that infiltrated kidneys of the control and experimental animals. Representative flow cytometry dot plots are shown. Values are mean ± SEM; n = 10 mice/group. ∗ p
Figure Legend Snippet: MSC-CM reduces influx of inflammatory DCs and CTLs in cisplatin-induced acute kidney injury and alters their cytokine profile. Total number of (a) IL-10-producing CD45+CD11c+ DCs, CD4+CD25+FoxP3+ T regulatory cells, (b) TNF- α +CD45+CD11c+ DCs, (c) IFN- γ - and IL-17-producing CD8+ CTL cells that infiltrated kidneys of the control and experimental animals. Representative flow cytometry dot plots are shown. Values are mean ± SEM; n = 10 mice/group. ∗ p

Techniques Used: CTL Assay, Flow Cytometry, Cytometry, Mouse Assay

MSCs attenuate cisplatin-induced acute kidney injury in iNOS-dependent manner. (a) Serum levels of BUN and creatinine. (b) Histological scores. (c) Representative H E and PAS-stained mouse kidney (magnifications ×200). (d) Serum levels of cytokines. Total numbers of (e) TNF- α -producing CD45+CD11c+ dendritic cells and IL-17-producing CD8+ cytotoxic T cells, (f) IL-10-producing CD45+CD11c+ DCs and CD4+CD25+FoxP3+ T regulatory cells. Values are mean ± SEM; n = 10 mice/group. ∗ p
Figure Legend Snippet: MSCs attenuate cisplatin-induced acute kidney injury in iNOS-dependent manner. (a) Serum levels of BUN and creatinine. (b) Histological scores. (c) Representative H E and PAS-stained mouse kidney (magnifications ×200). (d) Serum levels of cytokines. Total numbers of (e) TNF- α -producing CD45+CD11c+ dendritic cells and IL-17-producing CD8+ cytotoxic T cells, (f) IL-10-producing CD45+CD11c+ DCs and CD4+CD25+FoxP3+ T regulatory cells. Values are mean ± SEM; n = 10 mice/group. ∗ p

Techniques Used: Staining, Mouse Assay

MSCs significantly attenuate influx of immune cells and their capacity to produce nephrotoxic and inflammatory cytokines. Total number of (a) CD45+ leukocytes, (b) CD45+CD11b+ myeloid cells, CD45+F4/80+ macrophages, CD45+CD11c+ dendritic cells, CD45+CD11b+Ly6G+ neutrophils, CD45+CD4+ T helper cells, CD45+CD8+ cytotoxic T cells, (c) TNF- α +CD11c+ dendritic cells, (d) IFN- γ +CD4+ T helper cells, and IL-17+CD4+ T helper cells in cisplatin- and cisplatin + MSC-treated mice. (e) Total number and representative flow cytometry dot plots of IFN- γ - and IL-17-producing cytotoxic CD8+ T cells. Data presented as mean ± SEM; n = 10 mice/group. ∗ p
Figure Legend Snippet: MSCs significantly attenuate influx of immune cells and their capacity to produce nephrotoxic and inflammatory cytokines. Total number of (a) CD45+ leukocytes, (b) CD45+CD11b+ myeloid cells, CD45+F4/80+ macrophages, CD45+CD11c+ dendritic cells, CD45+CD11b+Ly6G+ neutrophils, CD45+CD4+ T helper cells, CD45+CD8+ cytotoxic T cells, (c) TNF- α +CD11c+ dendritic cells, (d) IFN- γ +CD4+ T helper cells, and IL-17+CD4+ T helper cells in cisplatin- and cisplatin + MSC-treated mice. (e) Total number and representative flow cytometry dot plots of IFN- γ - and IL-17-producing cytotoxic CD8+ T cells. Data presented as mean ± SEM; n = 10 mice/group. ∗ p

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

MSCs reduce cisplatin-induced nephrotoxicity via soluble factors. (a) Mice were euthanized 72 h after cisplatin administration, and blood urea nitrogen (BUN) and plasma creatinine levels are measured. (b) Histological examination was performed with H E staining. (c) H E and PAS staining images of representative kidney tissues are shown at the same magnifications (200x). Concentration of (d) cytokines, (e) kynurenine, and (f) NO in mouse serum. (g) Expression of IL-6 and TNF- α genes in mouse kidneys. (h) Total number of renal-infiltrated CD45+ leukocytes, CD45+CD11b+ myeloid cells, CD45+F4/80+ macrophages, CD45+CD11c+ dendritic cells, CD45+CD11b+Ly6G+ neutrophils, CD45+CD4+ T helper cells, and CD45+CD8+ cytotoxic T cells. Data presented as mean ± SEM; n = 10 mice/group. ∗ p
Figure Legend Snippet: MSCs reduce cisplatin-induced nephrotoxicity via soluble factors. (a) Mice were euthanized 72 h after cisplatin administration, and blood urea nitrogen (BUN) and plasma creatinine levels are measured. (b) Histological examination was performed with H E staining. (c) H E and PAS staining images of representative kidney tissues are shown at the same magnifications (200x). Concentration of (d) cytokines, (e) kynurenine, and (f) NO in mouse serum. (g) Expression of IL-6 and TNF- α genes in mouse kidneys. (h) Total number of renal-infiltrated CD45+ leukocytes, CD45+CD11b+ myeloid cells, CD45+F4/80+ macrophages, CD45+CD11c+ dendritic cells, CD45+CD11b+Ly6G+ neutrophils, CD45+CD4+ T helper cells, and CD45+CD8+ cytotoxic T cells. Data presented as mean ± SEM; n = 10 mice/group. ∗ p

Techniques Used: Mouse Assay, Staining, Concentration Assay, Expressing

MSCs attenuate cisplatin-induced acute kidney injury. (a) Blood urea nitrogen (BUN) and plasma creatinine levels are evaluated. (b) Histological scores (ranging between 0 and 4) were determinated and calculated on the percentage of tubules affected (0 ≤ 10%, 1 = 10–25%, 2 = 26–50%, 3 = 51–75%, and 4 ≥ 75%). (c) Representative H E- and PAS-stained mouse kidney. H E staining images of kidney tissue samples are shown at the same magnifications (×200). Concentration of (d) cytokines, (e) kynurenine, and (f) NO in mice sera. (g) IL-6 and TNF- α gene expression in mouse kidneys. Values are mean ± SEM; n = 10 mice/group. ∗ p
Figure Legend Snippet: MSCs attenuate cisplatin-induced acute kidney injury. (a) Blood urea nitrogen (BUN) and plasma creatinine levels are evaluated. (b) Histological scores (ranging between 0 and 4) were determinated and calculated on the percentage of tubules affected (0 ≤ 10%, 1 = 10–25%, 2 = 26–50%, 3 = 51–75%, and 4 ≥ 75%). (c) Representative H E- and PAS-stained mouse kidney. H E staining images of kidney tissue samples are shown at the same magnifications (×200). Concentration of (d) cytokines, (e) kynurenine, and (f) NO in mice sera. (g) IL-6 and TNF- α gene expression in mouse kidneys. Values are mean ± SEM; n = 10 mice/group. ∗ p

Techniques Used: Staining, Concentration Assay, Mouse Assay, Expressing

MSC-derived NO is important for activation of IDO in TNF- α -stimulated MSCs. (a) Expression of IDO and iNOS in nonstimulated and TNF- α -stimulated MSCs. (b) Concentration of kynurenine in supernatants of nonstimulated MSCs, TNF- α -stimulated MSCs, and TNF- α -stimulated MSCs cultured in the presence of L-NMMA. ∗ p
Figure Legend Snippet: MSC-derived NO is important for activation of IDO in TNF- α -stimulated MSCs. (a) Expression of IDO and iNOS in nonstimulated and TNF- α -stimulated MSCs. (b) Concentration of kynurenine in supernatants of nonstimulated MSCs, TNF- α -stimulated MSCs, and TNF- α -stimulated MSCs cultured in the presence of L-NMMA. ∗ p

Techniques Used: Derivative Assay, Activation Assay, Expressing, Concentration Assay, Cell Culture

5) Product Images from "Dual regulation of lin28a by Myc is necessary during zebrafish retina regeneration"

Article Title: Dual regulation of lin28a by Myc is necessary during zebrafish retina regeneration

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201802113

Her4.1 restricts the zone of MGPCs by suppressing lin28a expression. (A and B) RT-PCR (A) and qPCR (B) show decreased her4.1 induction and increased regeneration-associated genes’ levels with DAPT treatment relative to DMSO control in 2-dpi retina. UC, uninjured control. *, P
Figure Legend Snippet: Her4.1 restricts the zone of MGPCs by suppressing lin28a expression. (A and B) RT-PCR (A) and qPCR (B) show decreased her4.1 induction and increased regeneration-associated genes’ levels with DAPT treatment relative to DMSO control in 2-dpi retina. UC, uninjured control. *, P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Mycb regulates her4.1 gene transcription in injured retina. (A and B) RT-PCR (top) and qPCR (bottom) show increased her4.1 induction with MO-based mycb knockdown (A) or 10058-F4 (B) relative to control MO and DMSO, respectively, in 2-dpi retina. *, P
Figure Legend Snippet: Mycb regulates her4.1 gene transcription in injured retina. (A and B) RT-PCR (top) and qPCR (bottom) show increased her4.1 induction with MO-based mycb knockdown (A) or 10058-F4 (B) relative to control MO and DMSO, respectively, in 2-dpi retina. *, P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Myc-Ascl1a coexpression and interdependency during regeneration. (A and B) RT-PCR (top) and qPCR (bottom) show inhibition of mycb through MO (A), and 10058-F4 (B) down-regulates ascl1a and mycb induction, relative to the control at 2 dpi. *, P
Figure Legend Snippet: Myc-Ascl1a coexpression and interdependency during regeneration. (A and B) RT-PCR (top) and qPCR (bottom) show inhibition of mycb through MO (A), and 10058-F4 (B) down-regulates ascl1a and mycb induction, relative to the control at 2 dpi. *, P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Inhibition

Myc genes are rapidly induced in the injured retina. (A and B) RT-PCR (top) and qPCR (bottom) were used to assay injury-dependent myca (A), and mycb (B) gene expressions; n = 6 biological replicates. (C) FISH and IF microscopy show expression of myca mRNA and GFP in retina of 1016 tuba1a:gfp transgenic fish at 4 dpi. (D and E) ISH and IF microscopy show that myca (D) and mycb (E) mRNA is expressed in PCNA + MGPCs and neighboring cells at 4 dpi. The white arrows indicate colabeled cells in D and E, and white arrowheads in D and E identify myca − and mycb − but PCNA + cells near injury site. Black arrowheads indicate GCL-specific myca and mycb expression. (F) FISH microscopy shows coexpression of myca and mycb mRNA in BrdU + cells and vicinity. The white arrow indicates EdU + , myca + , and mycb + cells, and arrowheads mark myca + and mycb + cells. (G–J) A single 0.5-µm-thick Z section shows myca (G) and mycb (H) in 4-dpi retina; dotted outline in red shows PCNA + myca − / mycb − cells, green shows colabel with PCNA and myca / mycb , and yellow indicates myca + / mycb + but PCNA − cells; and the percentage colabeling with PCNA is quantified (I and J); n = 5 biological replicates. Bars, 10 µm; white asterisks mark the injury sites. ONL, outer nuclear layer; INL, inner nuclear layer (C–H).
Figure Legend Snippet: Myc genes are rapidly induced in the injured retina. (A and B) RT-PCR (top) and qPCR (bottom) were used to assay injury-dependent myca (A), and mycb (B) gene expressions; n = 6 biological replicates. (C) FISH and IF microscopy show expression of myca mRNA and GFP in retina of 1016 tuba1a:gfp transgenic fish at 4 dpi. (D and E) ISH and IF microscopy show that myca (D) and mycb (E) mRNA is expressed in PCNA + MGPCs and neighboring cells at 4 dpi. The white arrows indicate colabeled cells in D and E, and white arrowheads in D and E identify myca − and mycb − but PCNA + cells near injury site. Black arrowheads indicate GCL-specific myca and mycb expression. (F) FISH microscopy shows coexpression of myca and mycb mRNA in BrdU + cells and vicinity. The white arrow indicates EdU + , myca + , and mycb + cells, and arrowheads mark myca + and mycb + cells. (G–J) A single 0.5-µm-thick Z section shows myca (G) and mycb (H) in 4-dpi retina; dotted outline in red shows PCNA + myca − / mycb − cells, green shows colabel with PCNA and myca / mycb , and yellow indicates myca + / mycb + but PCNA − cells; and the percentage colabeling with PCNA is quantified (I and J); n = 5 biological replicates. Bars, 10 µm; white asterisks mark the injury sites. ONL, outer nuclear layer; INL, inner nuclear layer (C–H).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Microscopy, Expressing, Transgenic Assay, In Situ Hybridization

Mycb-mediated regulation of lin28a in MGPCs. (A and B) RT-PCR (top) and qPCR (bottom) show Myc inhibition using antisense MO (A) or 10058-F4 (B) induce lin28a in 2-dpi retina. *, P
Figure Legend Snippet: Mycb-mediated regulation of lin28a in MGPCs. (A and B) RT-PCR (top) and qPCR (bottom) show Myc inhibition using antisense MO (A) or 10058-F4 (B) induce lin28a in 2-dpi retina. *, P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Inhibition

6) Product Images from "A Novel Synthesized Sulfonamido-Based Gallate—JEZ-C as Potential Therapeutic Agents for Osteoarthritis"

Article Title: A Novel Synthesized Sulfonamido-Based Gallate—JEZ-C as Potential Therapeutic Agents for Osteoarthritis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0125930

Quantitative comparison of ECM-related gene expression of MMP-1 (A), MMP-13 (B) and TIMP-1 (C) by qRT-PCR. The chondrocytes were cultured with different concentrations of JEZ-C, GA and SMZ: Control (without IL-1β), Model (with IL-1β), JEZ-C (J-1: 6.25×10 −7 μg/ml; J-2: 6.25×10 −6 μg/ml; J-3: 6.25×10 −5 μg/ml), GA (G-1: 0.078 μg/ml; G-2: 0.125 μg/ml; G-3: 0.156μg/ml) and SMZ (S-1: 6.25×10 −6 μg/ml; S-2: 6.25×10 −5 μg/ml; S-3: 6.25×10 −4 μg/ml) on the induction by IL-1β for 6 days (n = 3 for each experiment). The gene expression levels in JEZ-C, GA and SMZ media relative to the control group were analysed by the 2 -ΔΔCT method using β-actin as the internal control. The data represent the mean±SD of three independent culture experiments. Bars with different letters are significantly different from each other at P﹤0.05.
Figure Legend Snippet: Quantitative comparison of ECM-related gene expression of MMP-1 (A), MMP-13 (B) and TIMP-1 (C) by qRT-PCR. The chondrocytes were cultured with different concentrations of JEZ-C, GA and SMZ: Control (without IL-1β), Model (with IL-1β), JEZ-C (J-1: 6.25×10 −7 μg/ml; J-2: 6.25×10 −6 μg/ml; J-3: 6.25×10 −5 μg/ml), GA (G-1: 0.078 μg/ml; G-2: 0.125 μg/ml; G-3: 0.156μg/ml) and SMZ (S-1: 6.25×10 −6 μg/ml; S-2: 6.25×10 −5 μg/ml; S-3: 6.25×10 −4 μg/ml) on the induction by IL-1β for 6 days (n = 3 for each experiment). The gene expression levels in JEZ-C, GA and SMZ media relative to the control group were analysed by the 2 -ΔΔCT method using β-actin as the internal control. The data represent the mean±SD of three independent culture experiments. Bars with different letters are significantly different from each other at P﹤0.05.

Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture

Quantitative comparison of ECM-related gene expression of aggrecan (a), collagen II (b), Sox9 (c) collagen I (d) and by qRT-PCR. The chondrocytes were cultured with different concentrations of JEZ-C, GA and SMZ: Control (K: 0 μg/ml), JEZ-C (J-1: 6.25×10 −7 μg/ml; J-2: 6.25×10 −6 μg/ml; J-3: 6.25×10 −5 μg/ml), GA (G-1: 0.078 μg/ml; G-2: 0.125 μg/ml; G-3: 0.156μg/ml) and SDM (S-1: 6.25×10 −6 μg/ml; S-2: 6.25×10 −5 μg/ml; S-3: 6.25×10 −4 μg/ml) for 6 d (n = 3 for each experiment). The gene expression levels in JEZ-C, GA and SMZ media relative to the control group were analyzed by the 2 -ΔΔCT method using β-actin as the internal control. The data represent the means ± SD of three independent culture experiments. *p
Figure Legend Snippet: Quantitative comparison of ECM-related gene expression of aggrecan (a), collagen II (b), Sox9 (c) collagen I (d) and by qRT-PCR. The chondrocytes were cultured with different concentrations of JEZ-C, GA and SMZ: Control (K: 0 μg/ml), JEZ-C (J-1: 6.25×10 −7 μg/ml; J-2: 6.25×10 −6 μg/ml; J-3: 6.25×10 −5 μg/ml), GA (G-1: 0.078 μg/ml; G-2: 0.125 μg/ml; G-3: 0.156μg/ml) and SDM (S-1: 6.25×10 −6 μg/ml; S-2: 6.25×10 −5 μg/ml; S-3: 6.25×10 −4 μg/ml) for 6 d (n = 3 for each experiment). The gene expression levels in JEZ-C, GA and SMZ media relative to the control group were analyzed by the 2 -ΔΔCT method using β-actin as the internal control. The data represent the means ± SD of three independent culture experiments. *p

Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture

7) Product Images from "Clinical and genomic landscape of gastric cancer with a mesenchymal phenotype"

Article Title: Clinical and genomic landscape of gastric cancer with a mesenchymal phenotype

Journal: Nature Communications

doi: 10.1038/s41467-018-04179-8

IGF1/IGF1R pathway is therapeutic target for MP subtype. a Expression of IGF1 in gastric cancer cell lines. Expression was measured by qRT-PCR. b Western blot analyses in gastric cancer cell lines of MP subtype cells (MKN74 and SNU1) and EP subtype cells (MKN28 and MKN45) using antibodies against phosphorylated IGF1R (active), IGF1R (total), and β-actin. c Sensitivity of gastric cancer cell lines to linsitinib, inhibitor of IGF1R. Cell growth was measured at 96 h after treatment of linsitinib as indicated ( n = 4). d Growth of SNU1-derived xenograft tumors in mice treated with linsitinib or vehicle control. SNU1 cells were xenografted subcutaneously into the flanks of mice. At 10 days after xenografting, linsitinib or control tartaric acid was orally administrated to mice. Tumor volume was measured on the indicated days. Error bars indicate s.e.m. e Tumors harvested after treatment of linsitinib or control vehicle. f Tumor weight after treatment of linsitinib. At 25 days after linsitinib treatment, mice were killed and tumor weights were measured ( n = 8 or 9 per treatment). Data are presented with means. P values were obtained by Student’s t -test
Figure Legend Snippet: IGF1/IGF1R pathway is therapeutic target for MP subtype. a Expression of IGF1 in gastric cancer cell lines. Expression was measured by qRT-PCR. b Western blot analyses in gastric cancer cell lines of MP subtype cells (MKN74 and SNU1) and EP subtype cells (MKN28 and MKN45) using antibodies against phosphorylated IGF1R (active), IGF1R (total), and β-actin. c Sensitivity of gastric cancer cell lines to linsitinib, inhibitor of IGF1R. Cell growth was measured at 96 h after treatment of linsitinib as indicated ( n = 4). d Growth of SNU1-derived xenograft tumors in mice treated with linsitinib or vehicle control. SNU1 cells were xenografted subcutaneously into the flanks of mice. At 10 days after xenografting, linsitinib or control tartaric acid was orally administrated to mice. Tumor volume was measured on the indicated days. Error bars indicate s.e.m. e Tumors harvested after treatment of linsitinib or control vehicle. f Tumor weight after treatment of linsitinib. At 25 days after linsitinib treatment, mice were killed and tumor weights were measured ( n = 8 or 9 per treatment). Data are presented with means. P values were obtained by Student’s t -test

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Mouse Assay

8) Product Images from "Soluble Forms of the Notch Ligands Delta1 and Jagged1 Promote in Vivo Tumorigenicity in NIH3T3 Fibroblasts with Distinct Phenotypes"

Article Title: Soluble Forms of the Notch Ligands Delta1 and Jagged1 Promote in Vivo Tumorigenicity in NIH3T3 Fibroblasts with Distinct Phenotypes

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2008.080006

Ligand expression alters Notch receptor activity and expression of downstream effectors in tumors derived from NIH3T3 cells. A: Quantitative RT-PCR (Taqman) was performed to determine expression levels of transcripts for each Notch receptor as indicated. cDNA was generated from total RNA isolated from at least three individual tumors from each group. Quantitative PCR was performed for each sample in triplicate and quantitative analysis of the real-time data was performed using the comparative Ct method (ABI SDS Software package) with β-actin used as the endogenous control. Data are presented as fold expression in comparison to NIH3T3 control (value of “1”) for each assay. Error bars represent the 95% maximum and minimum confidence limits. One-way analysis of variance was used to determine significant differences between samples in comparison to control for each expression assay. Significant differences between the target sample and control are indicated with * representing * P
Figure Legend Snippet: Ligand expression alters Notch receptor activity and expression of downstream effectors in tumors derived from NIH3T3 cells. A: Quantitative RT-PCR (Taqman) was performed to determine expression levels of transcripts for each Notch receptor as indicated. cDNA was generated from total RNA isolated from at least three individual tumors from each group. Quantitative PCR was performed for each sample in triplicate and quantitative analysis of the real-time data was performed using the comparative Ct method (ABI SDS Software package) with β-actin used as the endogenous control. Data are presented as fold expression in comparison to NIH3T3 control (value of “1”) for each assay. Error bars represent the 95% maximum and minimum confidence limits. One-way analysis of variance was used to determine significant differences between samples in comparison to control for each expression assay. Significant differences between the target sample and control are indicated with * representing * P

Techniques Used: Expressing, Activity Assay, Derivative Assay, Quantitative RT-PCR, Generated, Isolation, Real-time Polymerase Chain Reaction, Software

Notch ligand expression regulates tumor angiogenesis. A: Fixed and Masson’s trichrome-stained tumor sections (magnification ×200) were analyzed for the presence of vasculature within the specimens. Vessels can be seen as pink steaks (smaller vessels) or in more dilated vessels, as clear areas containing red blood cells. B: 5 μm, paraffin-embedded tumor sections were immunostained with anti-PECAM to visualize and quantify vessel structure. Ten random fields (magnification ×40) were captured for each sample and data are presented as number of stained area per ×40 field. Shown are means ± SEM. Significance was determined by one-way analysis of variance for each tumor type in comparison to NIH3T3 control. The number of PECAM-positive cells was found significantly different from control in Dl1 and sDl1 tumors with P values of 0.0007 and 0.002, respectively. C–D ) Plasma collected from tumor-bearing animals was processed for detection of positive regulators of angiogenesis ( C ) and angiogenic inhibitors ( D ) using an antibody array (Panomics) as described in Materials and Methods. Chemiluminescent signals from each array were captured by autoradiography and quantified by densitometry. Signals are reported as relative signal intensity. E: Total RNA was collected from tumor tissue to detect transcript levels of VEGF and VEGFR family members by qualitative RT-PCR. RT-PCR with actin primers was used as a control for cDNA synthesis. F: Expression of VEFGA in protein lysates isolated from tumors was quantified by ELISA per manufacturer’s instructions. VEGFA is reported as the average of four individually processed tumor lysates for each type and is normalized to total protein in the sample as determined by bicinchoninic acid assay. Shown are means ± SEM. Significance was determined by one-way analysis of variance for each tumor type in comparison to control. VEGFA expression was found to be significantly different from control in all tumor types with P values of 0.001(sJag1), 0.009 (sDl1), and 0.01 (Dl1).
Figure Legend Snippet: Notch ligand expression regulates tumor angiogenesis. A: Fixed and Masson’s trichrome-stained tumor sections (magnification ×200) were analyzed for the presence of vasculature within the specimens. Vessels can be seen as pink steaks (smaller vessels) or in more dilated vessels, as clear areas containing red blood cells. B: 5 μm, paraffin-embedded tumor sections were immunostained with anti-PECAM to visualize and quantify vessel structure. Ten random fields (magnification ×40) were captured for each sample and data are presented as number of stained area per ×40 field. Shown are means ± SEM. Significance was determined by one-way analysis of variance for each tumor type in comparison to NIH3T3 control. The number of PECAM-positive cells was found significantly different from control in Dl1 and sDl1 tumors with P values of 0.0007 and 0.002, respectively. C–D ) Plasma collected from tumor-bearing animals was processed for detection of positive regulators of angiogenesis ( C ) and angiogenic inhibitors ( D ) using an antibody array (Panomics) as described in Materials and Methods. Chemiluminescent signals from each array were captured by autoradiography and quantified by densitometry. Signals are reported as relative signal intensity. E: Total RNA was collected from tumor tissue to detect transcript levels of VEGF and VEGFR family members by qualitative RT-PCR. RT-PCR with actin primers was used as a control for cDNA synthesis. F: Expression of VEFGA in protein lysates isolated from tumors was quantified by ELISA per manufacturer’s instructions. VEGFA is reported as the average of four individually processed tumor lysates for each type and is normalized to total protein in the sample as determined by bicinchoninic acid assay. Shown are means ± SEM. Significance was determined by one-way analysis of variance for each tumor type in comparison to control. VEGFA expression was found to be significantly different from control in all tumor types with P values of 0.001(sJag1), 0.009 (sDl1), and 0.01 (Dl1).

Techniques Used: Expressing, Staining, Ab Array, Autoradiography, Reverse Transcription Polymerase Chain Reaction, Isolation, Enzyme-linked Immunosorbent Assay, Acid Assay

Expression of soluble forms of Notch ligands increases lipid accumulation in tumors. A: Tumors specimens were cryopreserved, sectioned, and stained with Oil Red O to detect neutral lipids. No internal lipid was detected in the control tumors ( A ), whereas lipid droplets were found throughout the sections of sDl1 tumors ( B ). sJag1 had low levels of spotty lipid accumulation marked by the formation of small droplets ( C and inset ) and Dl1 tumors ( D ), although predominantly negative throughout, had one to two small regions of lipid accumulation per tumor ( inset ). Scale bar = 50 μm. E: Magnetic resonance imaging and localized spectroscopy of tumors in vivo was performed to evaluate tumor growth and lipid content. Images are of representative of mice bearing a tumor derived from either sDl1 or sJag1 transfectants as indicated. Top panel represents axial images through the tumor area acquired from isoflurane-anesthetized mice in a 7.0T Bruker PharmaScan magnet. Tumors (a), intestines (b), spinal cord (c), muscle (d), and bladder (e) are indicated. The bottom panel represents localized spectroscopy with PRESS (TR 2000ms, TE 21.4ms) as acquired using a voxel of 1 mm 3 placed into the center of the tumor. Placement of the voxel had no influence on the spectrum. One spectrum is the sum of 800 fields with a total scan time of 26 minutes 40 seconds. Scans were done at 20 days after injection of cells. The water and fat peak are indicated. F: Quantitative RT-PCR was performed to determine expression levels of transcripts for genes important for lipid metabolism. cDNA was generated from total RNA isolated from at least three individual tumors from each group. Quantitative PCR was performed for each sample in triplicate and quantitative analysis of the real-time data was performed using the comparative Ct method (ABI SDS Software package) with β-actin as the endogenous control. Data are presented as fold expression in comparison to control for each assay. Error bars represent the 95% maximum and minimum confidence limits. One-way analysis of variance was used to determine significant differences between samples in comparison to control for each expression assay. Significant differences ( P
Figure Legend Snippet: Expression of soluble forms of Notch ligands increases lipid accumulation in tumors. A: Tumors specimens were cryopreserved, sectioned, and stained with Oil Red O to detect neutral lipids. No internal lipid was detected in the control tumors ( A ), whereas lipid droplets were found throughout the sections of sDl1 tumors ( B ). sJag1 had low levels of spotty lipid accumulation marked by the formation of small droplets ( C and inset ) and Dl1 tumors ( D ), although predominantly negative throughout, had one to two small regions of lipid accumulation per tumor ( inset ). Scale bar = 50 μm. E: Magnetic resonance imaging and localized spectroscopy of tumors in vivo was performed to evaluate tumor growth and lipid content. Images are of representative of mice bearing a tumor derived from either sDl1 or sJag1 transfectants as indicated. Top panel represents axial images through the tumor area acquired from isoflurane-anesthetized mice in a 7.0T Bruker PharmaScan magnet. Tumors (a), intestines (b), spinal cord (c), muscle (d), and bladder (e) are indicated. The bottom panel represents localized spectroscopy with PRESS (TR 2000ms, TE 21.4ms) as acquired using a voxel of 1 mm 3 placed into the center of the tumor. Placement of the voxel had no influence on the spectrum. One spectrum is the sum of 800 fields with a total scan time of 26 minutes 40 seconds. Scans were done at 20 days after injection of cells. The water and fat peak are indicated. F: Quantitative RT-PCR was performed to determine expression levels of transcripts for genes important for lipid metabolism. cDNA was generated from total RNA isolated from at least three individual tumors from each group. Quantitative PCR was performed for each sample in triplicate and quantitative analysis of the real-time data was performed using the comparative Ct method (ABI SDS Software package) with β-actin as the endogenous control. Data are presented as fold expression in comparison to control for each assay. Error bars represent the 95% maximum and minimum confidence limits. One-way analysis of variance was used to determine significant differences between samples in comparison to control for each expression assay. Significant differences ( P

Techniques Used: Expressing, Staining, Magnetic Resonance Imaging, Spectroscopy, In Vivo, Mouse Assay, Derivative Assay, Injection, Quantitative RT-PCR, Generated, Isolation, Real-time Polymerase Chain Reaction, Software

9) Product Images from "Transcriptome sequencing and development of an expression microarray platform for the domestic ferret"

Article Title: Transcriptome sequencing and development of an expression microarray platform for the domestic ferret

Journal: BMC Genomics

doi: 10.1186/1471-2164-11-251

Tissue-specific gene expression . Panel A shows a selection of transcripts identified by ANOVA analysis followed by Pavlidis Template Matching analysis. The genes are clustered according to their expression profile for the analyzed tissues (Pearson correlation, average linkage clustering). The expression value is indicated by a sliding scale, going from black, indicating no expression values, through blue and green to red for the most highly expressed genes. Panel B show the tissue specific expression verification using qRT-PCR of two of the genes showed in panel A: the ferret albumin gene, Alb , and the ferret pulmonary surfactant-associated protein C, Sftpc . The graphs depict the log 2 -transformed curves of the relative fluorescence against cycle number during the qRT-PCR amplification. The analysis was done using RNA extracted from liver, lung, blood and brain, using the Atf4 housekeeping gene as reference. The C(t) threshold line (indicated by a red solid line) is set automatically using the noise band as cut off.
Figure Legend Snippet: Tissue-specific gene expression . Panel A shows a selection of transcripts identified by ANOVA analysis followed by Pavlidis Template Matching analysis. The genes are clustered according to their expression profile for the analyzed tissues (Pearson correlation, average linkage clustering). The expression value is indicated by a sliding scale, going from black, indicating no expression values, through blue and green to red for the most highly expressed genes. Panel B show the tissue specific expression verification using qRT-PCR of two of the genes showed in panel A: the ferret albumin gene, Alb , and the ferret pulmonary surfactant-associated protein C, Sftpc . The graphs depict the log 2 -transformed curves of the relative fluorescence against cycle number during the qRT-PCR amplification. The analysis was done using RNA extracted from liver, lung, blood and brain, using the Atf4 housekeeping gene as reference. The C(t) threshold line (indicated by a red solid line) is set automatically using the noise band as cut off.

Techniques Used: Expressing, Selection, Quantitative RT-PCR, Transformation Assay, Fluorescence, Amplification

10) Product Images from "Elucidation of major contributors involved in nitrogen removal and transcription level of nitrogen-cycling genes in activated sludge from WWTPs"

Article Title: Elucidation of major contributors involved in nitrogen removal and transcription level of nitrogen-cycling genes in activated sludge from WWTPs

Journal: Scientific Reports

doi: 10.1038/srep44728

Relative transcription level of amoA, nirS, nirK genes in the 8 activated sludge samples revealed by qRT-PCR. Significant ( p
Figure Legend Snippet: Relative transcription level of amoA, nirS, nirK genes in the 8 activated sludge samples revealed by qRT-PCR. Significant ( p

Techniques Used: Quantitative RT-PCR

11) Product Images from "Tumor necrosis factor-inducible gene 6 promotes liver regeneration in mice with acute liver injury"

Article Title: Tumor necrosis factor-inducible gene 6 promotes liver regeneration in mice with acute liver injury

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-015-0019-z

Neutralization of TSG-6 by TSG-6 antibody attenuates the restoration effect of TSG-6 in liver of CCl 4 + TSG-6-treated mice. (A) H E staining shows the histomorphological changes in CCl 4 + TSG-6-treated mice with (CCl 4 + TSG-6 + TSG-6 antibody) (CCl 4 + TSG-6 + IgG) without TSG-6 antibody (CCl 4 + TSG-6) or IgG. The representative images were shown at × 40. (B) Relative liver weight/body weight of mice. (C) , (D) The values of AST and ALT are graphed. (E , F , G) QRT-PCR analysis for G6pc (E), the fibrotic markers (F), tgf-β, α–sma and collagen-α1, the inflammation markers (G), tnfα, il-1β, cxcl1 and cxcl2, of liver mRNA from the treated mice (n ≥4 mice / group). Mean ± SD results are graphed (ANOVA, * P
Figure Legend Snippet: Neutralization of TSG-6 by TSG-6 antibody attenuates the restoration effect of TSG-6 in liver of CCl 4 + TSG-6-treated mice. (A) H E staining shows the histomorphological changes in CCl 4 + TSG-6-treated mice with (CCl 4 + TSG-6 + TSG-6 antibody) (CCl 4 + TSG-6 + IgG) without TSG-6 antibody (CCl 4 + TSG-6) or IgG. The representative images were shown at × 40. (B) Relative liver weight/body weight of mice. (C) , (D) The values of AST and ALT are graphed. (E , F , G) QRT-PCR analysis for G6pc (E), the fibrotic markers (F), tgf-β, α–sma and collagen-α1, the inflammation markers (G), tnfα, il-1β, cxcl1 and cxcl2, of liver mRNA from the treated mice (n ≥4 mice / group). Mean ± SD results are graphed (ANOVA, * P

Techniques Used: Neutralization, Mouse Assay, Staining, AST Assay, Quantitative RT-PCR

Reduced fibrosis in the TSG-6 treated liver. (A) QRT-PCR analysis of liver mRNA for tgf-β, α–sma and collagen-α1 (n ≥4 mice/group). Mean ± SD results are graphed. (B) and (C) . Western blot analysis of TGF-β (25 kDa: processed form) (inducer of fibrosis) and α-SMA (fibrogenic marker) (GAPDH was used as an internal control) (n ≥4 mice/group). Data shown represent one of three experiments with similar results (B: Immunoblot/ C: Band density of TGF-β and α-SMA). Data represent the mean ± SD of three independent experiments. (D) IHC for α-SMA in liver sections from representative control, CCl 4 , CCl 4 + NC, and CCl 4 + TSG-6 mice (×40) (ANOVA, * P
Figure Legend Snippet: Reduced fibrosis in the TSG-6 treated liver. (A) QRT-PCR analysis of liver mRNA for tgf-β, α–sma and collagen-α1 (n ≥4 mice/group). Mean ± SD results are graphed. (B) and (C) . Western blot analysis of TGF-β (25 kDa: processed form) (inducer of fibrosis) and α-SMA (fibrogenic marker) (GAPDH was used as an internal control) (n ≥4 mice/group). Data shown represent one of three experiments with similar results (B: Immunoblot/ C: Band density of TGF-β and α-SMA). Data represent the mean ± SD of three independent experiments. (D) IHC for α-SMA in liver sections from representative control, CCl 4 , CCl 4 + NC, and CCl 4 + TSG-6 mice (×40) (ANOVA, * P

Techniques Used: Quantitative RT-PCR, Mouse Assay, Western Blot, Marker, Immunohistochemistry

TSG-6 blocks activation of hepatic stellate cells. (A) QRT-PCR analysis for tgf-β, vimentin and collagen-α1 in LX2 cells which were cultured in LX2 cell medium (LX2), NC-CM (LX2 + NC), TSG-6-CM with (LX2 + TSG-6 + Ab) or without TSG-6 antibody (LX2 + TSG-6). IgG was used as a negative control for TSG-6 antibody (LX2 + TSG-6 + IgG). Mean ± SD results are graphed. Data represent the mean ± SD of three independent experiments (ANOVA, * P
Figure Legend Snippet: TSG-6 blocks activation of hepatic stellate cells. (A) QRT-PCR analysis for tgf-β, vimentin and collagen-α1 in LX2 cells which were cultured in LX2 cell medium (LX2), NC-CM (LX2 + NC), TSG-6-CM with (LX2 + TSG-6 + Ab) or without TSG-6 antibody (LX2 + TSG-6). IgG was used as a negative control for TSG-6 antibody (LX2 + TSG-6 + IgG). Mean ± SD results are graphed. Data represent the mean ± SD of three independent experiments (ANOVA, * P

Techniques Used: Activation Assay, Quantitative RT-PCR, Cell Culture, Negative Control

Effects of TSG-6 on liver histomorphology and function in CCl 4 -treated mice. (A) H E staining shows the extensive cellular damage and infiltration of inflammatory cells in CCl 4 -treated mice with or without NC (negative control: mock transfected cell)-conditioned medium (CM). Those cellular injuries were reduced in CCl 4 mice treated with TSG-6-CM (CCl 4 + TSG-6). The representative images are shown at × 40 (CTRL: corn-oil-treated control mice/ CCl 4 :CCl 4 -treated mice/ CCl 4 + NC: CCl 4 -treated mice with NC-CM). (B) Relative liver weight / body weight of mice. (C) The values of AST and ALT are graphed. (D) QRT-PCR analysis for G6pc of liver mRNA from normal (CTRL), CCl 4 , CCl 4 with NC-CM (NC) or TSG-6-CM (TSG-6) (n ≥4 mice / group) (ANOVA, * P
Figure Legend Snippet: Effects of TSG-6 on liver histomorphology and function in CCl 4 -treated mice. (A) H E staining shows the extensive cellular damage and infiltration of inflammatory cells in CCl 4 -treated mice with or without NC (negative control: mock transfected cell)-conditioned medium (CM). Those cellular injuries were reduced in CCl 4 mice treated with TSG-6-CM (CCl 4 + TSG-6). The representative images are shown at × 40 (CTRL: corn-oil-treated control mice/ CCl 4 :CCl 4 -treated mice/ CCl 4 + NC: CCl 4 -treated mice with NC-CM). (B) Relative liver weight / body weight of mice. (C) The values of AST and ALT are graphed. (D) QRT-PCR analysis for G6pc of liver mRNA from normal (CTRL), CCl 4 , CCl 4 with NC-CM (NC) or TSG-6-CM (TSG-6) (n ≥4 mice / group) (ANOVA, * P

Techniques Used: Mouse Assay, Staining, Negative Control, Transfection, AST Assay, Quantitative RT-PCR

TSG-6 decreases the inflammation in the liver damaged with CCl 4 . QRT-PCR analysis of mouse liver for tnfα, il-1β, cxcl1 and cxcl2. Mean ± SD results are graphed (n ≥4 mice/group) (ANOVA, * P
Figure Legend Snippet: TSG-6 decreases the inflammation in the liver damaged with CCl 4 . QRT-PCR analysis of mouse liver for tnfα, il-1β, cxcl1 and cxcl2. Mean ± SD results are graphed (n ≥4 mice/group) (ANOVA, * P

Techniques Used: Quantitative RT-PCR, Mouse Assay

12) Product Images from "A New Mutation, hap1-2, Reveals a C Terminal Domain Function in AtMago Protein and Its Biological Effects in Male Gametophyte Development in Arabidopsis thaliana"

Article Title: A New Mutation, hap1-2, Reveals a C Terminal Domain Function in AtMago Protein and Its Biological Effects in Male Gametophyte Development in Arabidopsis thaliana

Journal: PLoS ONE

doi: 10.1371/journal.pone.0148200

Both AtMago and AtMagoΔC are detected in P-bodies. Scale bar = 50 μm. A zoom-in view is shown for each image with a scale bar of 10 μm.
Figure Legend Snippet: Both AtMago and AtMagoΔC are detected in P-bodies. Scale bar = 50 μm. A zoom-in view is shown for each image with a scale bar of 10 μm.

Techniques Used:

CTD of AtMago is required for AtMago-AtY14 interaction, but not for AtMago-AteIF4AIII. Western blots showing results from in vitro pull-down assay for AtMagoΔC-AtY14 (A), AtMagoΔC-AteIF4AIII (B), and various AtMago C terminal truncations-AtY14 (C).
Figure Legend Snippet: CTD of AtMago is required for AtMago-AtY14 interaction, but not for AtMago-AteIF4AIII. Western blots showing results from in vitro pull-down assay for AtMagoΔC-AtY14 (A), AtMagoΔC-AteIF4AIII (B), and various AtMago C terminal truncations-AtY14 (C).

Techniques Used: Western Blot, In Vitro, Pull Down Assay

Characterization and complementation of the HAP1/hap1-2 mutant. (A) HAP1 gene structure. Exons were shown as colored boxes; introns were shown as straight lines; UTRs were shown as gray boxes. The T-DNA insertion in hap1-1 and hap1-2 was indicated by red arrow. (B) rtPCR results showed the expression of a full-length transcript ( AtMago ), a truncated transcript ( AtMagoΔC ) and GAPDH in qrt , qrt;HAP1/hap1-2 , qrt;sr45-1 and qrt;upf3-1 mutant plants. Total RNAs were extracted from the inflorescence tissue. The exon composition of the transcript and the position of used primers were shown next to the corresponding rtPCR results. (C) GUS stained pollen grains from qrt , qrt;HAP1/hap1-1 and qrt;HAP1/hap1-2 showing the identity of mutant pollen grains. Scale bar = 20 μm. (D) Seed number per silique in qrt and qrt;HAP1/hap1-2 plants. Scale bar = 1 mm. The quantification was done with sixteen siliques. Student t-test was used for statistical analysis. Error bars represent standard deviations. (E) HAP1 gene complemented hap1-2 mutant phenotype. The mature plants, GUS-stained pollen grains and seed yield per plant were shown in qrt , qrt;HAP1/hap1-2 , HAP1;qrt; hap1-2 . (F) qPCR results showing that the AtMago gene expression level was fully recovered in the transgenic plant. GAPDH was used as control. Three biological replicates were used in the analysis. Error bar showed standard deviation. Statistical significance compared to qrt was measured by student t-test ( p
Figure Legend Snippet: Characterization and complementation of the HAP1/hap1-2 mutant. (A) HAP1 gene structure. Exons were shown as colored boxes; introns were shown as straight lines; UTRs were shown as gray boxes. The T-DNA insertion in hap1-1 and hap1-2 was indicated by red arrow. (B) rtPCR results showed the expression of a full-length transcript ( AtMago ), a truncated transcript ( AtMagoΔC ) and GAPDH in qrt , qrt;HAP1/hap1-2 , qrt;sr45-1 and qrt;upf3-1 mutant plants. Total RNAs were extracted from the inflorescence tissue. The exon composition of the transcript and the position of used primers were shown next to the corresponding rtPCR results. (C) GUS stained pollen grains from qrt , qrt;HAP1/hap1-1 and qrt;HAP1/hap1-2 showing the identity of mutant pollen grains. Scale bar = 20 μm. (D) Seed number per silique in qrt and qrt;HAP1/hap1-2 plants. Scale bar = 1 mm. The quantification was done with sixteen siliques. Student t-test was used for statistical analysis. Error bars represent standard deviations. (E) HAP1 gene complemented hap1-2 mutant phenotype. The mature plants, GUS-stained pollen grains and seed yield per plant were shown in qrt , qrt;HAP1/hap1-2 , HAP1;qrt; hap1-2 . (F) qPCR results showing that the AtMago gene expression level was fully recovered in the transgenic plant. GAPDH was used as control. Three biological replicates were used in the analysis. Error bar showed standard deviation. Statistical significance compared to qrt was measured by student t-test ( p

Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Real-time Polymerase Chain Reaction, Transgenic Assay, Standard Deviation

13) Product Images from "Development of a Neutralization Assay for Influenza Virus Using an Endpoint Assessment Based on Quantitative Reverse-Transcription PCR"

Article Title: Development of a Neutralization Assay for Influenza Virus Using an Endpoint Assessment Based on Quantitative Reverse-Transcription PCR

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056023

Replication kinetics of influenza virus assessed by qRT-PCR. Virus replication was assessed in the absence or presence of TPCK-trypsin (1 µg/mL). Virus (1000 TCID 50 in 100 µL) was placed into a well of a 96-well plate. After incubation for 1 h at 37°C, a suspension of MDCK-London cells (30,000 in 100 µL) was added. At the indicated times, experimental samples were prepared using SPR and subjected to qRT-PCR. The RNA copy numbers were normalized to the mean value observed for A/PR/8/34 at 6 hours in the presence of TPCK-trypsin. Each point represents the mean ± SEM (n = 3).
Figure Legend Snippet: Replication kinetics of influenza virus assessed by qRT-PCR. Virus replication was assessed in the absence or presence of TPCK-trypsin (1 µg/mL). Virus (1000 TCID 50 in 100 µL) was placed into a well of a 96-well plate. After incubation for 1 h at 37°C, a suspension of MDCK-London cells (30,000 in 100 µL) was added. At the indicated times, experimental samples were prepared using SPR and subjected to qRT-PCR. The RNA copy numbers were normalized to the mean value observed for A/PR/8/34 at 6 hours in the presence of TPCK-trypsin. Each point represents the mean ± SEM (n = 3).

Techniques Used: Quantitative RT-PCR, Incubation, SPR Assay

Influenza virus microneutralization assessed by qRT-PCR (qPCR-MN). ( A ) An inoculum containing 1000 TCID 50 of virus (Bris/07) was mixed with a dilution from a 2-fold dilution series of ferret antiserum in a well of a 96-well plate. After allowing the neutralization reaction to proceed for 1 hour at 37°C, trypsinized MDCK-London cells (30,000 per well) were added. TPCK-trypsin was present at 1 µg/mL. After 6 hours, experimental samples were prepared using SPR and subjected to qRT-PCR. The RNA copy numbers were normalized to the mean value obtained from infected wells in the absence of neutralizing serum (virus control wells). Each point represents the mean ± SEM (n = 3). The neutralization titer was defined as the reciprocal of the highest dilution factor of serum necessary to inhibit the PCR signal by 90%. ( B ) Same data as in (A); however, each experimental replicate was assessed independently. The mean of these curves would result in the curve depicted in (A).
Figure Legend Snippet: Influenza virus microneutralization assessed by qRT-PCR (qPCR-MN). ( A ) An inoculum containing 1000 TCID 50 of virus (Bris/07) was mixed with a dilution from a 2-fold dilution series of ferret antiserum in a well of a 96-well plate. After allowing the neutralization reaction to proceed for 1 hour at 37°C, trypsinized MDCK-London cells (30,000 per well) were added. TPCK-trypsin was present at 1 µg/mL. After 6 hours, experimental samples were prepared using SPR and subjected to qRT-PCR. The RNA copy numbers were normalized to the mean value obtained from infected wells in the absence of neutralizing serum (virus control wells). Each point represents the mean ± SEM (n = 3). The neutralization titer was defined as the reciprocal of the highest dilution factor of serum necessary to inhibit the PCR signal by 90%. ( B ) Same data as in (A); however, each experimental replicate was assessed independently. The mean of these curves would result in the curve depicted in (A).

Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Neutralization, SPR Assay, Infection, Polymerase Chain Reaction

14) Product Images from "Profligate Biotin Synthesis in ?-Proteobacteria - A Developing or Degenerating Regulatory System?"

Article Title: Profligate Biotin Synthesis in ?-Proteobacteria - A Developing or Degenerating Regulatory System?

Journal: Molecular microbiology

doi: 10.1111/mmi.12170

A. tumefaciens BioR functions as a repressor of bioBFDAZ expression A . qRT-PCR assay of BioR repression of bioBFDAZ operon expression in wild type A. tumefaciens NTL4 grown in defined M9 minimal media without added biotin. The qRT-PCR data were from no less than five independent tests and are expressed in means ± SD. B . qRT-PCR assay of BioR repression of bioBFDAZ operon expression upon addition of 1000 nM biotin to cultures of strain FYJ212 (Δ bioR:: Km) grown in M9 minimal medium. The qRT-PCR data were from no less than five independent tests and are expressed in means ± SD. C . Effects of deletion of bioR and BioR overexpression on β-galactosidase expression from a plasmid carrying a P bioB - lacZ transcriptional fusion. The strains were FYJ259 (WT), FYJ260 (Δ bioR ), and FYJ261 (Δ bioR carrying pSRK- bioR ), respectively. Mid-log phase cultures were grown in RB medium and β-galactosidase activities were measured from more than three independent experiments. The error bars indicate standard deviations. The plus sign denotes addition of 0.5 mM IPTG whereas the minus sign denotes no addition of IPTG. D . Effects of deletion of bioBFDA and bioR on β-galactosidase expression. The strains were FYJ290 (WT), FYJ291 (Δ bioR ), and FYJ292 (Δ bioR carrying pSRK- bioR ). The experiments were performed as in panel C. RB medium contains about 30 nM biotin.
Figure Legend Snippet: A. tumefaciens BioR functions as a repressor of bioBFDAZ expression A . qRT-PCR assay of BioR repression of bioBFDAZ operon expression in wild type A. tumefaciens NTL4 grown in defined M9 minimal media without added biotin. The qRT-PCR data were from no less than five independent tests and are expressed in means ± SD. B . qRT-PCR assay of BioR repression of bioBFDAZ operon expression upon addition of 1000 nM biotin to cultures of strain FYJ212 (Δ bioR:: Km) grown in M9 minimal medium. The qRT-PCR data were from no less than five independent tests and are expressed in means ± SD. C . Effects of deletion of bioR and BioR overexpression on β-galactosidase expression from a plasmid carrying a P bioB - lacZ transcriptional fusion. The strains were FYJ259 (WT), FYJ260 (Δ bioR ), and FYJ261 (Δ bioR carrying pSRK- bioR ), respectively. Mid-log phase cultures were grown in RB medium and β-galactosidase activities were measured from more than three independent experiments. The error bars indicate standard deviations. The plus sign denotes addition of 0.5 mM IPTG whereas the minus sign denotes no addition of IPTG. D . Effects of deletion of bioBFDA and bioR on β-galactosidase expression. The strains were FYJ290 (WT), FYJ291 (Δ bioR ), and FYJ292 (Δ bioR carrying pSRK- bioR ). The experiments were performed as in panel C. RB medium contains about 30 nM biotin.

Techniques Used: Expressing, Quantitative RT-PCR, Over Expression, Plasmid Preparation

Effects of biotin supplementation on expression of the bioBFDAZ operon and bioR A . Repression of bioBFDAZ operon expression in the wild type strain NTL4 by exogenous biotin supplementation The wild type strain NTL4 grown in M9 minimal media supplemented with different concentrations of biotin. RNA was isolated from these cultures and assayed by qRT-PCR. The data were obtained from more than six independent experiments and are expressed as mean ± SD. B . Effects of biotin supplementation of the bioR mutant on bioBFDAZ operon expression. Strain FYJ212 (Δ bioR:: Km) was grown in M9 minimal media with or without addition of 1000 nM biotin. The relative expression levels of bioBFDAZ operon was measured in three independent qRT-PCR tests and expressed in means ± SD. C and D . bioR expression in either the wild type strain NTL4 or the Δ bioBFDA strain grown on M9 minimal media with or without biotin supplementation. qRT-PCR assays of bioR expression in strains NTL4 ( Panel C ) or FYJ283 (Δ bioBFDA ) ( Panel D . The relative level of bioR expression was measured in three independent experiments and is expressed in mean ± SD.
Figure Legend Snippet: Effects of biotin supplementation on expression of the bioBFDAZ operon and bioR A . Repression of bioBFDAZ operon expression in the wild type strain NTL4 by exogenous biotin supplementation The wild type strain NTL4 grown in M9 minimal media supplemented with different concentrations of biotin. RNA was isolated from these cultures and assayed by qRT-PCR. The data were obtained from more than six independent experiments and are expressed as mean ± SD. B . Effects of biotin supplementation of the bioR mutant on bioBFDAZ operon expression. Strain FYJ212 (Δ bioR:: Km) was grown in M9 minimal media with or without addition of 1000 nM biotin. The relative expression levels of bioBFDAZ operon was measured in three independent qRT-PCR tests and expressed in means ± SD. C and D . bioR expression in either the wild type strain NTL4 or the Δ bioBFDA strain grown on M9 minimal media with or without biotin supplementation. qRT-PCR assays of bioR expression in strains NTL4 ( Panel C ) or FYJ283 (Δ bioBFDA ) ( Panel D . The relative level of bioR expression was measured in three independent experiments and is expressed in mean ± SD.

Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Mutagenesis

15) Product Images from "Clinical and genomic landscape of gastric cancer with a mesenchymal phenotype"

Article Title: Clinical and genomic landscape of gastric cancer with a mesenchymal phenotype

Journal: Nature Communications

doi: 10.1038/s41467-018-04179-8

IGF1/IGF1R pathway is therapeutic target for MP subtype. a Expression of IGF1 in gastric cancer cell lines. Expression was measured by qRT-PCR. b Western blot analyses in gastric cancer cell lines of MP subtype cells (MKN74 and SNU1) and EP subtype cells (MKN28 and MKN45) using antibodies against phosphorylated IGF1R (active), IGF1R (total), and β-actin. c Sensitivity of gastric cancer cell lines to linsitinib, inhibitor of IGF1R. Cell growth was measured at 96 h after treatment of linsitinib as indicated ( n = 4). d Growth of SNU1-derived xenograft tumors in mice treated with linsitinib or vehicle control. SNU1 cells were xenografted subcutaneously into the flanks of mice. At 10 days after xenografting, linsitinib or control tartaric acid was orally administrated to mice. Tumor volume was measured on the indicated days. Error bars indicate s.e.m. e Tumors harvested after treatment of linsitinib or control vehicle. f Tumor weight after treatment of linsitinib. At 25 days after linsitinib treatment, mice were killed and tumor weights were measured ( n = 8 or 9 per treatment). Data are presented with means. P values were obtained by Student’s t -test
Figure Legend Snippet: IGF1/IGF1R pathway is therapeutic target for MP subtype. a Expression of IGF1 in gastric cancer cell lines. Expression was measured by qRT-PCR. b Western blot analyses in gastric cancer cell lines of MP subtype cells (MKN74 and SNU1) and EP subtype cells (MKN28 and MKN45) using antibodies against phosphorylated IGF1R (active), IGF1R (total), and β-actin. c Sensitivity of gastric cancer cell lines to linsitinib, inhibitor of IGF1R. Cell growth was measured at 96 h after treatment of linsitinib as indicated ( n = 4). d Growth of SNU1-derived xenograft tumors in mice treated with linsitinib or vehicle control. SNU1 cells were xenografted subcutaneously into the flanks of mice. At 10 days after xenografting, linsitinib or control tartaric acid was orally administrated to mice. Tumor volume was measured on the indicated days. Error bars indicate s.e.m. e Tumors harvested after treatment of linsitinib or control vehicle. f Tumor weight after treatment of linsitinib. At 25 days after linsitinib treatment, mice were killed and tumor weights were measured ( n = 8 or 9 per treatment). Data are presented with means. P values were obtained by Student’s t -test

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Mouse Assay

16) Product Images from "Expression of Heat Shock Protein 70 Gene and Its Correlation with Inflammatory Markers in Essential Hypertension"

Article Title: Expression of Heat Shock Protein 70 Gene and Its Correlation with Inflammatory Markers in Essential Hypertension

Journal: PLoS ONE

doi: 10.1371/journal.pone.0151060

Semi-quantitative gel picture and Western blot analysis of Heat shock protein70. (A) represents the semi-quantitative analysis of heat shock protein70 (HSP70) gene and 18S rRNA (Housekeeping gene) in controls and patients respectively.(B) represents the western blots of heat shock protein70 (HSP70) and β-actin protein in controls and patients respectively.
Figure Legend Snippet: Semi-quantitative gel picture and Western blot analysis of Heat shock protein70. (A) represents the semi-quantitative analysis of heat shock protein70 (HSP70) gene and 18S rRNA (Housekeeping gene) in controls and patients respectively.(B) represents the western blots of heat shock protein70 (HSP70) and β-actin protein in controls and patients respectively.

Techniques Used: Western Blot

qRT-PCR analysis of Heat shock protein70 gene expressions at mRNA level and indirect ELISA analysis at protein level. (A) represents the scattered plot of levels of HSP70 gene expression at mRNA level in the individual study subjects by qRT-PCR. The relative HSP70 gene expression in patients and controls is expressed in delta-CT values. Results were expressed as densitometric ratio (HSP70 to 18S rRNA gene) in patients and controls group ± SD. P
Figure Legend Snippet: qRT-PCR analysis of Heat shock protein70 gene expressions at mRNA level and indirect ELISA analysis at protein level. (A) represents the scattered plot of levels of HSP70 gene expression at mRNA level in the individual study subjects by qRT-PCR. The relative HSP70 gene expression in patients and controls is expressed in delta-CT values. Results were expressed as densitometric ratio (HSP70 to 18S rRNA gene) in patients and controls group ± SD. P

Techniques Used: Quantitative RT-PCR, Indirect ELISA, Expressing

17) Product Images from "Transcriptome profiling of Camelina sativa to identify genes involved in triacylglycerol biosynthesis and accumulation in the developing seeds"

Article Title: Transcriptome profiling of Camelina sativa to identify genes involved in triacylglycerol biosynthesis and accumulation in the developing seeds

Journal: Biotechnology for Biofuels

doi: 10.1186/s13068-016-0555-5

Expression of TAG biosynthesis-associated genes in Camelina developing seeds and leaf measured by qRT-PCR. Relative combined expression of all three copies of CsWRl1, CsGPAT9, CsLPP1, CsLPP2, CsLPAT2, CsDGAT1, CsDGAT2, CsPDAT, CsWSD1, CsPDCT, CsOle1, CsOle4, and CsMGAT1. The descriptive gene names are available in supplemental tables. The bars represent the fold change in log2 scale as measured by real-time qPCR from cDNA derived from Camelina seeds at 10–15 (Cs-14) and 16–21 (Cs-21) days after flowering (DAF) and from leaf tissue (Cs-Leaf). The leaf sample was used as the calibrator for the remaining samples. Error bars represent the standard error ± SE of three biological replicates. The quantification of the genes is measured relative to the expression of the indigenous housekeeping gene β-actin
Figure Legend Snippet: Expression of TAG biosynthesis-associated genes in Camelina developing seeds and leaf measured by qRT-PCR. Relative combined expression of all three copies of CsWRl1, CsGPAT9, CsLPP1, CsLPP2, CsLPAT2, CsDGAT1, CsDGAT2, CsPDAT, CsWSD1, CsPDCT, CsOle1, CsOle4, and CsMGAT1. The descriptive gene names are available in supplemental tables. The bars represent the fold change in log2 scale as measured by real-time qPCR from cDNA derived from Camelina seeds at 10–15 (Cs-14) and 16–21 (Cs-21) days after flowering (DAF) and from leaf tissue (Cs-Leaf). The leaf sample was used as the calibrator for the remaining samples. Error bars represent the standard error ± SE of three biological replicates. The quantification of the genes is measured relative to the expression of the indigenous housekeeping gene β-actin

Techniques Used: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Derivative Assay

18) Product Images from "A New Mutation, hap1-2, Reveals a C Terminal Domain Function in AtMago Protein and Its Biological Effects in Male Gametophyte Development in Arabidopsis thaliana"

Article Title: A New Mutation, hap1-2, Reveals a C Terminal Domain Function in AtMago Protein and Its Biological Effects in Male Gametophyte Development in Arabidopsis thaliana

Journal: PLoS ONE

doi: 10.1371/journal.pone.0148200

Both AtMago and AtMagoΔC are detected in P-bodies. Scale bar = 50 μm. A zoom-in view is shown for each image with a scale bar of 10 μm.
Figure Legend Snippet: Both AtMago and AtMagoΔC are detected in P-bodies. Scale bar = 50 μm. A zoom-in view is shown for each image with a scale bar of 10 μm.

Techniques Used:

CTD of AtMago is required for AtMago-AtY14 interaction, but not for AtMago-AteIF4AIII. Western blots showing results from in vitro pull-down assay for AtMagoΔC-AtY14 (A), AtMagoΔC-AteIF4AIII (B), and various AtMago C terminal truncations-AtY14 (C).
Figure Legend Snippet: CTD of AtMago is required for AtMago-AtY14 interaction, but not for AtMago-AteIF4AIII. Western blots showing results from in vitro pull-down assay for AtMagoΔC-AtY14 (A), AtMagoΔC-AteIF4AIII (B), and various AtMago C terminal truncations-AtY14 (C).

Techniques Used: Western Blot, In Vitro, Pull Down Assay

Characterization and complementation of the HAP1/hap1-2 mutant. (A) HAP1 gene structure. Exons were shown as colored boxes; introns were shown as straight lines; UTRs were shown as gray boxes. The T-DNA insertion in hap1-1 and hap1-2 was indicated by red arrow. (B) rtPCR results showed the expression of a full-length transcript ( AtMago ), a truncated transcript ( AtMagoΔC ) and GAPDH in qrt , qrt;HAP1/hap1-2 , qrt;sr45-1 and qrt;upf3-1 mutant plants. Total RNAs were extracted from the inflorescence tissue. The exon composition of the transcript and the position of used primers were shown next to the corresponding rtPCR results. (C) GUS stained pollen grains from qrt , qrt;HAP1/hap1-1 and qrt;HAP1/hap1-2 showing the identity of mutant pollen grains. Scale bar = 20 μm. (D) Seed number per silique in qrt and qrt;HAP1/hap1-2 plants. Scale bar = 1 mm. The quantification was done with sixteen siliques. Student t-test was used for statistical analysis. Error bars represent standard deviations. (E) HAP1 gene complemented hap1-2 mutant phenotype. The mature plants, GUS-stained pollen grains and seed yield per plant were shown in qrt , qrt;HAP1/hap1-2 , HAP1;qrt; hap1-2 . (F) qPCR results showing that the AtMago gene expression level was fully recovered in the transgenic plant. GAPDH was used as control. Three biological replicates were used in the analysis. Error bar showed standard deviation. Statistical significance compared to qrt was measured by student t-test ( p
Figure Legend Snippet: Characterization and complementation of the HAP1/hap1-2 mutant. (A) HAP1 gene structure. Exons were shown as colored boxes; introns were shown as straight lines; UTRs were shown as gray boxes. The T-DNA insertion in hap1-1 and hap1-2 was indicated by red arrow. (B) rtPCR results showed the expression of a full-length transcript ( AtMago ), a truncated transcript ( AtMagoΔC ) and GAPDH in qrt , qrt;HAP1/hap1-2 , qrt;sr45-1 and qrt;upf3-1 mutant plants. Total RNAs were extracted from the inflorescence tissue. The exon composition of the transcript and the position of used primers were shown next to the corresponding rtPCR results. (C) GUS stained pollen grains from qrt , qrt;HAP1/hap1-1 and qrt;HAP1/hap1-2 showing the identity of mutant pollen grains. Scale bar = 20 μm. (D) Seed number per silique in qrt and qrt;HAP1/hap1-2 plants. Scale bar = 1 mm. The quantification was done with sixteen siliques. Student t-test was used for statistical analysis. Error bars represent standard deviations. (E) HAP1 gene complemented hap1-2 mutant phenotype. The mature plants, GUS-stained pollen grains and seed yield per plant were shown in qrt , qrt;HAP1/hap1-2 , HAP1;qrt; hap1-2 . (F) qPCR results showing that the AtMago gene expression level was fully recovered in the transgenic plant. GAPDH was used as control. Three biological replicates were used in the analysis. Error bar showed standard deviation. Statistical significance compared to qrt was measured by student t-test ( p

Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Real-time Polymerase Chain Reaction, Transgenic Assay, Standard Deviation

19) Product Images from "Regulation of colony stimulating factor-1 expression and ovarian cancer cell behavior in vitro by miR-128 and miR-152"

Article Title: Regulation of colony stimulating factor-1 expression and ovarian cancer cell behavior in vitro by miR-128 and miR-152

Journal: Molecular Cancer

doi: 10.1186/1476-4598-11-58

A) Splinted ligation of miR-128 in ovarian cancer cells. miR-128 RNA is detected in both NOSE.1 and Bix3 cells. B ) Splinted ligation of miR-152 in ovarian cancer cells. miR-152 RNA is detected in both Hey and SKOV3 cells. C ) miRNA expression pattern follows “host” gene expression pattern. R3HDM1 mRNA expression in ovarian cancer cells. D ) COPZ2 mRNA expression in ovarian cancer cells. GAPDH mRNA was used as a loading control in qRT-PCR. Bars represent mean ± SD from triplicate experiments.
Figure Legend Snippet: A) Splinted ligation of miR-128 in ovarian cancer cells. miR-128 RNA is detected in both NOSE.1 and Bix3 cells. B ) Splinted ligation of miR-152 in ovarian cancer cells. miR-152 RNA is detected in both Hey and SKOV3 cells. C ) miRNA expression pattern follows “host” gene expression pattern. R3HDM1 mRNA expression in ovarian cancer cells. D ) COPZ2 mRNA expression in ovarian cancer cells. GAPDH mRNA was used as a loading control in qRT-PCR. Bars represent mean ± SD from triplicate experiments.

Techniques Used: Ligation, Expressing, Quantitative RT-PCR

20) Product Images from "The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection"

Article Title: The glutamine synthetase of Trypanosoma cruzi is required for its resistance to ammonium accumulation and evasion of the parasitophorous vacuole during host-cell infection

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0006170

Expression profile of GS in T . cruzi . (A) Quantification of transcripts for the Tc GS gene in the different stages of T . cruzi . The mRNA was quantified by qRT-PCR using primers for a fragment of the gene. The Tc GAPDH gene was used as a normalizer (kept in-house). The expression ratio was calculated by the 2 -ΔΔCt method, in which E stage expression was defined as 1. The cDNA of CHO-K 1 cells was used as a negative control since this strain is used to obtain the intracellular forms of T . cruzi . The differences between the expression profiles were evaluated by a one-way ANOVA test (p
Figure Legend Snippet: Expression profile of GS in T . cruzi . (A) Quantification of transcripts for the Tc GS gene in the different stages of T . cruzi . The mRNA was quantified by qRT-PCR using primers for a fragment of the gene. The Tc GAPDH gene was used as a normalizer (kept in-house). The expression ratio was calculated by the 2 -ΔΔCt method, in which E stage expression was defined as 1. The cDNA of CHO-K 1 cells was used as a negative control since this strain is used to obtain the intracellular forms of T . cruzi . The differences between the expression profiles were evaluated by a one-way ANOVA test (p

Techniques Used: Expressing, Quantitative RT-PCR, Negative Control

21) Product Images from "The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis"

Article Title: The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18030576

GSDMB alternative splicing (AS) pattern. ( a ) Schematic representation of the GSDMB gene. Exons are represented by boxes and are approximately drawn to scale; untranslated regions (UTRs) are depicted by smaller boxes; introns are represented by lines. Constitutive exons are colored in light grey and alternative exons in dark grey. The three reverse-transcription (RT)-PCR assays (A, B, and C) performed to cover the GSDMB coding region are indicated; ( b ) An illustrative agarose gel (2%) shows the amplification products of the three RT-PCR assays. For “A” and “C” assays, the unique amplification product and their length, expressed as base pairs (bp), are schematized on the left. For the “B” assay, multiple specific amplification products were obtained, corresponding to several AS events involving exons 4 to 9, which were further analyzed by both fluorescent RT-PCR and direct sequencing (see further). MW: molecular-weight marker (pUC9- Hae III); ( c ) The left panel represents a demonstrative GeneMapper window showing the fluorescent RT-PCR products of the “B” assay (on RNA extracted from a healthy individual, heterozygous for the rs11078928 polymorphism), using the forward primer labelled with the HEX fluorophore. The filled peaks, shaded in grey, correspond to the RT-PCR products; empty peaks represent the size standard (ROX-500 HD). The vertical axis indicates the length of amplified products in bp, whereas the horizontal axis shows fluorescence units. The schematic representation and the name of the corresponding isoform product are indicated on the right side. The alternative exon 6* is depicted in black. F”: full length, “*” indicates the presence of exon 6* in the isoform, “∆” is followed by the skipped exon, separated by “-” or “,” if the exons are contiguous or not, respectively. The accession number of University of California, Santa Cruz (UCSC) Genome Browser-annotated isoforms is also indicated. The presence/absence in the transcript of a premature termination codon (PTC) is indicated by a “+”/“−”, respectively.
Figure Legend Snippet: GSDMB alternative splicing (AS) pattern. ( a ) Schematic representation of the GSDMB gene. Exons are represented by boxes and are approximately drawn to scale; untranslated regions (UTRs) are depicted by smaller boxes; introns are represented by lines. Constitutive exons are colored in light grey and alternative exons in dark grey. The three reverse-transcription (RT)-PCR assays (A, B, and C) performed to cover the GSDMB coding region are indicated; ( b ) An illustrative agarose gel (2%) shows the amplification products of the three RT-PCR assays. For “A” and “C” assays, the unique amplification product and their length, expressed as base pairs (bp), are schematized on the left. For the “B” assay, multiple specific amplification products were obtained, corresponding to several AS events involving exons 4 to 9, which were further analyzed by both fluorescent RT-PCR and direct sequencing (see further). MW: molecular-weight marker (pUC9- Hae III); ( c ) The left panel represents a demonstrative GeneMapper window showing the fluorescent RT-PCR products of the “B” assay (on RNA extracted from a healthy individual, heterozygous for the rs11078928 polymorphism), using the forward primer labelled with the HEX fluorophore. The filled peaks, shaded in grey, correspond to the RT-PCR products; empty peaks represent the size standard (ROX-500 HD). The vertical axis indicates the length of amplified products in bp, whereas the horizontal axis shows fluorescence units. The schematic representation and the name of the corresponding isoform product are indicated on the right side. The alternative exon 6* is depicted in black. F”: full length, “*” indicates the presence of exon 6* in the isoform, “∆” is followed by the skipped exon, separated by “-” or “,” if the exons are contiguous or not, respectively. The accession number of University of California, Santa Cruz (UCSC) Genome Browser-annotated isoforms is also indicated. The presence/absence in the transcript of a premature termination codon (PTC) is indicated by a “+”/“−”, respectively.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Sequencing, Molecular Weight, Marker, Fluorescence

Analysis of exon 6/6* AS in multiple sclerosis (MS) cases and healthy controls. ( a ) Schematic representation of the GSDMB genomic region comprised between exons 4 and 8. Exons are approximately drawn to scale; alternative exons are depicted in dark grey, as in Figure 1 . The dashed arrows below the scheme indicate the primer couple used in the fluorescent-competitive RT-PCR assay; the forward primer is labelled with the HEX fluorophore; ( b ) The left panel shows three GeneMapper windows, representing an example of the fluorescent products obtained for each of the rs11078928 genotypes. The peaks shaded in grey correspond to the RT-PCR products; those not shaded represent the size standard (ROX-500 HD). The schematic representation of the obtained products for each genotype is shown on the right. Exon 6* is depicted in black; ( c ) Boxplots showing the percentage of the Δ6 isoform with respect to the sum of Δ6, F*, and F isoforms measured on RNA extracted from 30 relapsing remitting (RR)-MS cases and 30 controls, grouped on the basis of the rs11078928 genotype. Boxes define the interquartile range; the thick line refers to the median. The number of subjects belonging to each group is also indicated (N). Significance levels of t -tests is shown above the boxplots (** p
Figure Legend Snippet: Analysis of exon 6/6* AS in multiple sclerosis (MS) cases and healthy controls. ( a ) Schematic representation of the GSDMB genomic region comprised between exons 4 and 8. Exons are approximately drawn to scale; alternative exons are depicted in dark grey, as in Figure 1 . The dashed arrows below the scheme indicate the primer couple used in the fluorescent-competitive RT-PCR assay; the forward primer is labelled with the HEX fluorophore; ( b ) The left panel shows three GeneMapper windows, representing an example of the fluorescent products obtained for each of the rs11078928 genotypes. The peaks shaded in grey correspond to the RT-PCR products; those not shaded represent the size standard (ROX-500 HD). The schematic representation of the obtained products for each genotype is shown on the right. Exon 6* is depicted in black; ( c ) Boxplots showing the percentage of the Δ6 isoform with respect to the sum of Δ6, F*, and F isoforms measured on RNA extracted from 30 relapsing remitting (RR)-MS cases and 30 controls, grouped on the basis of the rs11078928 genotype. Boxes define the interquartile range; the thick line refers to the median. The number of subjects belonging to each group is also indicated (N). Significance levels of t -tests is shown above the boxplots (** p

Techniques Used: Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction

Characterization of the GSDMB exonic circular RNA (ecircRNA). ( a ) Schematic representation of the formation of the GSDMB ecircRNA through a backsplicing event between exons 4 and 5. Exons are approximately drawn to scale; exon 4 is depicted in grey, exon 5 in black. The curved arrow joins the 5′ splice site of exon 5 to 3′ splice site of exon 4. Arrows below exon 5 indicate the divergent primer couple used to detect the putative circular RNA (circRNA). On the right, a schematic representation of the circRNA is depicted, with the circular black line representing the product amplified by the primer couple. Below the scheme, direct-sequencing electropherograms show the head-to-tail splice junction, indicated by an arrow, located between GSDMB exons 5 and 4; ( b ) Schematic representation of the linear splicing involving exons 4–5. Arrows below exons 4–5 indicate the primer couple used in RT-PCR assays. Electropherograms showing the junction between exons 4 and 5 is also reported; ( c ) Agarose gel (2%) with the results of the RNase R treatment. The expression of the GSDMB ecircRNA was evaluated by RT-PCR in untreated (−) or RNase R-treated (+) RNA of a healthy control, using the divergent primer couple within exon 5. Expression of total GSDMB linear mRNA was also evaluated, using the RT-PCR assay shown in Figure 2 (linear product; exons 9–11), the assay reported in ( b ) (linear product; exons 4 and 5), as well as an assay using a divergent primer couple on exon 10 (negative control; no circRNA detected). MW: molecular-weight marker (pUC9- Hae III); ( d ) Boxplots show expression levels of the GSDMB ecircRNA measured by semi-quantitative real-time RT-PCR in PBMCs of 30 MS cases and 30 healthy controls. Boxes define the interquartile range; the thick line refers to the median. Results were normalized to expression levels of the HMBS housekeeping gene. The number of subjects belonging to each group is also indicated (N). The significance level of t -test analysis is shown. ** p
Figure Legend Snippet: Characterization of the GSDMB exonic circular RNA (ecircRNA). ( a ) Schematic representation of the formation of the GSDMB ecircRNA through a backsplicing event between exons 4 and 5. Exons are approximately drawn to scale; exon 4 is depicted in grey, exon 5 in black. The curved arrow joins the 5′ splice site of exon 5 to 3′ splice site of exon 4. Arrows below exon 5 indicate the divergent primer couple used to detect the putative circular RNA (circRNA). On the right, a schematic representation of the circRNA is depicted, with the circular black line representing the product amplified by the primer couple. Below the scheme, direct-sequencing electropherograms show the head-to-tail splice junction, indicated by an arrow, located between GSDMB exons 5 and 4; ( b ) Schematic representation of the linear splicing involving exons 4–5. Arrows below exons 4–5 indicate the primer couple used in RT-PCR assays. Electropherograms showing the junction between exons 4 and 5 is also reported; ( c ) Agarose gel (2%) with the results of the RNase R treatment. The expression of the GSDMB ecircRNA was evaluated by RT-PCR in untreated (−) or RNase R-treated (+) RNA of a healthy control, using the divergent primer couple within exon 5. Expression of total GSDMB linear mRNA was also evaluated, using the RT-PCR assay shown in Figure 2 (linear product; exons 9–11), the assay reported in ( b ) (linear product; exons 4 and 5), as well as an assay using a divergent primer couple on exon 10 (negative control; no circRNA detected). MW: molecular-weight marker (pUC9- Hae III); ( d ) Boxplots show expression levels of the GSDMB ecircRNA measured by semi-quantitative real-time RT-PCR in PBMCs of 30 MS cases and 30 healthy controls. Boxes define the interquartile range; the thick line refers to the median. Results were normalized to expression levels of the HMBS housekeeping gene. The number of subjects belonging to each group is also indicated (N). The significance level of t -test analysis is shown. ** p

Techniques Used: Amplification, Sequencing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Negative Control, Molecular Weight, Marker, Quantitative RT-PCR, Mass Spectrometry

Evaluation of GSDMB susceptibility to nonsense-mediated mRNA decay (NMD). The upper panel shows a partial scheme of the GSDMB gene and the position of the primer couple used in the semi-quantitative real-time RT-PCR assay. The lower panel represents GSDMB total expression levels in HEK293 and HepG2 cells lines untreated (NT) or treated for 8 h with cycloheximide. Expression levels of two PRKCA transcripts, sensitive and insensitive to NMD, are also shown. The expression level of the untreated sample was set to 1. Bars represent means + SEM (standard error of the mean) of three independent experiments, each performed in triplicate. Significance levels of t -tests are shown. ** p
Figure Legend Snippet: Evaluation of GSDMB susceptibility to nonsense-mediated mRNA decay (NMD). The upper panel shows a partial scheme of the GSDMB gene and the position of the primer couple used in the semi-quantitative real-time RT-PCR assay. The lower panel represents GSDMB total expression levels in HEK293 and HepG2 cells lines untreated (NT) or treated for 8 h with cycloheximide. Expression levels of two PRKCA transcripts, sensitive and insensitive to NMD, are also shown. The expression level of the untreated sample was set to 1. Bars represent means + SEM (standard error of the mean) of three independent experiments, each performed in triplicate. Significance levels of t -tests are shown. ** p

Techniques Used: Quantitative RT-PCR, Expressing

22) Product Images from "Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction"

Article Title: Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction

Journal: Oncotarget

doi: 10.18632/oncotarget.11896

Human prostate cancer cells express LLT1 A. mRNA expression of LLT1 on prostate cancer cell lines PC3, DU145, LNCaP, 22Rv1, normal prostate cell PWR-1E and Jurkat (T cell line) was determined by qRT-PCR. LLT1 expression was determined by using LLT1 sequence specific primers and Taqman gene expression assays in an Eppendorf Realplex2 Mastercycler. Reactions were done in 20 μl triplicates using the ΔΔCT method, with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. Each bar represents a mean ± s.e. of three independent experiments. B. LLT1 protein expression was analyzed by Western blotting in a panel of prostate cancer cell lines including leukemic Jurkat cells. GAPDH was used as a loading control. C. A bar graph showing densitometric analysis of LLT1 protein expression normalized to GAPDH. Each bar represents the mean ± s.e. of three independent experiments.
Figure Legend Snippet: Human prostate cancer cells express LLT1 A. mRNA expression of LLT1 on prostate cancer cell lines PC3, DU145, LNCaP, 22Rv1, normal prostate cell PWR-1E and Jurkat (T cell line) was determined by qRT-PCR. LLT1 expression was determined by using LLT1 sequence specific primers and Taqman gene expression assays in an Eppendorf Realplex2 Mastercycler. Reactions were done in 20 μl triplicates using the ΔΔCT method, with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. Each bar represents a mean ± s.e. of three independent experiments. B. LLT1 protein expression was analyzed by Western blotting in a panel of prostate cancer cell lines including leukemic Jurkat cells. GAPDH was used as a loading control. C. A bar graph showing densitometric analysis of LLT1 protein expression normalized to GAPDH. Each bar represents the mean ± s.e. of three independent experiments.

Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Western Blot

23) Product Images from "Characterization of Cyclooxygenase-2 and its induction pathways in response to high lipid diet-induced inflammation in Larmichthys crocea"

Article Title: Characterization of Cyclooxygenase-2 and its induction pathways in response to high lipid diet-induced inflammation in Larmichthys crocea

Journal: Scientific Reports

doi: 10.1038/srep19921

Relative cox-2 , TNFα and IL-1β mRNA levels. Relative cox-2 , TNFα and IL-1β mRNA levels were evaluated by quantitative realtime PCR (qRT-PCR) and expressed relative to β-actin levels in the liver of experimental fish. Results are expressed as means ± S.E.M. (n = 3). * P
Figure Legend Snippet: Relative cox-2 , TNFα and IL-1β mRNA levels. Relative cox-2 , TNFα and IL-1β mRNA levels were evaluated by quantitative realtime PCR (qRT-PCR) and expressed relative to β-actin levels in the liver of experimental fish. Results are expressed as means ± S.E.M. (n = 3). * P

Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR, Fluorescence In Situ Hybridization

Tissue distribution of cox-2 in large yellow croaker. Relative cox-2 mRNA expression was determined by quantitative realtime PCR (qRT-PCR) and expressed relative to β-actin levels. Results are expressed as means ± S.E.M. (n = 3). Different letters above the bars denote significant differences among tissues at the P
Figure Legend Snippet: Tissue distribution of cox-2 in large yellow croaker. Relative cox-2 mRNA expression was determined by quantitative realtime PCR (qRT-PCR) and expressed relative to β-actin levels. Results are expressed as means ± S.E.M. (n = 3). Different letters above the bars denote significant differences among tissues at the P

Techniques Used: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR

24) Product Images from "Adiponectin modulates focal adhesion disassembly in activated hepatic stellate cells: implication for reversing hepatic fibrosis"

Article Title: Adiponectin modulates focal adhesion disassembly in activated hepatic stellate cells: implication for reversing hepatic fibrosis

Journal: The FASEB Journal

doi: 10.1096/fj.14-253229

Adiponectin attenuates CCl 4 -induced hepatic fibrosis in vivo. A ) Sirius red staining of collagen in liver sections from mice gavaged with CCl 4 followed by Ad-Adn delivery via tail vein. B ) Staining was quantified, and densitometric analysis was performed (original view ×40). C ) Serum aspartate aminotransferase (AST) levels from all 8 groups of mice. D ) Hepatic mRNA expression of integrin αv, β3, β1, vinculin, α-SMA, and α2(I) collagen by qRT-PCR. E ) Representative Western blot analysis of lysates obtained from liver tissues of WT and Ad −/− mice. * P
Figure Legend Snippet: Adiponectin attenuates CCl 4 -induced hepatic fibrosis in vivo. A ) Sirius red staining of collagen in liver sections from mice gavaged with CCl 4 followed by Ad-Adn delivery via tail vein. B ) Staining was quantified, and densitometric analysis was performed (original view ×40). C ) Serum aspartate aminotransferase (AST) levels from all 8 groups of mice. D ) Hepatic mRNA expression of integrin αv, β3, β1, vinculin, α-SMA, and α2(I) collagen by qRT-PCR. E ) Representative Western blot analysis of lysates obtained from liver tissues of WT and Ad −/− mice. * P

Techniques Used: In Vivo, Staining, Mouse Assay, AST Assay, Expressing, Quantitative RT-PCR, Western Blot

25) Product Images from "Potential Role of Hedgehog Pathway in Liver Response to Radiation"

Article Title: Potential Role of Hedgehog Pathway in Liver Response to Radiation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074141

Enhanced activation of the Hh signaling pathway in the liver post irradiation. (A) QRT-PCR analysis of liver mRNA from radiation-treated mice for ihh, shh, smo, and gli2 ((n ≥3 mice/group) Mean±SD results are graphed. (B) and (C). Western blot analysis of Ihh, Shh, Smo, and Gli2 (GAPDH was used as an internal control). Data shown represent one of three experiments with similar results (B: Immuoblot/C: Band density) (n ≥3 mice/group). Data represent the mean ± SD of three independent experiments (*p
Figure Legend Snippet: Enhanced activation of the Hh signaling pathway in the liver post irradiation. (A) QRT-PCR analysis of liver mRNA from radiation-treated mice for ihh, shh, smo, and gli2 ((n ≥3 mice/group) Mean±SD results are graphed. (B) and (C). Western blot analysis of Ihh, Shh, Smo, and Gli2 (GAPDH was used as an internal control). Data shown represent one of three experiments with similar results (B: Immuoblot/C: Band density) (n ≥3 mice/group). Data represent the mean ± SD of three independent experiments (*p

Techniques Used: Activation Assay, Irradiation, Quantitative RT-PCR, Mouse Assay, Western Blot

Hh inhibitor, GDC-0449, blocks hepatic Hh activity in the irradiated mice. (A) H E staining shows less fat accumulation in hepatocytes in liver from representative irradiated mice with GDC-0449 (IR+GDC) (X40). (B) Relative liver weight/body weight of mice. (C) The values of AST and ALT are graphed. (D) QRT-PCR analysis of liver mRNA from DMSO (DMSO), radiation treated mice with (IR+GDC) or without GDC-0449 (IR+DMSO) for smo, and gli2 ((n ≥4 mice/group). Mean±SD results are graphed. (E) and (F). Western blot analysis of Smo, and Gli2 (GAPDH was used as an internal control). Data shown represent one of three experiments with similar results (E: Immuoblot/F: Band density) (n ≥4 mice/group). Data represent the mean ± SD of three independent experiments (*p
Figure Legend Snippet: Hh inhibitor, GDC-0449, blocks hepatic Hh activity in the irradiated mice. (A) H E staining shows less fat accumulation in hepatocytes in liver from representative irradiated mice with GDC-0449 (IR+GDC) (X40). (B) Relative liver weight/body weight of mice. (C) The values of AST and ALT are graphed. (D) QRT-PCR analysis of liver mRNA from DMSO (DMSO), radiation treated mice with (IR+GDC) or without GDC-0449 (IR+DMSO) for smo, and gli2 ((n ≥4 mice/group). Mean±SD results are graphed. (E) and (F). Western blot analysis of Smo, and Gli2 (GAPDH was used as an internal control). Data shown represent one of three experiments with similar results (E: Immuoblot/F: Band density) (n ≥4 mice/group). Data represent the mean ± SD of three independent experiments (*p

Techniques Used: Activity Assay, Irradiation, Mouse Assay, Staining, AST Assay, Quantitative RT-PCR, Western Blot

Increased fibrosis and EMT in the irradiated liver. (A) QRT-PCR analysis of liver mRNA from radiation-treated mice for tfg- β, α -sma, collagen a1, n-cadherin, s100a4, and bmp7(n ≥3 mice/group). Mean±SD results are graphed. (B) (C). Western blot analysis of TGF-β (25 kDa: processed form) (inducer of fibrosis) and α-SMA (fibrogenic marker) expression (GAPDH was used as an internal control) (n≥3 mice/group). Data shown represent one of three experiments with similar results (B: Immunoblot/C: Band density of TGF-β and α-SMA). Data represent the mean ± SD of three independent experiments. (D) Sirius red staining in liver sections from representative control and irradiated mice (X40) (*p
Figure Legend Snippet: Increased fibrosis and EMT in the irradiated liver. (A) QRT-PCR analysis of liver mRNA from radiation-treated mice for tfg- β, α -sma, collagen a1, n-cadherin, s100a4, and bmp7(n ≥3 mice/group). Mean±SD results are graphed. (B) (C). Western blot analysis of TGF-β (25 kDa: processed form) (inducer of fibrosis) and α-SMA (fibrogenic marker) expression (GAPDH was used as an internal control) (n≥3 mice/group). Data shown represent one of three experiments with similar results (B: Immunoblot/C: Band density of TGF-β and α-SMA). Data represent the mean ± SD of three independent experiments. (D) Sirius red staining in liver sections from representative control and irradiated mice (X40) (*p

Techniques Used: Irradiation, Quantitative RT-PCR, Mouse Assay, Western Blot, Marker, Expressing, Staining

GDC-0449 treatment reduces the expression of EMT-stimulating genes in irradiated livers. QRT-PCR analysis of liver mRNA from radiation-treated mice for tgf-β, α-sma, collagen α1, n-cadherin, s100a4, and bmp7(n ≥4 mice/group). Mean±SD results are graphed (*p
Figure Legend Snippet: GDC-0449 treatment reduces the expression of EMT-stimulating genes in irradiated livers. QRT-PCR analysis of liver mRNA from radiation-treated mice for tgf-β, α-sma, collagen α1, n-cadherin, s100a4, and bmp7(n ≥4 mice/group). Mean±SD results are graphed (*p

Techniques Used: Expressing, Irradiation, Quantitative RT-PCR, Mouse Assay

26) Product Images from "Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species – A proof-of-concept using Alatospora pulchella"

Article Title: Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species – A proof-of-concept using Alatospora pulchella

Journal: PLoS ONE

doi: 10.1371/journal.pone.0174634

1a and 1b. Fluorescence profiles for the 6-carboxy-fluorescein fluorophore (FAM) as a function of PCR cycle and time. Each tested species and the negative controls are represented by a different color. A signal was classified as true positive if it was substantially higher than the initial fluorescence baseline [ 39 ], which was set automatically and individually for each sample by the qPCR device. Please note that the higher Ct value of A . pulchella in Fig 1b resulted from an extract with less template DNA.
Figure Legend Snippet: 1a and 1b. Fluorescence profiles for the 6-carboxy-fluorescein fluorophore (FAM) as a function of PCR cycle and time. Each tested species and the negative controls are represented by a different color. A signal was classified as true positive if it was substantially higher than the initial fluorescence baseline [ 39 ], which was set automatically and individually for each sample by the qPCR device. Please note that the higher Ct value of A . pulchella in Fig 1b resulted from an extract with less template DNA.

Techniques Used: Fluorescence, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

27) Product Images from "Retrovirus-Mediated Expression of E2A-PBX1 Blocks Lymphoid Fate but Permits Retention of Myeloid Potential in Early Hematopoietic Progenitors"

Article Title: Retrovirus-Mediated Expression of E2A-PBX1 Blocks Lymphoid Fate but Permits Retention of Myeloid Potential in Early Hematopoietic Progenitors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0130495

Ectopic expression of Hoxa9 , but not Bmi1 , in fetal liver progenitors antagonizes B-lymphoid differentiation. (A) Analysis by qRT-PCR of Hoxa9 and Bmi1 gene expression in E2A-PBX1- versus vector-infected FLPs. Values for each gene are expressed relative to expression in vector-infected cells. (B) Immunophenotype of FLPs transduced with Hoxa9 - versus Bmi1 -expressing retroviruses. Lin - FLPs were transduced with retroviruses expressing Bmi1 or Hoxa9 and then cultured under B-lymphoid conditions. Cells were analyzed by flow cytometry on day 14 post-infection.
Figure Legend Snippet: Ectopic expression of Hoxa9 , but not Bmi1 , in fetal liver progenitors antagonizes B-lymphoid differentiation. (A) Analysis by qRT-PCR of Hoxa9 and Bmi1 gene expression in E2A-PBX1- versus vector-infected FLPs. Values for each gene are expressed relative to expression in vector-infected cells. (B) Immunophenotype of FLPs transduced with Hoxa9 - versus Bmi1 -expressing retroviruses. Lin - FLPs were transduced with retroviruses expressing Bmi1 or Hoxa9 and then cultured under B-lymphoid conditions. Cells were analyzed by flow cytometry on day 14 post-infection.

Techniques Used: Expressing, Quantitative RT-PCR, Plasmid Preparation, Infection, Transduction, Cell Culture, Flow Cytometry, Cytometry

Forced expression of E2A-PBX in FLPs deregulates hematopoietic genes. (A) Hierarchical cluster analysis performed on expression data from gene expression microarrays indicates that the closest relationship to E2A-PBX1-expressing FLPs is with pro-B-cells that were differentiated from FLPs in vitro . (B) Confirmation by qRT-PCR of differential expression of genes identified initially using expression microarrays in E2A-PBX-expressing versus pro-B-cells derived from vector-infected FLPs. Data are from 2 independent samples. Error bars indicate one standard deviation; p-values are less than 0.005 for each comparison.
Figure Legend Snippet: Forced expression of E2A-PBX in FLPs deregulates hematopoietic genes. (A) Hierarchical cluster analysis performed on expression data from gene expression microarrays indicates that the closest relationship to E2A-PBX1-expressing FLPs is with pro-B-cells that were differentiated from FLPs in vitro . (B) Confirmation by qRT-PCR of differential expression of genes identified initially using expression microarrays in E2A-PBX-expressing versus pro-B-cells derived from vector-infected FLPs. Data are from 2 independent samples. Error bars indicate one standard deviation; p-values are less than 0.005 for each comparison.

Techniques Used: Expressing, In Vitro, Quantitative RT-PCR, Derivative Assay, Plasmid Preparation, Infection, Standard Deviation

B-lymphopoiesis is blocked at a point prior to the CLP stage in FLPs expressing E2A-PBX1. (A) Immunoblotting indicates markedly reduced or absent expression of the B-lymphopoietic transcription factors Ebf1 and Pax5 in FLPs expressing E2A-PBX1 relative to those infected with the empty retroviral vector. The apparent weak signal for Pax5 in the E2A-PBX1 lane may represent spillover between lanes. Anti-γ-tubulin serves as a loading control. (B) ChIP-qPCR results obtained from chromatin prepared from control, vector-infected FLPs indicate prevalence of the activating H3K4me3 mark relative to the silencing H3K27me3 mark at the Ebf1 and Pax5 promoters. Error bars represent 95% confidence limits. (C) Results from qRT-PCR indicating marked reduction or absence of the Ebf1 and Pax5 transcripts in FLPs expressing E2A-PBX1 relative to control cells. The asterisk indicates failure to observe a PCR product in either of the 2 independent samples. Error bars indicate one standard deviation. (D) ChIP-qPCR results obtained from chromatin prepared from FLPs expressing E2A-PBX1 indicate prevalence of the silencing H3K27me3 mark relative to the activating H3K4me3 mark at the Ebf1 and Pax5 promoters.
Figure Legend Snippet: B-lymphopoiesis is blocked at a point prior to the CLP stage in FLPs expressing E2A-PBX1. (A) Immunoblotting indicates markedly reduced or absent expression of the B-lymphopoietic transcription factors Ebf1 and Pax5 in FLPs expressing E2A-PBX1 relative to those infected with the empty retroviral vector. The apparent weak signal for Pax5 in the E2A-PBX1 lane may represent spillover between lanes. Anti-γ-tubulin serves as a loading control. (B) ChIP-qPCR results obtained from chromatin prepared from control, vector-infected FLPs indicate prevalence of the activating H3K4me3 mark relative to the silencing H3K27me3 mark at the Ebf1 and Pax5 promoters. Error bars represent 95% confidence limits. (C) Results from qRT-PCR indicating marked reduction or absence of the Ebf1 and Pax5 transcripts in FLPs expressing E2A-PBX1 relative to control cells. The asterisk indicates failure to observe a PCR product in either of the 2 independent samples. Error bars indicate one standard deviation. (D) ChIP-qPCR results obtained from chromatin prepared from FLPs expressing E2A-PBX1 indicate prevalence of the silencing H3K27me3 mark relative to the activating H3K4me3 mark at the Ebf1 and Pax5 promoters.

Techniques Used: Expressing, Infection, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Polymerase Chain Reaction, Standard Deviation

28) Product Images from "Tumor Cell-Derived Extracellular Vesicle-Coated Nanocarriers: An Efficient Theranostic Platform for the Cancer-Specific Delivery of Anti-miR-21 and Imaging Agents"

Article Title: Tumor Cell-Derived Extracellular Vesicle-Coated Nanocarriers: An Efficient Theranostic Platform for the Cancer-Specific Delivery of Anti-miR-21 and Imaging Agents

Journal: ACS nano

doi: 10.1021/acsnano.8b02587

TEVs preparation and characterization. Fluorescence microscopy images showing: (a) transient transfection of Cy5-anti-miRNA-21 into the donor 4T1 cells; (b) Cy5-anti-miRNA-21-packed donor 4T1 cell derived TEVs; (c) flow-cytometry analysis of Cy5-anti-miRNA-21 packed donor 4T1 cell derived TEVs; (d) size distribution of TEVs packed with Cy5-anti-miRNA-21 measured by NTA; (e) transmission electron microscopy image of 4T1-TEVs (scale bar: 50 nm); (f) fluorescence microscopy images showing 4T1-TEVs packed with Cy5-antimiRNA- 21 (red), labeled with DiO (green), and merged images of DiO-labeled TEVs packed with Cy5-anti-miRNA-21; (g) quantification of packed anti-miRNA-21 and endogenous miRNA-21 in donor and naive 4T1 cell derived TEVs by qRT-PCR; (h) quantification of selective endogenous miRNAs profile in TEVs purified from donor 4T1 cells; (i) quantification of endogenous miRNA-21 level in 4T1 cells delivered by different concentrations of Cy5-anti-miRNA-21 TEVs; (j) quantification of endogenous anti-miRNA-21 level in 4T1 cells delivered by different concentration of Cy5-anti-miRNA-21 TEVs; and (k) Western blot analysis showing the expression of therapeutic miRNA target proteins (PTEN, PDCD4, and Bcl2) in 4T1 cells transfected with Cy5-anti-miRNA-21 TEVs.
Figure Legend Snippet: TEVs preparation and characterization. Fluorescence microscopy images showing: (a) transient transfection of Cy5-anti-miRNA-21 into the donor 4T1 cells; (b) Cy5-anti-miRNA-21-packed donor 4T1 cell derived TEVs; (c) flow-cytometry analysis of Cy5-anti-miRNA-21 packed donor 4T1 cell derived TEVs; (d) size distribution of TEVs packed with Cy5-anti-miRNA-21 measured by NTA; (e) transmission electron microscopy image of 4T1-TEVs (scale bar: 50 nm); (f) fluorescence microscopy images showing 4T1-TEVs packed with Cy5-antimiRNA- 21 (red), labeled with DiO (green), and merged images of DiO-labeled TEVs packed with Cy5-anti-miRNA-21; (g) quantification of packed anti-miRNA-21 and endogenous miRNA-21 in donor and naive 4T1 cell derived TEVs by qRT-PCR; (h) quantification of selective endogenous miRNAs profile in TEVs purified from donor 4T1 cells; (i) quantification of endogenous miRNA-21 level in 4T1 cells delivered by different concentrations of Cy5-anti-miRNA-21 TEVs; (j) quantification of endogenous anti-miRNA-21 level in 4T1 cells delivered by different concentration of Cy5-anti-miRNA-21 TEVs; and (k) Western blot analysis showing the expression of therapeutic miRNA target proteins (PTEN, PDCD4, and Bcl2) in 4T1 cells transfected with Cy5-anti-miRNA-21 TEVs.

Techniques Used: Fluorescence, Microscopy, Transfection, Derivative Assay, Flow Cytometry, Cytometry, Transmission Assay, Electron Microscopy, Labeling, Quantitative RT-PCR, Purification, Concentration Assay, Western Blot, Expressing

Evaluation of anti-miR-21 transfection and loading efficiencies in transfected 4T1 cells, TEVs isolated from 4T1 cells, TEVs after extrusion with and without GIONs, and from 4T1 transfected with TEVs and TEV-GIONs as absolute copy numbers. (a) Standard plot of antimiR-21 in different copy numbers and the corresponding fitting curve derived by gene specific qRT-PCR amplification. (b) Schematic illustration explaining different stages of the procedure in which anti-miR-21 copy numbers were quantified for evaluation. (c) qRT-PCR analysis showing the amplification efficiency of anti-miR-21 isolated at different stages of TEVs processing. (d)A summarized table showing the quantitative values of the absolute number of copies of anti-miR-21 estimated in TEVs and TEV-GIONs at different stages of their evaluation.
Figure Legend Snippet: Evaluation of anti-miR-21 transfection and loading efficiencies in transfected 4T1 cells, TEVs isolated from 4T1 cells, TEVs after extrusion with and without GIONs, and from 4T1 transfected with TEVs and TEV-GIONs as absolute copy numbers. (a) Standard plot of antimiR-21 in different copy numbers and the corresponding fitting curve derived by gene specific qRT-PCR amplification. (b) Schematic illustration explaining different stages of the procedure in which anti-miR-21 copy numbers were quantified for evaluation. (c) qRT-PCR analysis showing the amplification efficiency of anti-miR-21 isolated at different stages of TEVs processing. (d)A summarized table showing the quantitative values of the absolute number of copies of anti-miR-21 estimated in TEVs and TEV-GIONs at different stages of their evaluation.

Techniques Used: Transfection, Isolation, Derivative Assay, Quantitative RT-PCR, Amplification

29) Product Images from "Transcriptome profiling of Camelina sativa to identify genes involved in triacylglycerol biosynthesis and accumulation in the developing seeds"

Article Title: Transcriptome profiling of Camelina sativa to identify genes involved in triacylglycerol biosynthesis and accumulation in the developing seeds

Journal: Biotechnology for Biofuels

doi: 10.1186/s13068-016-0555-5

Expression of TAG biosynthesis-associated genes in Camelina developing seeds and leaf measured by qRT-PCR. Relative combined expression of all three copies of CsWRl1, CsGPAT9, CsLPP1, CsLPP2, CsLPAT2, CsDGAT1, CsDGAT2, CsPDAT, CsWSD1, CsPDCT, CsOle1, CsOle4, and CsMGAT1. The descriptive gene names are available in supplemental tables. The bars represent the fold change in log2 scale as measured by real-time qPCR from cDNA derived from Camelina seeds at 10–15 (Cs-14) and 16–21 (Cs-21) days after flowering (DAF) and from leaf tissue (Cs-Leaf). The leaf sample was used as the calibrator for the remaining samples. Error bars represent the standard error ± SE of three biological replicates. The quantification of the genes is measured relative to the expression of the indigenous housekeeping gene β-actin
Figure Legend Snippet: Expression of TAG biosynthesis-associated genes in Camelina developing seeds and leaf measured by qRT-PCR. Relative combined expression of all three copies of CsWRl1, CsGPAT9, CsLPP1, CsLPP2, CsLPAT2, CsDGAT1, CsDGAT2, CsPDAT, CsWSD1, CsPDCT, CsOle1, CsOle4, and CsMGAT1. The descriptive gene names are available in supplemental tables. The bars represent the fold change in log2 scale as measured by real-time qPCR from cDNA derived from Camelina seeds at 10–15 (Cs-14) and 16–21 (Cs-21) days after flowering (DAF) and from leaf tissue (Cs-Leaf). The leaf sample was used as the calibrator for the remaining samples. Error bars represent the standard error ± SE of three biological replicates. The quantification of the genes is measured relative to the expression of the indigenous housekeeping gene β-actin

Techniques Used: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Derivative Assay

30) Product Images from "Comparative transcriptome and metabolome analysis suggests bottlenecks that limit seed and oil yields in transgenic Camelina sativa expressing diacylglycerol acyltransferase 1 and glycerol-3-phosphate dehydrogenase"

Article Title: Comparative transcriptome and metabolome analysis suggests bottlenecks that limit seed and oil yields in transgenic Camelina sativa expressing diacylglycerol acyltransferase 1 and glycerol-3-phosphate dehydrogenase

Journal: Biotechnology for Biofuels

doi: 10.1186/s13068-018-1326-2

The gene expression analysis for the selected genes showing differential regulation in Camelina transgenic lines. Data are the fold changes (FC) in expression measured by using both RNA-Seq and qRT-PCR techniques ( a , b ) in both DGAT1 and GPD1, respectively, relative to WT. The fold change values used in the analysis are presented in Additional file 1 : Table S15. Data shown in c , d indicate the relative gene expression for the selected genes measured by qRT-PCR in both DGAT1 and GPD1 lines, respectively, relative to WT. The genes shown here are non-specific lipid transfer 4-like ( NSLT - L ), glycerol-3-phosphate sn -2-acyltransferase 1 ( GPAT1 ), oleosin 5 ( OLE5 ), 3-ketoacyl-synthase 18-like ( KCS18 ), TAG-lipase 2-like ( TAGL2 - L ), acyl CoA thioesterase 13-like ( ACOT13 - L ), cruciferin 3 ( CRU3 ), acyl-CoA:diacylglycerol acyltransferase 1 ( DGAT1 ), oleosin 1 ( OLE1 ), glycerol-3-phosphate acyltransferase 9 ( GPAT9 ), lysophosphatidyl acyltransferase 2 ( LPAT2 ), glycerol-3-phosphate transporter 1 ( GLPT1 ), lysophosphatidyl acyltransferase 5 ( LPAT5 ), glucose-6-phosphate l-epimerase ( G6Pe ), diacylglycerol kinase 3-like isoform X1 ( DAGK ), 3-keto acyl-synthase 6 ( KCS6 ), acyl-activating enzyme 7 ( Acylae7 ), glycerol-3-phosphate acyltransferase 5 ( GPAT5 )
Figure Legend Snippet: The gene expression analysis for the selected genes showing differential regulation in Camelina transgenic lines. Data are the fold changes (FC) in expression measured by using both RNA-Seq and qRT-PCR techniques ( a , b ) in both DGAT1 and GPD1, respectively, relative to WT. The fold change values used in the analysis are presented in Additional file 1 : Table S15. Data shown in c , d indicate the relative gene expression for the selected genes measured by qRT-PCR in both DGAT1 and GPD1 lines, respectively, relative to WT. The genes shown here are non-specific lipid transfer 4-like ( NSLT - L ), glycerol-3-phosphate sn -2-acyltransferase 1 ( GPAT1 ), oleosin 5 ( OLE5 ), 3-ketoacyl-synthase 18-like ( KCS18 ), TAG-lipase 2-like ( TAGL2 - L ), acyl CoA thioesterase 13-like ( ACOT13 - L ), cruciferin 3 ( CRU3 ), acyl-CoA:diacylglycerol acyltransferase 1 ( DGAT1 ), oleosin 1 ( OLE1 ), glycerol-3-phosphate acyltransferase 9 ( GPAT9 ), lysophosphatidyl acyltransferase 2 ( LPAT2 ), glycerol-3-phosphate transporter 1 ( GLPT1 ), lysophosphatidyl acyltransferase 5 ( LPAT5 ), glucose-6-phosphate l-epimerase ( G6Pe ), diacylglycerol kinase 3-like isoform X1 ( DAGK ), 3-keto acyl-synthase 6 ( KCS6 ), acyl-activating enzyme 7 ( Acylae7 ), glycerol-3-phosphate acyltransferase 5 ( GPAT5 )

Techniques Used: Expressing, Transgenic Assay, RNA Sequencing Assay, Quantitative RT-PCR

31) Product Images from "Andrographolide Enhances Proliferation and Prevents Dedifferentiation of Rabbit Articular Chondrocytes: An In Vitro Study"

Article Title: Andrographolide Enhances Proliferation and Prevents Dedifferentiation of Rabbit Articular Chondrocytes: An In Vitro Study

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2015/984850

Quantitative comparison of extracellular-matrix-related gene expression by qRT-PCR. Chondrocytes were cultured with 0 (Control), 1.5 (T1), 3 (T2), and 6 μ M (T3) ANDRO for 2 days (a), 4 days (b), and 6 days (c) ( n = 3 for each group at a certain time point). Gene expression levels in AND groups relative to the control group were analyzed by the 2 −ΔΔCT method using GAPDH as the internal control. Isoforms of collagen type I, type II, and type X studied are COL1A1, COL2A1, and COL10A1, respectively. The data reported as mean ± SD. ∗, ∧ indicate P
Figure Legend Snippet: Quantitative comparison of extracellular-matrix-related gene expression by qRT-PCR. Chondrocytes were cultured with 0 (Control), 1.5 (T1), 3 (T2), and 6 μ M (T3) ANDRO for 2 days (a), 4 days (b), and 6 days (c) ( n = 3 for each group at a certain time point). Gene expression levels in AND groups relative to the control group were analyzed by the 2 −ΔΔCT method using GAPDH as the internal control. Isoforms of collagen type I, type II, and type X studied are COL1A1, COL2A1, and COL10A1, respectively. The data reported as mean ± SD. ∗, ∧ indicate P

Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture

32) Product Images from "Inertial Focusing for Tumor Antigen-Dependent and -Independent Sorting of Rare Circulating Tumor Cells"

Article Title: Inertial Focusing for Tumor Antigen-Dependent and -Independent Sorting of Rare Circulating Tumor Cells

Journal: Science translational medicine

doi: 10.1126/scitranslmed.3005616

CTC isolation by pos CTC-iChip in cancer patients. ( A ) CTCs isolated from castrate-resistant prostate cancer (CRPC) patients were enumerated and compared with blood specimens processed from healthy donors. ( B ) EpCAM-based isolation using pos CTC-iChip was compared with the Cell-Search system. Clinical samples were metastatic cancer patients of prostate ( n = 19), breast ( n = 12), pancreas ( n = 6), colorectal ( n = 2), and lung ( n = 2). All counts were normalized to 7.5 ml. ( C ) For enumeration of CTCs from CRPC patients, CK8/18/19 staining was used (green). CD45 antigen (red) was used to identify contaminating leukocytes. Scale bars, 10 μm. ( D ) A CTC from a CRPC patient was stained for prostate-specific antigen (PSA) (red), prostate-specific membrane antigen (PSMA) (yellow), and DAPI (blue) to demonstrate dual immunofluorescence staining for PSAs. ( E ) Validation of EML4-ALK RT-PCR assay was completed with cell lines. pos CTC-iChip products of whole blood from a healthy donor (HD) spiked with 0, 10, and 100 H3122 cells (expressing EML4-ALK variant 1) per 10 ml were subjected to RT-PCR for detection of the EML4-ALK fusion. Product isolated from healthy donor blood spiked with 500 VCaP cells/ml was processed as a negative control. ( F ) pos CTC-iChip products from patient samples known to harbor the EML4-ALK translocation by FISH were similarly processed as in (E), and the bands were sequenced to confirm the presence of the fusion transcript. A representative sequence trace from patient 3 shows the translocation breakpoint between exon 13 of EML4 and exon 20 of ALK . CTC analysis of three patients whose cancer lacks the translocation was used to establish specificity: a prostate cancer patient (lane 1), an EGFR mutant lung cancer patient (lane 2), and a HER2 -amplified lung cancer patient (lane 6).
Figure Legend Snippet: CTC isolation by pos CTC-iChip in cancer patients. ( A ) CTCs isolated from castrate-resistant prostate cancer (CRPC) patients were enumerated and compared with blood specimens processed from healthy donors. ( B ) EpCAM-based isolation using pos CTC-iChip was compared with the Cell-Search system. Clinical samples were metastatic cancer patients of prostate ( n = 19), breast ( n = 12), pancreas ( n = 6), colorectal ( n = 2), and lung ( n = 2). All counts were normalized to 7.5 ml. ( C ) For enumeration of CTCs from CRPC patients, CK8/18/19 staining was used (green). CD45 antigen (red) was used to identify contaminating leukocytes. Scale bars, 10 μm. ( D ) A CTC from a CRPC patient was stained for prostate-specific antigen (PSA) (red), prostate-specific membrane antigen (PSMA) (yellow), and DAPI (blue) to demonstrate dual immunofluorescence staining for PSAs. ( E ) Validation of EML4-ALK RT-PCR assay was completed with cell lines. pos CTC-iChip products of whole blood from a healthy donor (HD) spiked with 0, 10, and 100 H3122 cells (expressing EML4-ALK variant 1) per 10 ml were subjected to RT-PCR for detection of the EML4-ALK fusion. Product isolated from healthy donor blood spiked with 500 VCaP cells/ml was processed as a negative control. ( F ) pos CTC-iChip products from patient samples known to harbor the EML4-ALK translocation by FISH were similarly processed as in (E), and the bands were sequenced to confirm the presence of the fusion transcript. A representative sequence trace from patient 3 shows the translocation breakpoint between exon 13 of EML4 and exon 20 of ALK . CTC analysis of three patients whose cancer lacks the translocation was used to establish specificity: a prostate cancer patient (lane 1), an EGFR mutant lung cancer patient (lane 2), and a HER2 -amplified lung cancer patient (lane 6).

Techniques Used: Isolation, Staining, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Expressing, Variant Assay, Negative Control, Translocation Assay, Fluorescence In Situ Hybridization, Sequencing, Mutagenesis, Amplification

Heterogeneity of RNA expression between CTCs isolated from a prostate cancer patient. ( A ) Micromanipulation of single CTCs isolated from a blood specimen of a patient with prostate cancer using the neg CTC-iChip and stained in solution with anti-EpCAM (green) and anti-CD45 (red) antibodies. Top panel shows a bright-field image merged. Wide arrow points to an EpCAM + /CD45 − CTC. Thin arrow points to EpCAM − /CD45 + leukocytes. Arrowhead denotes an erythrocyte. Dashed line outlines the micromanipulator needle tip. Bottom two panels show distinct imaging channels. Scale bar, 20 μm. ( B ) EpCAM and bright-field images of 15 single prostate cancer CTCs from a single patient selected for transcriptional profiling. Scale bar, 10 μm. ( C ) Heat map of normalized gene expression (−Δ C t ) of 43 genes in each of the single CTCs measured by microfluidic qRT-PCR. Columns list each individual prostate CTC, and rows show the panel of genes assayed, grouped thematically. The red asterisk highlights the gene expression patterns of PSA and PSMA, which provide a measure of AR signaling activity. NTC, no-template control. ( D ) Table listing the proportional distribution of various gene groups expressed in single CTCs isolated from the prostate cancer patient.
Figure Legend Snippet: Heterogeneity of RNA expression between CTCs isolated from a prostate cancer patient. ( A ) Micromanipulation of single CTCs isolated from a blood specimen of a patient with prostate cancer using the neg CTC-iChip and stained in solution with anti-EpCAM (green) and anti-CD45 (red) antibodies. Top panel shows a bright-field image merged. Wide arrow points to an EpCAM + /CD45 − CTC. Thin arrow points to EpCAM − /CD45 + leukocytes. Arrowhead denotes an erythrocyte. Dashed line outlines the micromanipulator needle tip. Bottom two panels show distinct imaging channels. Scale bar, 20 μm. ( B ) EpCAM and bright-field images of 15 single prostate cancer CTCs from a single patient selected for transcriptional profiling. Scale bar, 10 μm. ( C ) Heat map of normalized gene expression (−Δ C t ) of 43 genes in each of the single CTCs measured by microfluidic qRT-PCR. Columns list each individual prostate CTC, and rows show the panel of genes assayed, grouped thematically. The red asterisk highlights the gene expression patterns of PSA and PSMA, which provide a measure of AR signaling activity. NTC, no-template control. ( D ) Table listing the proportional distribution of various gene groups expressed in single CTCs isolated from the prostate cancer patient.

Techniques Used: RNA Expression, Isolation, Micromanipulation, Staining, Imaging, Expressing, Quantitative RT-PCR, Activity Assay

33) Product Images from "Transforming Growth Factor-?1 and -?2 in Gastric Precancer and Cancer and Roles in Tumor-Cell Interactions with Peripheral Blood Mononuclear Cells In Vitro"

Article Title: Transforming Growth Factor-?1 and -?2 in Gastric Precancer and Cancer and Roles in Tumor-Cell Interactions with Peripheral Blood Mononuclear Cells In Vitro

Journal: PLoS ONE

doi: 10.1371/journal.pone.0054249

TGF-β1 and TGF-β2 mRNA profiles in gastric precancer and carcinoma. (A) TGF-β1 mRNA levels in the sequence from controls ( n = 20), precancer (PC) ( n = 21), early gastric cancer (EGC) ( n = 22), to advanced gastric cancer (AGC) ( n = 30). Data are given as means ± SD of transcript levels normalized to GAPDH. (B) Corresponding TGF-β2 mRNA levels in the same sequence. (C) and (D) TGF-β1 levels were upregulated and TGF-β2 levels were downregulated in tumor tissues, compared to peritumoral tissues from the same patients. Levels were normalized to GAPDH. Data from qRT-PCR in 20 paired cases are shown. (E) and (F) Significant positive correlations between TGF-β1 and Smad2/Smad7, using a bivariate correlation model. Data represent the transcript levels in 36 cases of GC after normalization to GAPDH. (G)Serum concentrations of TGF-β1 and TGF-β2 measured by ELISA were significantly higher in early and advanced GC compared to controls ( F = 4.745 and P = 0.018; F = 4.939 and P = 0.015, respectively). There was no significant difference between early and advanced GC. Ctrl : controls volunteers; EGC : early gastric cancer; AGC : advanced gastric cancer.
Figure Legend Snippet: TGF-β1 and TGF-β2 mRNA profiles in gastric precancer and carcinoma. (A) TGF-β1 mRNA levels in the sequence from controls ( n = 20), precancer (PC) ( n = 21), early gastric cancer (EGC) ( n = 22), to advanced gastric cancer (AGC) ( n = 30). Data are given as means ± SD of transcript levels normalized to GAPDH. (B) Corresponding TGF-β2 mRNA levels in the same sequence. (C) and (D) TGF-β1 levels were upregulated and TGF-β2 levels were downregulated in tumor tissues, compared to peritumoral tissues from the same patients. Levels were normalized to GAPDH. Data from qRT-PCR in 20 paired cases are shown. (E) and (F) Significant positive correlations between TGF-β1 and Smad2/Smad7, using a bivariate correlation model. Data represent the transcript levels in 36 cases of GC after normalization to GAPDH. (G)Serum concentrations of TGF-β1 and TGF-β2 measured by ELISA were significantly higher in early and advanced GC compared to controls ( F = 4.745 and P = 0.018; F = 4.939 and P = 0.015, respectively). There was no significant difference between early and advanced GC. Ctrl : controls volunteers; EGC : early gastric cancer; AGC : advanced gastric cancer.

Techniques Used: Sequencing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

34) Product Images from "Expression and accumulation of the two-domain odorant-binding protein AaegOBP45 in the ovaries of blood-fed Aedes aegypti"

Article Title: Expression and accumulation of the two-domain odorant-binding protein AaegOBP45 in the ovaries of blood-fed Aedes aegypti

Journal: Parasites & Vectors

doi: 10.1186/1756-3305-6-364

Gene amplification analyses of transcription profiles of AAEL010714. A) mRNA accumulation profiles in developmental stages and times after adult females feed on blood: fourth instar larvae (L4), white pupae (WP), male pupae (MP), female pupae (FM), newly-emerged males (EM), newly-emerged females (EF), sugar meal adult females (S), and adult females 24, 48 and 72 hPBM. β-actin mRNA levels were used as a control. B) Tissue-specific expression profile. All tissues, midgut (MG), ovaries (OV) and fat bodies (FB), were dissected from Ae. aegypti females at 48 hPBM. The same RNA samples were also analyzed by 18S rRNA-specific primers (Additional file 1 - [ 25 ]) as a control. C) Expression profile in female heads dissected at 48 hPBM. β-actin specific primers were used as controls on the same RNA samples. D) Quantification of accumulated of AAEL010714 transcripts by qRT-PCR in adult females 36, 48, 60, 72, 84 and 96 hPBM. All data represent mean values and error bars indicate standard error of the mean; *: p
Figure Legend Snippet: Gene amplification analyses of transcription profiles of AAEL010714. A) mRNA accumulation profiles in developmental stages and times after adult females feed on blood: fourth instar larvae (L4), white pupae (WP), male pupae (MP), female pupae (FM), newly-emerged males (EM), newly-emerged females (EF), sugar meal adult females (S), and adult females 24, 48 and 72 hPBM. β-actin mRNA levels were used as a control. B) Tissue-specific expression profile. All tissues, midgut (MG), ovaries (OV) and fat bodies (FB), were dissected from Ae. aegypti females at 48 hPBM. The same RNA samples were also analyzed by 18S rRNA-specific primers (Additional file 1 - [ 25 ]) as a control. C) Expression profile in female heads dissected at 48 hPBM. β-actin specific primers were used as controls on the same RNA samples. D) Quantification of accumulated of AAEL010714 transcripts by qRT-PCR in adult females 36, 48, 60, 72, 84 and 96 hPBM. All data represent mean values and error bars indicate standard error of the mean; *: p

Techniques Used: Amplification, Expressing, Quantitative RT-PCR

35) Product Images from "In vitro model for the analysis of synovial fibroblast-mediated degradation of intact cartilage"

Article Title: In vitro model for the analysis of synovial fibroblast-mediated degradation of intact cartilage

Journal: Arthritis Research & Therapy

doi: 10.1186/ar2618

Expression of bovine MMP-1 and MMP-3 mRNA. Cartilage from (a, b) monoculture (n = 5, with two replicates each) and (c, d) human mRNA/protein in synovial fibroblasts (SFB) after co-culture with cartilage (n = 5, with two replicates each) with/without stimulation with TNF-α, IL-1β or TNF-α/IL-1β (14 days) were used.gene expression values (means ± standard error of the mean (SEM)), as determined by quantitiative PCR, are expressed as percentage of the values in non-stimulated samples (100%). In addition, (e, f) the values for human MMP-1 and MMP-3 protein secreted by SFB into the supernatant of co-cultures are shown. The protein levels, as measured in the supernatant by ELISA, are expressed as means +/- SEM. § p ≤ 0.05 Mann-Whitney U Test compared with non-stimulated control; * p ≤ 0.05 Mann-Whitney U Test compared with stimulation with TNF-α.
Figure Legend Snippet: Expression of bovine MMP-1 and MMP-3 mRNA. Cartilage from (a, b) monoculture (n = 5, with two replicates each) and (c, d) human mRNA/protein in synovial fibroblasts (SFB) after co-culture with cartilage (n = 5, with two replicates each) with/without stimulation with TNF-α, IL-1β or TNF-α/IL-1β (14 days) were used.gene expression values (means ± standard error of the mean (SEM)), as determined by quantitiative PCR, are expressed as percentage of the values in non-stimulated samples (100%). In addition, (e, f) the values for human MMP-1 and MMP-3 protein secreted by SFB into the supernatant of co-cultures are shown. The protein levels, as measured in the supernatant by ELISA, are expressed as means +/- SEM. § p ≤ 0.05 Mann-Whitney U Test compared with non-stimulated control; * p ≤ 0.05 Mann-Whitney U Test compared with stimulation with TNF-α.

Techniques Used: Expressing, Co-Culture Assay, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Caseinolytic activity. Supernatants of cartilage monocultures (n = 5, with two replicates each) and co-cultures with synovial fibroblasts (SFB) (n = 5, with two replicates each) with/without stimulation with TNF-α, IL-1β or TNF-α/IL-1β (14 days) were used. In order to assess the total caseinolytic activity (lower panel), the bands for both the active and the latent forms were used for quantification. Means +/- standard error of the mean are plotted. § p ≤ 0.05 Mann-Whitney U Test compared with non-stimulated control; + p ≤ 0.05 Mann-Whitney U Test compared with cartilage monoculture. Parallel analysis of the supernatants by western blot revealed that bovine/human matrix metalloproteases (MMP) 1 and MMP-3 (upper and middle panel) are responsible for the caseinolytic enzyme activity in culture supernatants.
Figure Legend Snippet: Caseinolytic activity. Supernatants of cartilage monocultures (n = 5, with two replicates each) and co-cultures with synovial fibroblasts (SFB) (n = 5, with two replicates each) with/without stimulation with TNF-α, IL-1β or TNF-α/IL-1β (14 days) were used. In order to assess the total caseinolytic activity (lower panel), the bands for both the active and the latent forms were used for quantification. Means +/- standard error of the mean are plotted. § p ≤ 0.05 Mann-Whitney U Test compared with non-stimulated control; + p ≤ 0.05 Mann-Whitney U Test compared with cartilage monoculture. Parallel analysis of the supernatants by western blot revealed that bovine/human matrix metalloproteases (MMP) 1 and MMP-3 (upper and middle panel) are responsible for the caseinolytic enzyme activity in culture supernatants.

Techniques Used: Activity Assay, MANN-WHITNEY, Western Blot

36) Product Images from "Upregulation of long noncoding RNA CCAT1-L promotes epithelial–mesenchymal transition in gastric adenocarcinoma"

Article Title: Upregulation of long noncoding RNA CCAT1-L promotes epithelial–mesenchymal transition in gastric adenocarcinoma

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S170553

CCAT1-L knockdown effectively inhibited tumor growth both in vitro and in vivo. Notes: ( A ) Verification of CCAT1-L knockdown by specific siRNA (CCAT-L siRNA #1 and CCAT-L siRNA #3) and control siRNA using qRT-PCR (n=3). ( B , C ) Colony formation analysis of cells after siRNA transfection. ( D ) Picture of MKN-28 tumors harvested from different treatment groups. ( E ) In vivo tumor growth of MKN-28 cells transfected with CCAT1-L siRNA #1 or control siRNA. ( F ) Survival curve of mice bearing MKN-28 tumors transfected with CCAT1-L siRNA or control siRNA (n=10). *** P
Figure Legend Snippet: CCAT1-L knockdown effectively inhibited tumor growth both in vitro and in vivo. Notes: ( A ) Verification of CCAT1-L knockdown by specific siRNA (CCAT-L siRNA #1 and CCAT-L siRNA #3) and control siRNA using qRT-PCR (n=3). ( B , C ) Colony formation analysis of cells after siRNA transfection. ( D ) Picture of MKN-28 tumors harvested from different treatment groups. ( E ) In vivo tumor growth of MKN-28 cells transfected with CCAT1-L siRNA #1 or control siRNA. ( F ) Survival curve of mice bearing MKN-28 tumors transfected with CCAT1-L siRNA or control siRNA (n=10). *** P

Techniques Used: In Vitro, In Vivo, Quantitative RT-PCR, Transfection, Mouse Assay

The expression of CCAT1-L mRNA and c-MYC mRNA. Notes: ( A ) qRT-PCR detection of CCAT1-L expression in gastric adenocarcinoma and adjacent normal tissue (n=60; *** P
Figure Legend Snippet: The expression of CCAT1-L mRNA and c-MYC mRNA. Notes: ( A ) qRT-PCR detection of CCAT1-L expression in gastric adenocarcinoma and adjacent normal tissue (n=60; *** P

Techniques Used: Expressing, Quantitative RT-PCR

37) Product Images from "Cell distribution and cytokine levels in induced sputum from healthy subjects and patients with asthma after using different nebulizer techniques"

Article Title: Cell distribution and cytokine levels in induced sputum from healthy subjects and patients with asthma after using different nebulizer techniques

Journal: BMC Pulmonary Medicine

doi: 10.1186/s12890-018-0683-8

IL-5 mRNA expression measured after the use of different nebulizers. Data are presented as medians. In the controls, p = 0.3927 when comparing the Omron and Akita nebulizers. In the patients with asthma, p = 0.4307. qRT-PCR revealed a significant elevation in IL-5 levels in patients with asthma compared with levels in controls
Figure Legend Snippet: IL-5 mRNA expression measured after the use of different nebulizers. Data are presented as medians. In the controls, p = 0.3927 when comparing the Omron and Akita nebulizers. In the patients with asthma, p = 0.4307. qRT-PCR revealed a significant elevation in IL-5 levels in patients with asthma compared with levels in controls

Techniques Used: Expressing, Quantitative RT-PCR

38) Product Images from "Histone Deacetylase Inhibitors Enhance the Therapeutic Potential of Reovirus in Multiple Myeloma"

Article Title: Histone Deacetylase Inhibitors Enhance the Therapeutic Potential of Reovirus in Multiple Myeloma

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-15-0240-T

MM cell lines variably express the high affinity RV protein receptor JAM-1. A) Western blot showing total protein expression of JAM-1 in MM cell lines L-363, RPMI, H929, and U266. GAPDH expression was used as a loading control. The data presented are representative of 3 biological replicates. B) Cell surface expression of the RV receptor JAM-1 in MM cell lines H929, L-363, U266, and RPMI was determined by flow cytometry using PE-conjugated JAM-1 antibody. C) JAM-1 mRNA expression affects the efficiency of RV infection. L363 cells were treated with RV at MOI of 10 for 9 hrs and viral genome expression was determined by qRT-PCR and normalized to GAPDH. Data are expressed in relative fold change (f.c.) compared to the controls. D) MM cell lines more susceptible to RV infection (RPMI and U266) show higher levels of the RV genome at early time-points of infection (10, 20 and 45min), compared to more resistant L-363 and H929 cells. Cells were washed in PBS to remove unbound virus and in trypsin to remove bound un-internalized virus. RV genome expression was determined by q-RT-PCR and normalized against GAPDH.
Figure Legend Snippet: MM cell lines variably express the high affinity RV protein receptor JAM-1. A) Western blot showing total protein expression of JAM-1 in MM cell lines L-363, RPMI, H929, and U266. GAPDH expression was used as a loading control. The data presented are representative of 3 biological replicates. B) Cell surface expression of the RV receptor JAM-1 in MM cell lines H929, L-363, U266, and RPMI was determined by flow cytometry using PE-conjugated JAM-1 antibody. C) JAM-1 mRNA expression affects the efficiency of RV infection. L363 cells were treated with RV at MOI of 10 for 9 hrs and viral genome expression was determined by qRT-PCR and normalized to GAPDH. Data are expressed in relative fold change (f.c.) compared to the controls. D) MM cell lines more susceptible to RV infection (RPMI and U266) show higher levels of the RV genome at early time-points of infection (10, 20 and 45min), compared to more resistant L-363 and H929 cells. Cells were washed in PBS to remove unbound virus and in trypsin to remove bound un-internalized virus. RV genome expression was determined by q-RT-PCR and normalized against GAPDH.

Techniques Used: Western Blot, Expressing, Flow Cytometry, Cytometry, Infection, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

HDACi increase JAM-1 expression in MM cells. A) L363 and H929 cell lines were treated with 0.01% DMSO as control, 0.1μM AR-42, or 0.2μM AR-42 for 24 hrs and JAM-1 protein was measured by immunoblot with GAPDH as a loading control. Immunoblots displayed are representative of 3 independent experiments. B) L363 and H929 cells were treated as in (A) and JAM-1 mRNA expression was measured by qRT-PCR, using GAPDH for normalization. C) Increased cell surface expression of JAM-1 in H929 (top) and L-363 (bottom) MM cell lines following 24-hr treatments with 0.2μM AR-42, 1μM SAHA, or 0.01μM LBH, as compared to control (0.5% DMSO), measured by flow cytometry using PE-conjugated anti-JAM-1 antibody. The LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit was used to ensure data were collected from live cells. D) HDACi treatment of primary plasma cells isolated from MM patients increases JAM-1 cell surface expression. CD138+ cells were isolated by positive selection from bone marrow aspirates of 2 MM patients (MM Pt1 and MM Pt2) and treated for 24 hrs with 0.5μM SAHA, 0.008μM LBH, or control (0.01% DMSO). Cell surface JAM-1 expression was then determined by flow cytometry with PE-conjugated anti-JAM-1 antibody. E) AR-42 increases JAM-1 mRNA expression in CD138+ cells from MM patients. As part of a phase I clinical trial, patients received 40mg AR-42 orally Mon-Wed-Fri, in cycles of 28 days, followed by a 7-day break. RNA was isolated from CD138+ cells purified by positive selection from bone marrow aspirates before AR-42 treatment and 15 days after receiving the last dose of AR-42. JAM-1 mRNA expression was determined by qRT-PCR and normalized for GAPDH.
Figure Legend Snippet: HDACi increase JAM-1 expression in MM cells. A) L363 and H929 cell lines were treated with 0.01% DMSO as control, 0.1μM AR-42, or 0.2μM AR-42 for 24 hrs and JAM-1 protein was measured by immunoblot with GAPDH as a loading control. Immunoblots displayed are representative of 3 independent experiments. B) L363 and H929 cells were treated as in (A) and JAM-1 mRNA expression was measured by qRT-PCR, using GAPDH for normalization. C) Increased cell surface expression of JAM-1 in H929 (top) and L-363 (bottom) MM cell lines following 24-hr treatments with 0.2μM AR-42, 1μM SAHA, or 0.01μM LBH, as compared to control (0.5% DMSO), measured by flow cytometry using PE-conjugated anti-JAM-1 antibody. The LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit was used to ensure data were collected from live cells. D) HDACi treatment of primary plasma cells isolated from MM patients increases JAM-1 cell surface expression. CD138+ cells were isolated by positive selection from bone marrow aspirates of 2 MM patients (MM Pt1 and MM Pt2) and treated for 24 hrs with 0.5μM SAHA, 0.008μM LBH, or control (0.01% DMSO). Cell surface JAM-1 expression was then determined by flow cytometry with PE-conjugated anti-JAM-1 antibody. E) AR-42 increases JAM-1 mRNA expression in CD138+ cells from MM patients. As part of a phase I clinical trial, patients received 40mg AR-42 orally Mon-Wed-Fri, in cycles of 28 days, followed by a 7-day break. RNA was isolated from CD138+ cells purified by positive selection from bone marrow aspirates before AR-42 treatment and 15 days after receiving the last dose of AR-42. JAM-1 mRNA expression was determined by qRT-PCR and normalized for GAPDH.

Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining, Isolation, Selection, Purification

JAM-1 expression is epigenetically regulated in MM cells. A) L363 cells were treated for 24 hrs with indicated concentrations of JQ-12, RGFP966, Etinostat, AR-42, SAHA and LBH or DMSO as control. Whole cell extracts were analyzed by western blot using JAM-1 antibody (top). Staining with anti-Acetylated histone 3 antibody (H3AcK) (middle) was used to confirm activity of all drugs and GAPDH (bottom) served as a loading control. B) JAM-1 mRNA fold change ±SD in L363 cells after treatment with HDACi. GADPH was used for normalization proposes. C) L363 cells were treated with 0.01μM LBH or 0.01% DMSO for 24 hrs and analyzed by ChIP assay using H3AcK27, anti-RNA Polymerase II (PolII), anti-H3AcK, and anti-H4AcK antibodies. The association between acetylated histones and RNA polymerase II with the JAM-1 promoter were visualized by agarose gel electrophoresis. D) Immunoprecipitated chromatin obtained in the ChIP experiment in (C) was also tested by qRT-PCR and demonstrated increased levels of the JAM-1 promoter in association with acetylated H3AcK27 and Pol II. Control primers for JAM-1 exon 3 and for GAPDH were used for normalization purposes.
Figure Legend Snippet: JAM-1 expression is epigenetically regulated in MM cells. A) L363 cells were treated for 24 hrs with indicated concentrations of JQ-12, RGFP966, Etinostat, AR-42, SAHA and LBH or DMSO as control. Whole cell extracts were analyzed by western blot using JAM-1 antibody (top). Staining with anti-Acetylated histone 3 antibody (H3AcK) (middle) was used to confirm activity of all drugs and GAPDH (bottom) served as a loading control. B) JAM-1 mRNA fold change ±SD in L363 cells after treatment with HDACi. GADPH was used for normalization proposes. C) L363 cells were treated with 0.01μM LBH or 0.01% DMSO for 24 hrs and analyzed by ChIP assay using H3AcK27, anti-RNA Polymerase II (PolII), anti-H3AcK, and anti-H4AcK antibodies. The association between acetylated histones and RNA polymerase II with the JAM-1 promoter were visualized by agarose gel electrophoresis. D) Immunoprecipitated chromatin obtained in the ChIP experiment in (C) was also tested by qRT-PCR and demonstrated increased levels of the JAM-1 promoter in association with acetylated H3AcK27 and Pol II. Control primers for JAM-1 exon 3 and for GAPDH were used for normalization purposes.

Techniques Used: Expressing, Western Blot, Staining, Activity Assay, Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Immunoprecipitation, Quantitative RT-PCR

39) Product Images from "Control of Flowering and Cell Fate by LIF2, an RNA Binding Partner of the Polycomb Complex Component LHP1"

Article Title: Control of Flowering and Cell Fate by LIF2, an RNA Binding Partner of the Polycomb Complex Component LHP1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0016592

H3K27me3 and LHP1 distribution on FLC. (A) Expression at various developmental stages in wild-type and lif2 plants. Semi-quantitative RT-PCR were performed on seven-day-old in vitro seedlings, rosette leaves after bolting (RL), floral buds just after bolting (FB1) and floral buds after the production of 10 siliques (FB2). The EF-1α gene expression was used as a control. (B) Schematic representation of the FLC locus and the 8 amplified regions used in chromatin immunoprecipitation (ChIP) assays. (C) ChIP analysis to determine the relative level of H3K27me3 at the indicated FLC regions in wild-type and lif2 seedlings. Immunoprecipitated DNA was analyzed by real-time qPCR, and enrichment was calculated as percentage of INPUT. All ChIP experiments were normalized for histone H3 occupancy and normed by using ChIP results on an AGAMOUS control region. Data in the graphs are the average of at least two qPCR assays from three independent ChIP experiments; the bars represent standard error. (D–F) Complementation of the lhp1 mutant by the expression of the genomic LHP1:MYC-tagged construct. (D) Plant phenotypes, (E) total number of rosette leaves, and (F) protein levels. (G) ChIP assays to determine the relative level of LHP1 binding at the indicated FLC regions in wild-type and lif2 backgrounds expressing the LHP1:MYC-tagged construct. A CONSTANS (CO) region was used as a negative control [23] . Immunoprecipitated DNA was analyzed by real-time qPCR, and enrichment was calculated as percentage of INPUT and normalized relative to a Col-0 control, represented as a dashed line. Data in the graphs are the average of at least two qPCR assays from three independent ChIP experiments; the bars represent standard error.
Figure Legend Snippet: H3K27me3 and LHP1 distribution on FLC. (A) Expression at various developmental stages in wild-type and lif2 plants. Semi-quantitative RT-PCR were performed on seven-day-old in vitro seedlings, rosette leaves after bolting (RL), floral buds just after bolting (FB1) and floral buds after the production of 10 siliques (FB2). The EF-1α gene expression was used as a control. (B) Schematic representation of the FLC locus and the 8 amplified regions used in chromatin immunoprecipitation (ChIP) assays. (C) ChIP analysis to determine the relative level of H3K27me3 at the indicated FLC regions in wild-type and lif2 seedlings. Immunoprecipitated DNA was analyzed by real-time qPCR, and enrichment was calculated as percentage of INPUT. All ChIP experiments were normalized for histone H3 occupancy and normed by using ChIP results on an AGAMOUS control region. Data in the graphs are the average of at least two qPCR assays from three independent ChIP experiments; the bars represent standard error. (D–F) Complementation of the lhp1 mutant by the expression of the genomic LHP1:MYC-tagged construct. (D) Plant phenotypes, (E) total number of rosette leaves, and (F) protein levels. (G) ChIP assays to determine the relative level of LHP1 binding at the indicated FLC regions in wild-type and lif2 backgrounds expressing the LHP1:MYC-tagged construct. A CONSTANS (CO) region was used as a negative control [23] . Immunoprecipitated DNA was analyzed by real-time qPCR, and enrichment was calculated as percentage of INPUT and normalized relative to a Col-0 control, represented as a dashed line. Data in the graphs are the average of at least two qPCR assays from three independent ChIP experiments; the bars represent standard error.

Techniques Used: Expressing, Quantitative RT-PCR, In Vitro, Amplification, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Mutagenesis, Construct, Binding Assay, Negative Control

40) Product Images from "Retigeric Acid B Exhibits Antitumor Activity through Suppression of Nuclear Factor-?B Signaling in Prostate Cancer Cells in Vitro and in Vivo"

Article Title: Retigeric Acid B Exhibits Antitumor Activity through Suppression of Nuclear Factor-?B Signaling in Prostate Cancer Cells in Vitro and in Vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038000

The effects of RB on function of NF-κB-dependent gene expresion and cell invasion in vitro . (A) RB dosage-dependently inhibited the mRNA expression of Bcl-x L , Bcl-2, survivin, and cyclin D1 as analyzed by QRT-PCR. GAPDH was used for normalization. Results are the mean ± SD of three independent experiments, each performed in triplicate. p
Figure Legend Snippet: The effects of RB on function of NF-κB-dependent gene expresion and cell invasion in vitro . (A) RB dosage-dependently inhibited the mRNA expression of Bcl-x L , Bcl-2, survivin, and cyclin D1 as analyzed by QRT-PCR. GAPDH was used for normalization. Results are the mean ± SD of three independent experiments, each performed in triplicate. p

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR

The effect of RB on p65 expression and phosphorylation in PCa cells. (A) and (B) RB slightly inhibited expression of p65 in PC3 and DU145 cells, while significantly for the Ser-536 phosphorylation of p65 ( p–p65 ), in both dosage-dependent and time-dependent manner. Lysates from whole cells treated with RB of different concentrations for 24 h (A) or with 10 µM RB for indicated times were used for western blot (B). GAPDH served as the loading control. Protein amount was normalized to the amount of GAPDH, and was quantified by densitometry of X-ray films. Results of one of at least three independent experiments are shown. (C) and (D) RB moderately down-regulated p65 mRNA level in PC3 and DU145 cells as detected by QRT-PCR assay. The procedure was performed as described in Materials and Methods . Results shown are representatives of three independent experiments, p
Figure Legend Snippet: The effect of RB on p65 expression and phosphorylation in PCa cells. (A) and (B) RB slightly inhibited expression of p65 in PC3 and DU145 cells, while significantly for the Ser-536 phosphorylation of p65 ( p–p65 ), in both dosage-dependent and time-dependent manner. Lysates from whole cells treated with RB of different concentrations for 24 h (A) or with 10 µM RB for indicated times were used for western blot (B). GAPDH served as the loading control. Protein amount was normalized to the amount of GAPDH, and was quantified by densitometry of X-ray films. Results of one of at least three independent experiments are shown. (C) and (D) RB moderately down-regulated p65 mRNA level in PC3 and DU145 cells as detected by QRT-PCR assay. The procedure was performed as described in Materials and Methods . Results shown are representatives of three independent experiments, p

Techniques Used: Expressing, Western Blot, Quantitative RT-PCR

41) Product Images from "Comparison of human coagulation factor VIII expression directed by cytomegalovirus and mammary gland-specific promoters in HC11 cells and transgenic mice"

Article Title: Comparison of human coagulation factor VIII expression directed by cytomegalovirus and mammary gland-specific promoters in HC11 cells and transgenic mice

Journal: Blood Coagulation & Fibrinolysis

doi: 10.1097/MBC.0000000000000318

Real-time RT-PCR analysis of HC-11 cell strains transfected with hFVIIIBD expression vectors. (a) The hFVIIIBD copy number in the HC-11 cell strain transfected with two different hFVIIIBD vectors with the P1A3 promoter or the CMV promoter. (b) The β-actin copy number in the HC-11 cell strain transfected with two different hFVIIIBD vectors with the P1A3 promoter or the CMV promoter. No. 1 represents HC-11 cells transfected with CMV-hFVIIIBD. No. 2 represents HC-11 cells transfected with P1A3-hFVIIIBD. No. 3 represents HC-11 cells transfected with pcDNA 3.1 (negative controls). (c) The relative hFVIIIBD expression of the three different vectors in HC-11 cells. No. 1 represents the CMV-hFVIIIBD vector-transfected HC-11 cells. No. 2 represents the P1A3-hFVIIIBD vector-transfected HC-11 cells. No. 3 represents pcDNA 3.1(+) vector-transfected HC-11 cells. CMV, cytomegalovirus; hFVIII, human factor VIII; RT-PCR, reverse transcription PCR.
Figure Legend Snippet: Real-time RT-PCR analysis of HC-11 cell strains transfected with hFVIIIBD expression vectors. (a) The hFVIIIBD copy number in the HC-11 cell strain transfected with two different hFVIIIBD vectors with the P1A3 promoter or the CMV promoter. (b) The β-actin copy number in the HC-11 cell strain transfected with two different hFVIIIBD vectors with the P1A3 promoter or the CMV promoter. No. 1 represents HC-11 cells transfected with CMV-hFVIIIBD. No. 2 represents HC-11 cells transfected with P1A3-hFVIIIBD. No. 3 represents HC-11 cells transfected with pcDNA 3.1 (negative controls). (c) The relative hFVIIIBD expression of the three different vectors in HC-11 cells. No. 1 represents the CMV-hFVIIIBD vector-transfected HC-11 cells. No. 2 represents the P1A3-hFVIIIBD vector-transfected HC-11 cells. No. 3 represents pcDNA 3.1(+) vector-transfected HC-11 cells. CMV, cytomegalovirus; hFVIII, human factor VIII; RT-PCR, reverse transcription PCR.

Techniques Used: Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

RT-PCR assessment of hFVIIIBD in hFVIIIBD transgenic and WT mice. RT-PCR results in different organs of hFVIIIBD transgenic and WT mice. (a) hFVIIIBD expression in CMV-hFVIIIBD transgenic mice. (b) hFVIIIBD expression in P1A3-hFVIIIBD transgenic mice. (c) hFVIIIBD expression in WT mice. M: 100 bp marker ladder, lane 1: positive control, lane 2: negative control, lane 3: blank control, and lanes 4–9 indicate mammary glands, heart, liver, spleen, lung, and kidney, respectively. CMV, cytomegalovirus; hFVIII, human factor VIII; RT-PCR, reverse transcription PCR; WT, wild type.
Figure Legend Snippet: RT-PCR assessment of hFVIIIBD in hFVIIIBD transgenic and WT mice. RT-PCR results in different organs of hFVIIIBD transgenic and WT mice. (a) hFVIIIBD expression in CMV-hFVIIIBD transgenic mice. (b) hFVIIIBD expression in P1A3-hFVIIIBD transgenic mice. (c) hFVIIIBD expression in WT mice. M: 100 bp marker ladder, lane 1: positive control, lane 2: negative control, lane 3: blank control, and lanes 4–9 indicate mammary glands, heart, liver, spleen, lung, and kidney, respectively. CMV, cytomegalovirus; hFVIII, human factor VIII; RT-PCR, reverse transcription PCR; WT, wild type.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Mouse Assay, Expressing, Marker, Positive Control, Negative Control, Polymerase Chain Reaction

Real-time PCR for the assessment of hFVIIIBD or β-actin in hFVIIIBD transgenic and WT mice. Real-time PCR results in different organs of hFVIIIBD transgenic and WT mice. The ratios of hFVIIIBD/β-actin in different organs of hFVIIIBD transgenic and WT mice are shown ( n = 4). (a) CMV-hFVIIIBD transgenic mice. (b) P1A3-hFVIIIBD transgenic mice. (c) Wild-type mice. CMV, cytomegalovirus; hFVIII, human factor VIII; mamma, mammary gland; RT-PCR, reverse transcription PCR; WT, wild type.
Figure Legend Snippet: Real-time PCR for the assessment of hFVIIIBD or β-actin in hFVIIIBD transgenic and WT mice. Real-time PCR results in different organs of hFVIIIBD transgenic and WT mice. The ratios of hFVIIIBD/β-actin in different organs of hFVIIIBD transgenic and WT mice are shown ( n = 4). (a) CMV-hFVIIIBD transgenic mice. (b) P1A3-hFVIIIBD transgenic mice. (c) Wild-type mice. CMV, cytomegalovirus; hFVIII, human factor VIII; mamma, mammary gland; RT-PCR, reverse transcription PCR; WT, wild type.

Techniques Used: Real-time Polymerase Chain Reaction, Transgenic Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

42) Product Images from "Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome"

Article Title: Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks1324

Recovery from EtBr treatment stimulates replication of high-molecular weight mtDNA structures. ( A ) 2D-IMAGE analysis of wild-type MEFs cultured for 24 h in 500 ng/ml of EtBr. Medium was changed to omit EtBr and cells harvested at 1 ( B ), 3 ( C ) or 6 h ( D ) post-washout. ( E ) mtDNA content during washout as determined by qPCR (left) or total mtDNA hybridization signal for panels (A–D). (F) Distribution of signal across nine selected topoisomers as mtDNA levels recover. ( G ) Relative abundance of selected topoisomers from ( F ) after setting unrecovered sample equal to 1. ( H ) Model for template–product relationship for early and late recovery from EtBr-mediated mtDNA replication arrest. Solid arrows indicate template–product synthesis, and dashed arrows indicate conversion of one topoisomer to another. Location of nick in 1n NC is arbitrary.
Figure Legend Snippet: Recovery from EtBr treatment stimulates replication of high-molecular weight mtDNA structures. ( A ) 2D-IMAGE analysis of wild-type MEFs cultured for 24 h in 500 ng/ml of EtBr. Medium was changed to omit EtBr and cells harvested at 1 ( B ), 3 ( C ) or 6 h ( D ) post-washout. ( E ) mtDNA content during washout as determined by qPCR (left) or total mtDNA hybridization signal for panels (A–D). (F) Distribution of signal across nine selected topoisomers as mtDNA levels recover. ( G ) Relative abundance of selected topoisomers from ( F ) after setting unrecovered sample equal to 1. ( H ) Model for template–product relationship for early and late recovery from EtBr-mediated mtDNA replication arrest. Solid arrows indicate template–product synthesis, and dashed arrows indicate conversion of one topoisomer to another. Location of nick in 1n NC is arbitrary.

Techniques Used: Molecular Weight, Cell Culture, Real-time Polymerase Chain Reaction, Hybridization

43) Product Images from "Effect of cyclooxygenase inhibition on cholesterol efflux proteins and atheromatous foam cell transformation in THP-1 human macrophages: a possible mechanism for increased cardiovascular risk"

Article Title: Effect of cyclooxygenase inhibition on cholesterol efflux proteins and atheromatous foam cell transformation in THP-1 human macrophages: a possible mechanism for increased cardiovascular risk

Journal: Arthritis Research & Therapy

doi: 10.1186/ar2109

QRT-PCR for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 cells exposed to prostaglandins. (a) 27-Hydroxylase message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. ** p
Figure Legend Snippet: QRT-PCR for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 cells exposed to prostaglandins. (a) 27-Hydroxylase message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 1 , E 2 , and D 2 . THP-1 human monocytes were exposed to the following conditions represented by the six bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 1 (0.1 μM) + NS398 (50 μM), (4) PGE 2 (0.1 μM) + NS398 (50 μM), (5) PGE 1 (0.1 μM) + PGE 2 (0.1 μM) + NS398 (50 μM), and (6) PGD 2 (14 μM) + NS398 (50 μM) (all 18-hour exposures). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. ** p

Techniques Used: Quantitative RT-PCR, Expressing, Amplification

COX inhibition impairs reverse cholesterol transport. COX-1/2 inhibition downregulates 27-hydroxylase and ABCA1, thereby decreasing cholesterol efflux, in turn promoting the accumulation of cholesterol in macrophages that transform into foam cells. This effect is restored by the addition of prostaglandins. AA, arachidonic acid; ABCA1, ATP-binding cassette transporter A1; COX, cyclooxygenase; PG, prostaglandin; TXA, thromboxane A.
Figure Legend Snippet: COX inhibition impairs reverse cholesterol transport. COX-1/2 inhibition downregulates 27-hydroxylase and ABCA1, thereby decreasing cholesterol efflux, in turn promoting the accumulation of cholesterol in macrophages that transform into foam cells. This effect is restored by the addition of prostaglandins. AA, arachidonic acid; ABCA1, ATP-binding cassette transporter A1; COX, cyclooxygenase; PG, prostaglandin; TXA, thromboxane A.

Techniques Used: Inhibition, Binding Assay

QRT-PCR for 27-hydroxylase and ABCA1 message in indomethacin-treated THP-1 cells. (a) 27-Hydroxylase mRNA expression is decreased by the non-specific COX inhibitor indomethacin in a dose-dependent fashion in THP-1 monocytes. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of indomethacin for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by QRT-PCR as described. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 mRNA expression is decreased by the non-specific COX inhibitor indomethacin in a dose-dependent fashion in THP-1 monocytes. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of indomethacin for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by QRT-PCR as described. Signals obtained from the amplification of GAPDH message were used as internal controls. At 50 mM indomethacin concentration, cell death was statistically significant (16.8% ± 1.0%). ABCA1, ATP-binding cassette transporter A1; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; QRT-PCR, quantitative real-time polymerase chain reaction. * p
Figure Legend Snippet: QRT-PCR for 27-hydroxylase and ABCA1 message in indomethacin-treated THP-1 cells. (a) 27-Hydroxylase mRNA expression is decreased by the non-specific COX inhibitor indomethacin in a dose-dependent fashion in THP-1 monocytes. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of indomethacin for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by QRT-PCR as described. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 mRNA expression is decreased by the non-specific COX inhibitor indomethacin in a dose-dependent fashion in THP-1 monocytes. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of indomethacin for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by QRT-PCR as described. Signals obtained from the amplification of GAPDH message were used as internal controls. At 50 mM indomethacin concentration, cell death was statistically significant (16.8% ± 1.0%). ABCA1, ATP-binding cassette transporter A1; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; QRT-PCR, quantitative real-time polymerase chain reaction. * p

Techniques Used: Quantitative RT-PCR, Expressing, Cell Culture, Isolation, Amplification, Concentration Assay, Binding Assay, Real-time Polymerase Chain Reaction

Detection and quantitation of cholesterol 27-hydroxylase in THP-1 cells exposed to SC560. (a) 27-Hydroxylase mRNA expression in THP-1 monocytes is decreased by the specific COX-1 inhibitor SC560. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of SC560 for 24 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by quantitative real-time polymerase chain reaction as described. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) Decrease in 27-hydroxylase protein expression in THP-1 monocytes treated with the COX-1 inhibitor SC560. Cultured THP-1 human monocytes were untreated or exposed to SC560 for 24 hours. Total cell protein was isolated and 27-hydroxylase detected with specific rabbit polyclonal anti-human 27-hydroxylase antibody. Western blotting was performed with an anti-β-actin antibody to confirm equal protein loading. COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SC560, 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazol. ** p
Figure Legend Snippet: Detection and quantitation of cholesterol 27-hydroxylase in THP-1 cells exposed to SC560. (a) 27-Hydroxylase mRNA expression in THP-1 monocytes is decreased by the specific COX-1 inhibitor SC560. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of SC560 for 24 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by quantitative real-time polymerase chain reaction as described. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) Decrease in 27-hydroxylase protein expression in THP-1 monocytes treated with the COX-1 inhibitor SC560. Cultured THP-1 human monocytes were untreated or exposed to SC560 for 24 hours. Total cell protein was isolated and 27-hydroxylase detected with specific rabbit polyclonal anti-human 27-hydroxylase antibody. Western blotting was performed with an anti-β-actin antibody to confirm equal protein loading. COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SC560, 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazol. ** p

Techniques Used: Quantitation Assay, Expressing, Cell Culture, Isolation, Amplification, Real-time Polymerase Chain Reaction, Western Blot

QRT-PCR for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 macrophages exposed to prostaglandins or TXA 2 . (a) 27-Hydroxylase message in THP-1 macrophages is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 2 and D 2 , but not TXA 2 . THP-1 human macrophages were exposed to the following conditions represented by the five bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 2 (0.1 μM) + NS398 (50 μM), (4) PGD 2 (14 μM) + NS398 (50 μM), and (5) TXA 2 (3 μM) + NS398 (50 μM) (24-hour exposures to NS398 alone followed by addition of indicated PG or TXA 2 for a further 24 hours). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 in THP-1 macrophages and this decrease is reversed by prostaglandins E 2 and D 2 , but not TXA 2 . THP-1 human macrophages were exposed to the following conditions represented by the five bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 2 (0.1 μM) + NS398 (50 μM), (4) PGD 2 (14 μM) + NS398 (50 μM), and (5) TXA 2 (3 μM) + NS398 (50 μM) (24-hour exposures to NS398 alone followed by addition of indicated PG or TXA 2 for a further 24 hours). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. * p
Figure Legend Snippet: QRT-PCR for 27-hydroxylase and ABCA1 message in NS398-treated THP-1 macrophages exposed to prostaglandins or TXA 2 . (a) 27-Hydroxylase message in THP-1 macrophages is decreased by the COX-2 inhibitor NS398 and this decrease is reversed by prostaglandins E 2 and D 2 , but not TXA 2 . THP-1 human macrophages were exposed to the following conditions represented by the five bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 2 (0.1 μM) + NS398 (50 μM), (4) PGD 2 (14 μM) + NS398 (50 μM), and (5) TXA 2 (3 μM) + NS398 (50 μM) (24-hour exposures to NS398 alone followed by addition of indicated PG or TXA 2 for a further 24 hours). Cells were extracted for total RNA and were evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) ABCA1 message is decreased by the COX-2 inhibitor NS398 in THP-1 macrophages and this decrease is reversed by prostaglandins E 2 and D 2 , but not TXA 2 . THP-1 human macrophages were exposed to the following conditions represented by the five bars (from left to right): (1) RPMI 1640, (2) NS398 (50 μM), (3) PGE 2 (0.1 μM) + NS398 (50 μM), (4) PGD 2 (14 μM) + NS398 (50 μM), and (5) TXA 2 (3 μM) + NS398 (50 μM) (24-hour exposures to NS398 alone followed by addition of indicated PG or TXA 2 for a further 24 hours). Cells were extracted for total RNA and were evaluated for ABCA1 mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. * p

Techniques Used: Quantitative RT-PCR, Expressing, Amplification

Detection and quantitation of cholesterol 27-hydroxylase in THP-1 cells exposed to NS398. (a) Dose-dependent decrease in 27-hydroxylase mRNA expression in THP-1 monocytes treated with the COX-2 inhibitor NS398. Cultured THP-1 monocytic cells were untreated or exposed to NS398 for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by quantitative real-time polymerase chain reaction as described. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) Dose-dependent decrease in 27-hydroxylase protein expression in THP-1 monocytes treated with the COX-2 inhibitor NS398. Cultured THP-1 monocytic cells were untreated or exposed to NS398 for 18 hours. Total cell protein was isolated and 27-hydroxylase detected with specific rabbit polyclonal anti-human 27-hydroxylase antibody. Western blotting was performed with an anti-β-actin antibody to confirm equal protein loading. At 100 mM NS398 concentration, cell death was statistically significant (14.8% ± 6.3%). COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS398, N -(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide. ** p
Figure Legend Snippet: Detection and quantitation of cholesterol 27-hydroxylase in THP-1 cells exposed to NS398. (a) Dose-dependent decrease in 27-hydroxylase mRNA expression in THP-1 monocytes treated with the COX-2 inhibitor NS398. Cultured THP-1 monocytic cells were untreated or exposed to NS398 for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by quantitative real-time polymerase chain reaction as described. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) Dose-dependent decrease in 27-hydroxylase protein expression in THP-1 monocytes treated with the COX-2 inhibitor NS398. Cultured THP-1 monocytic cells were untreated or exposed to NS398 for 18 hours. Total cell protein was isolated and 27-hydroxylase detected with specific rabbit polyclonal anti-human 27-hydroxylase antibody. Western blotting was performed with an anti-β-actin antibody to confirm equal protein loading. At 100 mM NS398 concentration, cell death was statistically significant (14.8% ± 6.3%). COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS398, N -(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide. ** p

Techniques Used: Quantitation Assay, Expressing, Cell Culture, Isolation, Amplification, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay

44) Product Images from "Substrate specificity and transport mechanism of amino-acid transceptor Slimfast from Aedes aegypti"

Article Title: Substrate specificity and transport mechanism of amino-acid transceptor Slimfast from Aedes aegypti

Journal: Nature Communications

doi: 10.1038/ncomms9546

Aa Slif expression and regulation in selected mosquito tissues and organs. The transcript levels were determined using quantitative PCR (qPCR). The data represent relative quantities of Aa Slif transcript that were normalized with qPCR levels of ribosomal protein S7 (rpS7) mRNA in the same tissue sample set. Bars are means+s.e.m. for n =3 replicates collected from different sets of mosquitoes (analysis of variance followed by Tukey's HSD post hoc test). The RNA samples were isolated from whole body and various body parts and organs of adult female mosquitoes grouped as shown by the colour-coding insert. The horizontal scale indicates group-specific conditions: non-blood fed (NBF, control), 3, 12, 24, 48, 72 and 96 h post blood meal (PBM).
Figure Legend Snippet: Aa Slif expression and regulation in selected mosquito tissues and organs. The transcript levels were determined using quantitative PCR (qPCR). The data represent relative quantities of Aa Slif transcript that were normalized with qPCR levels of ribosomal protein S7 (rpS7) mRNA in the same tissue sample set. Bars are means+s.e.m. for n =3 replicates collected from different sets of mosquitoes (analysis of variance followed by Tukey's HSD post hoc test). The RNA samples were isolated from whole body and various body parts and organs of adult female mosquitoes grouped as shown by the colour-coding insert. The horizontal scale indicates group-specific conditions: non-blood fed (NBF, control), 3, 12, 24, 48, 72 and 96 h post blood meal (PBM).

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

45) Product Images from "Differential Immunometabolic Phenotype in Th1 and Th2 Dominant Mouse Strains in Response to High-Fat Feeding"

Article Title: Differential Immunometabolic Phenotype in Th1 and Th2 Dominant Mouse Strains in Response to High-Fat Feeding

Journal: PLoS ONE

doi: 10.1371/journal.pone.0134089

Liver gene expression in chow and HFD-fed C57Bl/6 and BALB/c mice. (A) Expression of genes related to lipid metabolism in liver in chow and HFD-fed C57Bl/6 and BALB/c mice. Expression of LXRα and PPARγ was significantly lower in C57Bl/6 mice mice on HFD. (B) Expression of profibrogenic genes in liver in chow and HFD-fed C57Bl/6 and BALB/c mice. Expression of procollagen and TGF-β was significantly higher in C57Bl/6 mice on both diets. The results are shown as the means ± SEM of 9–10 animals per group. *p
Figure Legend Snippet: Liver gene expression in chow and HFD-fed C57Bl/6 and BALB/c mice. (A) Expression of genes related to lipid metabolism in liver in chow and HFD-fed C57Bl/6 and BALB/c mice. Expression of LXRα and PPARγ was significantly lower in C57Bl/6 mice mice on HFD. (B) Expression of profibrogenic genes in liver in chow and HFD-fed C57Bl/6 and BALB/c mice. Expression of procollagen and TGF-β was significantly higher in C57Bl/6 mice on both diets. The results are shown as the means ± SEM of 9–10 animals per group. *p

Techniques Used: Expressing, Mouse Assay

46) Product Images from "Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction"

Article Title: Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction

Journal: Oncotarget

doi: 10.18632/oncotarget.11896

Human prostate cancer cells express LLT1 A. mRNA expression of LLT1 on prostate cancer cell lines PC3, DU145, LNCaP, 22Rv1, normal prostate cell PWR-1E and Jurkat (T cell line) was determined by qRT-PCR. LLT1 expression was determined by using LLT1 sequence specific primers and Taqman gene expression assays in an Eppendorf Realplex2 Mastercycler. Reactions were done in 20 μl triplicates using the ΔΔCT method, with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. Each bar represents a mean ± s.e. of three independent experiments. B. LLT1 protein expression was analyzed by Western blotting in a panel of prostate cancer cell lines including leukemic Jurkat cells. GAPDH was used as a loading control. C. A bar graph showing densitometric analysis of LLT1 protein expression normalized to GAPDH. Each bar represents the mean ± s.e. of three independent experiments.
Figure Legend Snippet: Human prostate cancer cells express LLT1 A. mRNA expression of LLT1 on prostate cancer cell lines PC3, DU145, LNCaP, 22Rv1, normal prostate cell PWR-1E and Jurkat (T cell line) was determined by qRT-PCR. LLT1 expression was determined by using LLT1 sequence specific primers and Taqman gene expression assays in an Eppendorf Realplex2 Mastercycler. Reactions were done in 20 μl triplicates using the ΔΔCT method, with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. Each bar represents a mean ± s.e. of three independent experiments. B. LLT1 protein expression was analyzed by Western blotting in a panel of prostate cancer cell lines including leukemic Jurkat cells. GAPDH was used as a loading control. C. A bar graph showing densitometric analysis of LLT1 protein expression normalized to GAPDH. Each bar represents the mean ± s.e. of three independent experiments.

Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Western Blot

47) Product Images from "Expression of cytochrome P450 CYP81A6 in rice: tissue specificity, protein subcellular localization, and response to herbicide application *"

Article Title: Expression of cytochrome P450 CYP81A6 in rice: tissue specificity, protein subcellular localization, and response to herbicide application *

Journal: Journal of Zhejiang University. Science. B

doi: 10.1631/jzus.B1400168

Expression of CYP81A6 in wild-type rice cultivar Jiazhe B (W) and its knockout mutant Jiazhe mB (M) through qRT-PCR analyses
Figure Legend Snippet: Expression of CYP81A6 in wild-type rice cultivar Jiazhe B (W) and its knockout mutant Jiazhe mB (M) through qRT-PCR analyses

Techniques Used: Expressing, Knock-Out, Mutagenesis, Quantitative RT-PCR

48) Product Images from "Profiling expression changes caused by a segmental aneuploid in maize"

Article Title: Profiling expression changes caused by a segmental aneuploid in maize

Journal: BMC Genomics

doi: 10.1186/1471-2164-9-7

Knox10 gene is the only knotted-like gene ectopically expressed in leaves of DpDf plants . Expression of nine knotted-like maize genes was tested in meristematic and leaf tissue in wild type and DpDf plants using RT-PCR. Leaf and meristematic tissue was isolated from three seedlings for each of the genotypes. Mez1 gene was used as a control to equalize the cDNA concentrations for all the samples. The tissue type and genotype are indicated above the gel images. The first lane is a genomic DNA control while the last lane is a no template control (NTC). Control reactions were also performed on a no reverse-transcriptase control (data not shown).
Figure Legend Snippet: Knox10 gene is the only knotted-like gene ectopically expressed in leaves of DpDf plants . Expression of nine knotted-like maize genes was tested in meristematic and leaf tissue in wild type and DpDf plants using RT-PCR. Leaf and meristematic tissue was isolated from three seedlings for each of the genotypes. Mez1 gene was used as a control to equalize the cDNA concentrations for all the samples. The tissue type and genotype are indicated above the gel images. The first lane is a genomic DNA control while the last lane is a no template control (NTC). Control reactions were also performed on a no reverse-transcriptase control (data not shown).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation

Validation of severe expression changes detected by expression profiling . For six of the genes with the greatest alteration in gene expression changes, the expression levels were validated in two tissue types from three different wild type and three different DpDf sibling plants by semi-quantitative RT-PCR. L16798, BM380426, BM269210 and AI783234 are examples of increased expression in DpDf plants while AY107007 and BM073880 are examples of decreased expression in DpDf plants. Mez1 is used as a loading control. The first lane is a genomic DNA control while the last lane is a no template control (NTC).
Figure Legend Snippet: Validation of severe expression changes detected by expression profiling . For six of the genes with the greatest alteration in gene expression changes, the expression levels were validated in two tissue types from three different wild type and three different DpDf sibling plants by semi-quantitative RT-PCR. L16798, BM380426, BM269210 and AI783234 are examples of increased expression in DpDf plants while AY107007 and BM073880 are examples of decreased expression in DpDf plants. Mez1 is used as a loading control. The first lane is a genomic DNA control while the last lane is a no template control (NTC).

Techniques Used: Expressing, Quantitative RT-PCR

49) Product Images from "Reversal of epigenetic promoter silencing in Friedreich ataxia by a class I histone deacetylase inhibitor"

Article Title: Reversal of epigenetic promoter silencing in Friedreich ataxia by a class I histone deacetylase inhibitor

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw107

Treatment with 109 significantly increases FXN promoter function in FRDA. Treatment with 109 ( A and C ), but not 966 ( B and D ), resulted in > 3-fold increased accumulation of nascent FXN transcript at various time points assayed (0.5–4 h) after initiation of metabolic labeling. Quantitative RT-PCR was performed both immediately downstream of the transcription start site (i.e. upstream of the expanded GAA-TR sequence; Ex1 in Figure 1A ) and downstream of the expanded GAA-TR sequence (Ex3–Ex4 in Figure 1A ), with both locations showing a similar > 3-fold increase in nascent FXN transcript accumulation in response to 109 treatment. Nascent transcript levels were quantified by real-time PCR relative to expression of the control TBP gene (for 109 treated samples [A and C]) or the control OAZ1 gene (for 966 treated samples [B and D]) from total biotinylated RNA using the ΔΔCt method. Graphs represent cumulative data from two complete experiments using three lymphoblastoid cell lines from individuals with FRDA who are homozygous for the expanded GAA-TR sequence, treated with 109, 966 or no drug (only DMSO), each assayed in triplicate. Error bars represent +/−SEM. *** P
Figure Legend Snippet: Treatment with 109 significantly increases FXN promoter function in FRDA. Treatment with 109 ( A and C ), but not 966 ( B and D ), resulted in > 3-fold increased accumulation of nascent FXN transcript at various time points assayed (0.5–4 h) after initiation of metabolic labeling. Quantitative RT-PCR was performed both immediately downstream of the transcription start site (i.e. upstream of the expanded GAA-TR sequence; Ex1 in Figure 1A ) and downstream of the expanded GAA-TR sequence (Ex3–Ex4 in Figure 1A ), with both locations showing a similar > 3-fold increase in nascent FXN transcript accumulation in response to 109 treatment. Nascent transcript levels were quantified by real-time PCR relative to expression of the control TBP gene (for 109 treated samples [A and C]) or the control OAZ1 gene (for 966 treated samples [B and D]) from total biotinylated RNA using the ΔΔCt method. Graphs represent cumulative data from two complete experiments using three lymphoblastoid cell lines from individuals with FRDA who are homozygous for the expanded GAA-TR sequence, treated with 109, 966 or no drug (only DMSO), each assayed in triplicate. Error bars represent +/−SEM. *** P

Techniques Used: Labeling, Quantitative RT-PCR, Sequencing, Real-time Polymerase Chain Reaction, Expressing

50) Product Images from "Upregulation of long noncoding RNA CCAT1-L promotes epithelial–mesenchymal transition in gastric adenocarcinoma"

Article Title: Upregulation of long noncoding RNA CCAT1-L promotes epithelial–mesenchymal transition in gastric adenocarcinoma

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S170553

CCAT1-L knockdown effectively inhibited tumor growth both in vitro and in vivo. Notes: ( A ) Verification of CCAT1-L knockdown by specific siRNA (CCAT-L siRNA #1 and CCAT-L siRNA #3) and control siRNA using qRT-PCR (n=3). ( B , C ) Colony formation analysis of cells after siRNA transfection. ( D ) Picture of MKN-28 tumors harvested from different treatment groups. ( E ) In vivo tumor growth of MKN-28 cells transfected with CCAT1-L siRNA #1 or control siRNA. ( F ) Survival curve of mice bearing MKN-28 tumors transfected with CCAT1-L siRNA or control siRNA (n=10). *** P
Figure Legend Snippet: CCAT1-L knockdown effectively inhibited tumor growth both in vitro and in vivo. Notes: ( A ) Verification of CCAT1-L knockdown by specific siRNA (CCAT-L siRNA #1 and CCAT-L siRNA #3) and control siRNA using qRT-PCR (n=3). ( B , C ) Colony formation analysis of cells after siRNA transfection. ( D ) Picture of MKN-28 tumors harvested from different treatment groups. ( E ) In vivo tumor growth of MKN-28 cells transfected with CCAT1-L siRNA #1 or control siRNA. ( F ) Survival curve of mice bearing MKN-28 tumors transfected with CCAT1-L siRNA or control siRNA (n=10). *** P

Techniques Used: In Vitro, In Vivo, Quantitative RT-PCR, Transfection, Mouse Assay

The expression of CCAT1-L mRNA and c-MYC mRNA. Notes: ( A ) qRT-PCR detection of CCAT1-L expression in gastric adenocarcinoma and adjacent normal tissue (n=60; *** P
Figure Legend Snippet: The expression of CCAT1-L mRNA and c-MYC mRNA. Notes: ( A ) qRT-PCR detection of CCAT1-L expression in gastric adenocarcinoma and adjacent normal tissue (n=60; *** P

Techniques Used: Expressing, Quantitative RT-PCR

51) Product Images from "Differential Immunometabolic Phenotype in Th1 and Th2 Dominant Mouse Strains in Response to High-Fat Feeding"

Article Title: Differential Immunometabolic Phenotype in Th1 and Th2 Dominant Mouse Strains in Response to High-Fat Feeding

Journal: PLoS ONE

doi: 10.1371/journal.pone.0134089

Liver gene expression in chow and HFD-fed C57Bl/6 and BALB/c mice. (A) Expression of genes related to lipid metabolism in liver in chow and HFD-fed C57Bl/6 and BALB/c mice. Expression of LXRα and PPARγ was significantly lower in C57Bl/6 mice mice on HFD. (B) Expression of profibrogenic genes in liver in chow and HFD-fed C57Bl/6 and BALB/c mice. Expression of procollagen and TGF-β was significantly higher in C57Bl/6 mice on both diets. The results are shown as the means ± SEM of 9–10 animals per group. *p
Figure Legend Snippet: Liver gene expression in chow and HFD-fed C57Bl/6 and BALB/c mice. (A) Expression of genes related to lipid metabolism in liver in chow and HFD-fed C57Bl/6 and BALB/c mice. Expression of LXRα and PPARγ was significantly lower in C57Bl/6 mice mice on HFD. (B) Expression of profibrogenic genes in liver in chow and HFD-fed C57Bl/6 and BALB/c mice. Expression of procollagen and TGF-β was significantly higher in C57Bl/6 mice on both diets. The results are shown as the means ± SEM of 9–10 animals per group. *p

Techniques Used: Expressing, Mouse Assay

52) Product Images from "Tumor necrosis factor-inducible gene 6 protein ameliorates chronic liver damage by promoting autophagy formation in mice"

Article Title: Tumor necrosis factor-inducible gene 6 protein ameliorates chronic liver damage by promoting autophagy formation in mice

Journal: Experimental & Molecular Medicine

doi: 10.1038/emm.2017.140

TSG-6 protects hepatocytes from tunicamycin-induced cell death by increasing autophagy influx. ( a ) Cell viability of AML12 cells, a normal murine hepatocyte cell line, treated with TSG-6 was analyzed using MTS assay. After being exposed to TM (5 ng ml −1 ) for 24 h, AML12 cells were treated with vehicle (TM), 3-MA (TM+3-MA) or TSG-6 with (TM+TSG-6+3-MA) or without 3-MA (TM+TSG-6) for 24 and 48 h. As a CON, AML12 cells were treated with equal volumes of vehicle without TM. The mean±s.e.m. results obtained from three identical experiments are plotted. ( b ) qRT-PCR analysis of atg3 , atg7 and lc3 in these cells. Data represent the mean±s.e.m. of three independent experiments. ( c ) Western blot assay and ( d ) cumulative densitometry analyses for LC3-II and RAB7 in these cell groups. GAPDH was used as an internal CON. Data shown represent one of three experiments with similar results, and the mean±s.e.m. results obtained from three identical experiments are shown (* P
Figure Legend Snippet: TSG-6 protects hepatocytes from tunicamycin-induced cell death by increasing autophagy influx. ( a ) Cell viability of AML12 cells, a normal murine hepatocyte cell line, treated with TSG-6 was analyzed using MTS assay. After being exposed to TM (5 ng ml −1 ) for 24 h, AML12 cells were treated with vehicle (TM), 3-MA (TM+3-MA) or TSG-6 with (TM+TSG-6+3-MA) or without 3-MA (TM+TSG-6) for 24 and 48 h. As a CON, AML12 cells were treated with equal volumes of vehicle without TM. The mean±s.e.m. results obtained from three identical experiments are plotted. ( b ) qRT-PCR analysis of atg3 , atg7 and lc3 in these cells. Data represent the mean±s.e.m. of three independent experiments. ( c ) Western blot assay and ( d ) cumulative densitometry analyses for LC3-II and RAB7 in these cell groups. GAPDH was used as an internal CON. Data shown represent one of three experiments with similar results, and the mean±s.e.m. results obtained from three identical experiments are shown (* P

Techniques Used: MTS Assay, Quantitative RT-PCR, Western Blot

3-MA suppresses TSG-6-mediated autophagy in livers of MCDE-fed mice. ( a ) qRT-PCR analysis of atg3 , atg7 and lc3 in livers from the M+V, M+TSG-6, M+TSG-6+3-MA and M+3-MA groups. Mean±s.d. results are plotted ( n ⩾4 mice per group) (* P
Figure Legend Snippet: 3-MA suppresses TSG-6-mediated autophagy in livers of MCDE-fed mice. ( a ) qRT-PCR analysis of atg3 , atg7 and lc3 in livers from the M+V, M+TSG-6, M+TSG-6+3-MA and M+3-MA groups. Mean±s.d. results are plotted ( n ⩾4 mice per group) (* P

Techniques Used: Mouse Assay, Quantitative RT-PCR

TSG-6 increases expression of autophagy markers in livers of MCDE-fed mice. ( a ) qRT-PCR analysis of atg3 , atg7 , lc3 and lamp2a in livers from CON, M+V, and M+TSG-6-treated mice results are plotted (mean±s.d. n ⩾4 mice per group). ( b ) Western blot analysis and ( c ) cumulative densitometry analyses for LC3 (LC3-II: 14 kDa, processed form) (inducer of autophagosomes), LAMP2A (lysosome membrane protein) and RAB7 (autophagosome-lysosome docking marker) in livers of three representative mice from each group. GAPDH was used as an internal CON. Data shown represent one of three experiments with similar results. The results are displayed as the mean±s.d. ( n ⩾4 mice per group) (* P
Figure Legend Snippet: TSG-6 increases expression of autophagy markers in livers of MCDE-fed mice. ( a ) qRT-PCR analysis of atg3 , atg7 , lc3 and lamp2a in livers from CON, M+V, and M+TSG-6-treated mice results are plotted (mean±s.d. n ⩾4 mice per group). ( b ) Western blot analysis and ( c ) cumulative densitometry analyses for LC3 (LC3-II: 14 kDa, processed form) (inducer of autophagosomes), LAMP2A (lysosome membrane protein) and RAB7 (autophagosome-lysosome docking marker) in livers of three representative mice from each group. GAPDH was used as an internal CON. Data shown represent one of three experiments with similar results. The results are displayed as the mean±s.d. ( n ⩾4 mice per group) (* P

Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Western Blot, Marker

53) Product Images from "Molecular and functional heterogeneity of early postnatal porcine satellite cell populations is associated with bioenergetic profile"

Article Title: Molecular and functional heterogeneity of early postnatal porcine satellite cell populations is associated with bioenergetic profile

Journal: Scientific Reports

doi: 10.1038/srep45052

Experimental workflow. P40/50 and P50/70 cells were isolated from muscles of 4-day-old piglets. Gene expression (via qRT-PCR) was analysed in freshly isolated cells. Proliferation rate was measured when cells were passaged at day 4, 8 and 14. Cell morphology and size during proliferation, growth behaviour, protein expression (via flow cytometry and immunofluorescence), bioenergetic profile and differentiation potential were monitored at given time points.
Figure Legend Snippet: Experimental workflow. P40/50 and P50/70 cells were isolated from muscles of 4-day-old piglets. Gene expression (via qRT-PCR) was analysed in freshly isolated cells. Proliferation rate was measured when cells were passaged at day 4, 8 and 14. Cell morphology and size during proliferation, growth behaviour, protein expression (via flow cytometry and immunofluorescence), bioenergetic profile and differentiation potential were monitored at given time points.

Techniques Used: Isolation, Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Immunofluorescence

54) Product Images from "A New Mutation, hap1-2, Reveals a C Terminal Domain Function in AtMago Protein and Its Biological Effects in Male Gametophyte Development in Arabidopsis thaliana"

Article Title: A New Mutation, hap1-2, Reveals a C Terminal Domain Function in AtMago Protein and Its Biological Effects in Male Gametophyte Development in Arabidopsis thaliana

Journal: PLoS ONE

doi: 10.1371/journal.pone.0148200

Characterization and complementation of the HAP1/hap1-2 mutant. (A) HAP1 gene structure. Exons were shown as colored boxes; introns were shown as straight lines; UTRs were shown as gray boxes. The T-DNA insertion in hap1-1 and hap1-2 was indicated by red arrow. (B) rtPCR results showed the expression of a full-length transcript ( AtMago ), a truncated transcript ( AtMagoΔC ) and GAPDH in qrt , qrt;HAP1/hap1-2 , qrt;sr45-1 and qrt;upf3-1 mutant plants. Total RNAs were extracted from the inflorescence tissue. The exon composition of the transcript and the position of used primers were shown next to the corresponding rtPCR results. (C) GUS stained pollen grains from qrt , qrt;HAP1/hap1-1 and qrt;HAP1/hap1-2 showing the identity of mutant pollen grains. Scale bar = 20 μm. (D) Seed number per silique in qrt and qrt;HAP1/hap1-2 plants. Scale bar = 1 mm. The quantification was done with sixteen siliques. Student t-test was used for statistical analysis. Error bars represent standard deviations. (E) HAP1 gene complemented hap1-2 mutant phenotype. The mature plants, GUS-stained pollen grains and seed yield per plant were shown in qrt , qrt;HAP1/hap1-2 , HAP1;qrt; hap1-2 . (F) qPCR results showing that the AtMago gene expression level was fully recovered in the transgenic plant. GAPDH was used as control. Three biological replicates were used in the analysis. Error bar showed standard deviation. Statistical significance compared to qrt was measured by student t-test ( p
Figure Legend Snippet: Characterization and complementation of the HAP1/hap1-2 mutant. (A) HAP1 gene structure. Exons were shown as colored boxes; introns were shown as straight lines; UTRs were shown as gray boxes. The T-DNA insertion in hap1-1 and hap1-2 was indicated by red arrow. (B) rtPCR results showed the expression of a full-length transcript ( AtMago ), a truncated transcript ( AtMagoΔC ) and GAPDH in qrt , qrt;HAP1/hap1-2 , qrt;sr45-1 and qrt;upf3-1 mutant plants. Total RNAs were extracted from the inflorescence tissue. The exon composition of the transcript and the position of used primers were shown next to the corresponding rtPCR results. (C) GUS stained pollen grains from qrt , qrt;HAP1/hap1-1 and qrt;HAP1/hap1-2 showing the identity of mutant pollen grains. Scale bar = 20 μm. (D) Seed number per silique in qrt and qrt;HAP1/hap1-2 plants. Scale bar = 1 mm. The quantification was done with sixteen siliques. Student t-test was used for statistical analysis. Error bars represent standard deviations. (E) HAP1 gene complemented hap1-2 mutant phenotype. The mature plants, GUS-stained pollen grains and seed yield per plant were shown in qrt , qrt;HAP1/hap1-2 , HAP1;qrt; hap1-2 . (F) qPCR results showing that the AtMago gene expression level was fully recovered in the transgenic plant. GAPDH was used as control. Three biological replicates were used in the analysis. Error bar showed standard deviation. Statistical significance compared to qrt was measured by student t-test ( p

Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Real-time Polymerase Chain Reaction, Transgenic Assay, Standard Deviation

55) Product Images from "A Novel Synthesized Sulfonamido-Based Gallate—JEZ-C as Potential Therapeutic Agents for Osteoarthritis"

Article Title: A Novel Synthesized Sulfonamido-Based Gallate—JEZ-C as Potential Therapeutic Agents for Osteoarthritis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0125930

Quantitative comparison of ECM-related gene expression of MMP-1 (A), MMP-13 (B) and TIMP-1 (C) by qRT-PCR. The chondrocytes were cultured with different concentrations of JEZ-C, GA and SMZ: Control (without IL-1β), Model (with IL-1β), JEZ-C (J-1: 6.25×10 −7 μg/ml; J-2: 6.25×10 −6 μg/ml; J-3: 6.25×10 −5 μg/ml), GA (G-1: 0.078 μg/ml; G-2: 0.125 μg/ml; G-3: 0.156μg/ml) and SMZ (S-1: 6.25×10 −6 μg/ml; S-2: 6.25×10 −5 μg/ml; S-3: 6.25×10 −4 μg/ml) on the induction by IL-1β for 6 days (n = 3 for each experiment). The gene expression levels in JEZ-C, GA and SMZ media relative to the control group were analysed by the 2 -ΔΔCT method using β-actin as the internal control. The data represent the mean±SD of three independent culture experiments. Bars with different letters are significantly different from each other at P﹤0.05.
Figure Legend Snippet: Quantitative comparison of ECM-related gene expression of MMP-1 (A), MMP-13 (B) and TIMP-1 (C) by qRT-PCR. The chondrocytes were cultured with different concentrations of JEZ-C, GA and SMZ: Control (without IL-1β), Model (with IL-1β), JEZ-C (J-1: 6.25×10 −7 μg/ml; J-2: 6.25×10 −6 μg/ml; J-3: 6.25×10 −5 μg/ml), GA (G-1: 0.078 μg/ml; G-2: 0.125 μg/ml; G-3: 0.156μg/ml) and SMZ (S-1: 6.25×10 −6 μg/ml; S-2: 6.25×10 −5 μg/ml; S-3: 6.25×10 −4 μg/ml) on the induction by IL-1β for 6 days (n = 3 for each experiment). The gene expression levels in JEZ-C, GA and SMZ media relative to the control group were analysed by the 2 -ΔΔCT method using β-actin as the internal control. The data represent the mean±SD of three independent culture experiments. Bars with different letters are significantly different from each other at P﹤0.05.

Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture

Quantitative comparison of ECM-related gene expression of aggrecan (a), collagen II (b), Sox9 (c) collagen I (d) and by qRT-PCR. The chondrocytes were cultured with different concentrations of JEZ-C, GA and SMZ: Control (K: 0 μg/ml), JEZ-C (J-1: 6.25×10 −7 μg/ml; J-2: 6.25×10 −6 μg/ml; J-3: 6.25×10 −5 μg/ml), GA (G-1: 0.078 μg/ml; G-2: 0.125 μg/ml; G-3: 0.156μg/ml) and SDM (S-1: 6.25×10 −6 μg/ml; S-2: 6.25×10 −5 μg/ml; S-3: 6.25×10 −4 μg/ml) for 6 d (n = 3 for each experiment). The gene expression levels in JEZ-C, GA and SMZ media relative to the control group were analyzed by the 2 -ΔΔCT method using β-actin as the internal control. The data represent the means ± SD of three independent culture experiments. *p
Figure Legend Snippet: Quantitative comparison of ECM-related gene expression of aggrecan (a), collagen II (b), Sox9 (c) collagen I (d) and by qRT-PCR. The chondrocytes were cultured with different concentrations of JEZ-C, GA and SMZ: Control (K: 0 μg/ml), JEZ-C (J-1: 6.25×10 −7 μg/ml; J-2: 6.25×10 −6 μg/ml; J-3: 6.25×10 −5 μg/ml), GA (G-1: 0.078 μg/ml; G-2: 0.125 μg/ml; G-3: 0.156μg/ml) and SDM (S-1: 6.25×10 −6 μg/ml; S-2: 6.25×10 −5 μg/ml; S-3: 6.25×10 −4 μg/ml) for 6 d (n = 3 for each experiment). The gene expression levels in JEZ-C, GA and SMZ media relative to the control group were analyzed by the 2 -ΔΔCT method using β-actin as the internal control. The data represent the means ± SD of three independent culture experiments. *p

Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture

56) Product Images from "Identification and Functional Demonstration of miRNAs in the Fungus Cryptococcus neoformans"

Article Title: Identification and Functional Demonstration of miRNAs in the Fungus Cryptococcus neoformans

Journal: PLoS ONE

doi: 10.1371/journal.pone.0052734

miR-mediated gene silencing requires RNAi machinery in C. neoformans . (A) URA5-miR1/2 restored the growth of B4500FOA ( ura5 ) on MIN agar in the RNAi-deficient mutant strains, NE465, NE493, NE473 and NE475, i.e. gene silencing of URA5-miRs that was observed in Fig. 4 did not occur in these mutants. And these transformants failed to grow on plates containing 5-FOA (the panels of MINFOA agar). MIN or MINFOA agar was supplemented with 100 µg/ml hygromycin B for the selection of all transformants of the reporters. The drug was left out for B4500 and B4500FOA. The transformants of miR1-m and miR2-m, together with the wild type B4500 and B4500FOA, served as control. (B) qRT-PCR confirmation of restored expression of URA5 in RNAi-deficient mutants, NE465, NE493, NE473 and NE475, which are originally ura5 defective strains.
Figure Legend Snippet: miR-mediated gene silencing requires RNAi machinery in C. neoformans . (A) URA5-miR1/2 restored the growth of B4500FOA ( ura5 ) on MIN agar in the RNAi-deficient mutant strains, NE465, NE493, NE473 and NE475, i.e. gene silencing of URA5-miRs that was observed in Fig. 4 did not occur in these mutants. And these transformants failed to grow on plates containing 5-FOA (the panels of MINFOA agar). MIN or MINFOA agar was supplemented with 100 µg/ml hygromycin B for the selection of all transformants of the reporters. The drug was left out for B4500 and B4500FOA. The transformants of miR1-m and miR2-m, together with the wild type B4500 and B4500FOA, served as control. (B) qRT-PCR confirmation of restored expression of URA5 in RNAi-deficient mutants, NE465, NE493, NE473 and NE475, which are originally ura5 defective strains.

Techniques Used: Mutagenesis, Selection, Quantitative RT-PCR, Expressing

57) Product Images from "Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction"

Article Title: Overexpression of LLT1 (OCIL, CLEC2D) on prostate cancer cells inhibits NK cell-mediated killing through LLT1-NKRP1A (CD161) interaction

Journal: Oncotarget

doi: 10.18632/oncotarget.11896

Prostate cancer tissues show increased expression of LLT1 as compared to normal prostate tissues The deparaffinized prostate cancer (A, C) and normal prostate tissue (B, D) sections were processed for antigen retrieval and stained with LLT1 Ab (mouse anti-human CLEC2D Ab, Lifespan Biosciences, Seattle, WA) and counter stained with anti-Mouse-IgG-Dylight 594 Ab ( red ). Sections were also stained with the nuclear stain DAPI ( blue ) indicated by the blue stain and imaged on an Olympus AX70 fluorescent microscope. Merged images of LLT1 Ab and DAPI are shown. LLT1 expression is indicated by the red/pink stain. The sections were imaged at 20x and 100x magnifications.
Figure Legend Snippet: Prostate cancer tissues show increased expression of LLT1 as compared to normal prostate tissues The deparaffinized prostate cancer (A, C) and normal prostate tissue (B, D) sections were processed for antigen retrieval and stained with LLT1 Ab (mouse anti-human CLEC2D Ab, Lifespan Biosciences, Seattle, WA) and counter stained with anti-Mouse-IgG-Dylight 594 Ab ( red ). Sections were also stained with the nuclear stain DAPI ( blue ) indicated by the blue stain and imaged on an Olympus AX70 fluorescent microscope. Merged images of LLT1 Ab and DAPI are shown. LLT1 expression is indicated by the red/pink stain. The sections were imaged at 20x and 100x magnifications.

Techniques Used: Expressing, Staining, Microscopy

Prostate cancer cell lines display increased cell surface expression of LLT1 A-F. Surface expression of LLT1 on prostate cancer cells DU145, LNCaP, PC3 and 22Rv1, normal prostate cell PWR-1E and Jurkat (T cell line) was determined by flow cytometry using mouse anti-human LLT1 mAb (clone# 2E5) and a PE conjugated goat anti-mouse IgG polyclonal secondary antibody. An isotype control antibody (mIgG1-PE mAb) (R D Systems, Minneapolis, MN) was used as negative control. Dotted histogram represents isotype control (mIgG1-PE mAb) staining and filled histogram shows LLT1 expression. MFIR is the mean fluorescence intensity ratio.
Figure Legend Snippet: Prostate cancer cell lines display increased cell surface expression of LLT1 A-F. Surface expression of LLT1 on prostate cancer cells DU145, LNCaP, PC3 and 22Rv1, normal prostate cell PWR-1E and Jurkat (T cell line) was determined by flow cytometry using mouse anti-human LLT1 mAb (clone# 2E5) and a PE conjugated goat anti-mouse IgG polyclonal secondary antibody. An isotype control antibody (mIgG1-PE mAb) (R D Systems, Minneapolis, MN) was used as negative control. Dotted histogram represents isotype control (mIgG1-PE mAb) staining and filled histogram shows LLT1 expression. MFIR is the mean fluorescence intensity ratio.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Negative Control, Staining, Fluorescence

Prostate cancer cells overexpress LLT1 on the cell surface as compared to intracellular LLT1 expression in normal prostate cells Normal prostate cells, PWR-1E and metatstatic prostate cancer cells, PC3 were fixed, blocked, and incubated with mouse anti-human LLT1 antibody with or without permeabilization, followed by anti-mouse Alexa Fluor 488 secondary antibody ( green ). DNA was counterstained with DAPI ( blue ). The slides were examined using LSM 510 Meta confocal microscope system.
Figure Legend Snippet: Prostate cancer cells overexpress LLT1 on the cell surface as compared to intracellular LLT1 expression in normal prostate cells Normal prostate cells, PWR-1E and metatstatic prostate cancer cells, PC3 were fixed, blocked, and incubated with mouse anti-human LLT1 antibody with or without permeabilization, followed by anti-mouse Alexa Fluor 488 secondary antibody ( green ). DNA was counterstained with DAPI ( blue ). The slides were examined using LSM 510 Meta confocal microscope system.

Techniques Used: Expressing, Incubation, Microscopy

Blocking LLT1 on prostate cancer cells enhances NK cell-mediated lysis of prostate cancer cells The cell surface expression of LLT1 on a panel of prostate cancer (A-D) and normal prostate (E) cells was blocked either with a mouse anti-human LLT1 mAb (LLT1) or mouse IgG1 isotype control mAb (cAb) and subsequently labeled with radioactive 51 Cr. The labeled cells were incubated with primary NK cells from a healthy individual and the cytolytic activity was determined by the standard 4 hr radioactive 51 Cr release assay at an Effector to target (E:T) ratios of 25:1, 5:1 and 1:1. Assays were performed in triplicates and the results are representative of two independent experiments. Each data point is the mean value of the repeated experiments and the error bars refer to the means SD generated from the two independent assays. Student's t-test was used to compare cytotoxicity of primary NK cells against prostate cancer cells blocked with LLT1 mAb and the cells incubated with isotype control Ab (mouse IgG1). (*, p
Figure Legend Snippet: Blocking LLT1 on prostate cancer cells enhances NK cell-mediated lysis of prostate cancer cells The cell surface expression of LLT1 on a panel of prostate cancer (A-D) and normal prostate (E) cells was blocked either with a mouse anti-human LLT1 mAb (LLT1) or mouse IgG1 isotype control mAb (cAb) and subsequently labeled with radioactive 51 Cr. The labeled cells were incubated with primary NK cells from a healthy individual and the cytolytic activity was determined by the standard 4 hr radioactive 51 Cr release assay at an Effector to target (E:T) ratios of 25:1, 5:1 and 1:1. Assays were performed in triplicates and the results are representative of two independent experiments. Each data point is the mean value of the repeated experiments and the error bars refer to the means SD generated from the two independent assays. Student's t-test was used to compare cytotoxicity of primary NK cells against prostate cancer cells blocked with LLT1 mAb and the cells incubated with isotype control Ab (mouse IgG1). (*, p

Techniques Used: Blocking Assay, Lysis, Expressing, Labeling, Incubation, Activity Assay, Release Assay, Generated

Human prostate cancer cells express LLT1 A. mRNA expression of LLT1 on prostate cancer cell lines PC3, DU145, LNCaP, 22Rv1, normal prostate cell PWR-1E and Jurkat (T cell line) was determined by qRT-PCR. LLT1 expression was determined by using LLT1 sequence specific primers and Taqman gene expression assays in an Eppendorf Realplex2 Mastercycler. Reactions were done in 20 μl triplicates using the ΔΔCT method, with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. Each bar represents a mean ± s.e. of three independent experiments. B. LLT1 protein expression was analyzed by Western blotting in a panel of prostate cancer cell lines including leukemic Jurkat cells. GAPDH was used as a loading control. C. A bar graph showing densitometric analysis of LLT1 protein expression normalized to GAPDH. Each bar represents the mean ± s.e. of three independent experiments.
Figure Legend Snippet: Human prostate cancer cells express LLT1 A. mRNA expression of LLT1 on prostate cancer cell lines PC3, DU145, LNCaP, 22Rv1, normal prostate cell PWR-1E and Jurkat (T cell line) was determined by qRT-PCR. LLT1 expression was determined by using LLT1 sequence specific primers and Taqman gene expression assays in an Eppendorf Realplex2 Mastercycler. Reactions were done in 20 μl triplicates using the ΔΔCT method, with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. Each bar represents a mean ± s.e. of three independent experiments. B. LLT1 protein expression was analyzed by Western blotting in a panel of prostate cancer cell lines including leukemic Jurkat cells. GAPDH was used as a loading control. C. A bar graph showing densitometric analysis of LLT1 protein expression normalized to GAPDH. Each bar represents the mean ± s.e. of three independent experiments.

Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Western Blot

58) Product Images from "Targeting Inflammatory T Helper Cells via Retinoic Acid-Related Orphan Receptor Gamma t Is Ineffective to Prevent Allo-Response-Driven Colitis"

Article Title: Targeting Inflammatory T Helper Cells via Retinoic Acid-Related Orphan Receptor Gamma t Is Ineffective to Prevent Allo-Response-Driven Colitis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01138

Retinoic acid-related orphan receptor gamma t-independent intestinal graft-versus-host disease (GvHD) is controlled by basic leucine zipper transcription factor ATF-like (BATF). (A) Transcript levels of Batf in colonic tissue of allo-HSCT BALB/c mice with severe signs of intestinal GvHD following transplantation of allogeneic CD3 + T cells derived from of Batf −/− and Rorc −/− mice were measured by quantitative real-time PCR (qPCR) at day 15 ( Batf −/− n = 12; Rorc −/− n = 2) and day 30 ( Batf −/− n = 12; Rorc −/− n = 4). Data represent pooled data from two independent experiments. Gene expression levels detected in colonic tissues of no T cells (noT) controls at day 15 were arbitrarily set down to 1 and all other gene expression levels were normalized to the expression level detected within noT controls. (B) Transcript levels of Rorc in colonic tissue of irradiated BALB/c mice transplanted with T cell depleted bone marrow alone (noT, n = 11) or followed by injection of allogeneic CD3 + T cells of wild-type (WT) ( n = 12) mice were measured by qPCR at day 30. (C) Transcript levels of RORC in colonic tissue biopsies from allo-HCT patients were measured by qPCR. Samples were grouped into indicated categories based on the absence (−GvHD; n = 30) or presence (+GvHD; n = 22) of GvHD-associated histopathological lesions or by the absence (−apoptosis; n = 32) or presence (+apoptosis; n = 20) of GvHD-related epithelial cell apoptosis. Data are shown as normalized relative RORC expression levels calculated from a standard curve. (D–G) Experimental GvHD induction was achieved as described in Figures 1 and 2 . To this end, allogeneic hematopoietic stem cell transplantation BALB/c mice were transplanted with allogeneic CD3 + T cells derived from WT ( n = 4), Rorc −/− ( n = 3), or Rorc −/− Batf −/− ( n = 3) mice or received no T cells (noT) as a control ( n = 4). (D) Mice were assessed three times a week for signs and severity of systemic GvHD. Data were analyzed by two-way ANOVA testing followed by Bonferroni’s multiple comparisons posttest. **** p
Figure Legend Snippet: Retinoic acid-related orphan receptor gamma t-independent intestinal graft-versus-host disease (GvHD) is controlled by basic leucine zipper transcription factor ATF-like (BATF). (A) Transcript levels of Batf in colonic tissue of allo-HSCT BALB/c mice with severe signs of intestinal GvHD following transplantation of allogeneic CD3 + T cells derived from of Batf −/− and Rorc −/− mice were measured by quantitative real-time PCR (qPCR) at day 15 ( Batf −/− n = 12; Rorc −/− n = 2) and day 30 ( Batf −/− n = 12; Rorc −/− n = 4). Data represent pooled data from two independent experiments. Gene expression levels detected in colonic tissues of no T cells (noT) controls at day 15 were arbitrarily set down to 1 and all other gene expression levels were normalized to the expression level detected within noT controls. (B) Transcript levels of Rorc in colonic tissue of irradiated BALB/c mice transplanted with T cell depleted bone marrow alone (noT, n = 11) or followed by injection of allogeneic CD3 + T cells of wild-type (WT) ( n = 12) mice were measured by qPCR at day 30. (C) Transcript levels of RORC in colonic tissue biopsies from allo-HCT patients were measured by qPCR. Samples were grouped into indicated categories based on the absence (−GvHD; n = 30) or presence (+GvHD; n = 22) of GvHD-associated histopathological lesions or by the absence (−apoptosis; n = 32) or presence (+apoptosis; n = 20) of GvHD-related epithelial cell apoptosis. Data are shown as normalized relative RORC expression levels calculated from a standard curve. (D–G) Experimental GvHD induction was achieved as described in Figures 1 and 2 . To this end, allogeneic hematopoietic stem cell transplantation BALB/c mice were transplanted with allogeneic CD3 + T cells derived from WT ( n = 4), Rorc −/− ( n = 3), or Rorc −/− Batf −/− ( n = 3) mice or received no T cells (noT) as a control ( n = 4). (D) Mice were assessed three times a week for signs and severity of systemic GvHD. Data were analyzed by two-way ANOVA testing followed by Bonferroni’s multiple comparisons posttest. **** p

Techniques Used: Mouse Assay, Transplantation Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Irradiation, Injection

Retinoic acid-related orphan receptor gamma t (RORγt)-dependent T helper (Th17) cells are dispensable for acute intestinal graft-versus-host disease (GvHD). (A) Naïve CD4 + T cells were isolated from the spleen by negative selection using magnetic microbeads and in vitro polarized into inflammatory Th17 cells by co-culturing anti-CD3/anti-CD28 activated T cells in the presence of anti-IFNγ antibodies and either without (−) or with (+) recombinant interleukin (IL)-1β, IL-6, and IL-23. After 5 days, T cells were harvested, RNA was isolated, and transcribed into cDNA followed by quantitative real-time PCR (qPCR) analyses of Il23r transcript levels. Gene expression levels detected within T cells cultured under drift conditions were arbitrarily set down to 1 and all other gene expression levels were normalized to the expression level of this control. Data are combined from two individual experiments and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons posttest. ** p
Figure Legend Snippet: Retinoic acid-related orphan receptor gamma t (RORγt)-dependent T helper (Th17) cells are dispensable for acute intestinal graft-versus-host disease (GvHD). (A) Naïve CD4 + T cells were isolated from the spleen by negative selection using magnetic microbeads and in vitro polarized into inflammatory Th17 cells by co-culturing anti-CD3/anti-CD28 activated T cells in the presence of anti-IFNγ antibodies and either without (−) or with (+) recombinant interleukin (IL)-1β, IL-6, and IL-23. After 5 days, T cells were harvested, RNA was isolated, and transcribed into cDNA followed by quantitative real-time PCR (qPCR) analyses of Il23r transcript levels. Gene expression levels detected within T cells cultured under drift conditions were arbitrarily set down to 1 and all other gene expression levels were normalized to the expression level of this control. Data are combined from two individual experiments and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons posttest. ** p

Techniques Used: Isolation, Selection, In Vitro, Recombinant, Real-time Polymerase Chain Reaction, Expressing, Cell Culture

Genetic depletion of retinoic acid-related orphan receptor gamma t-dependent T helper cells preserves the granulocyte-macrophage colony-stimulating factor (GM-CSF) expressing donor T cell pool. Experimental graft-versus-host disease (GvHD) induction was performed as described in Figures 1 and 2 . (A) Gene expression levels in total colonic tissue of wild-type (WT) and Il23r −/− donor T cell receiving mice of Ifng, Csf2 (WT n = 14; Il23r −/− n = 15) and Il17a (WT n = 9; Il23r −/− n = 5) transcripts were analyzed by quantitative real-time PCR (qPCR). (B) Gene expression levels in total colonic tissue of WT and Rorc −/− donor T cell receiving mice of Ifng, Csf2 (WT n = 17; Rorc −/− n = 14) and Il17a (WT n = 7; Rorc −/− n = 8) transcripts were analyzed and quantitated by qPCR around day 30, i.e., when allogeneic hematopoietic stem cell transplantation mice displayed fully established signs of intestinal GvHD. Data represent pooled data from two (A) and at least three (B) individual experiments, respectively. (C,D) In addition, lamina propria mononuclear cells of WT ( n = 6) and Il23r −/− ( n = 6) T cell receiving mice (C) or WT ( n = 4) and Rorc −/− ( n = 3) T cell receiving mice (D) were assessed by flow cytometry for the frequencies and absolute numbers of GM-CSF-, IFNγ- or interleukin 17a (IL-17a)-producing CD4 + donor T cells, respectively, employing intracellular cytokine staining techniques. Displayed data are derived from one representative of two independent experiments and were analyzed by Student’s t -test. * p
Figure Legend Snippet: Genetic depletion of retinoic acid-related orphan receptor gamma t-dependent T helper cells preserves the granulocyte-macrophage colony-stimulating factor (GM-CSF) expressing donor T cell pool. Experimental graft-versus-host disease (GvHD) induction was performed as described in Figures 1 and 2 . (A) Gene expression levels in total colonic tissue of wild-type (WT) and Il23r −/− donor T cell receiving mice of Ifng, Csf2 (WT n = 14; Il23r −/− n = 15) and Il17a (WT n = 9; Il23r −/− n = 5) transcripts were analyzed by quantitative real-time PCR (qPCR). (B) Gene expression levels in total colonic tissue of WT and Rorc −/− donor T cell receiving mice of Ifng, Csf2 (WT n = 17; Rorc −/− n = 14) and Il17a (WT n = 7; Rorc −/− n = 8) transcripts were analyzed and quantitated by qPCR around day 30, i.e., when allogeneic hematopoietic stem cell transplantation mice displayed fully established signs of intestinal GvHD. Data represent pooled data from two (A) and at least three (B) individual experiments, respectively. (C,D) In addition, lamina propria mononuclear cells of WT ( n = 6) and Il23r −/− ( n = 6) T cell receiving mice (C) or WT ( n = 4) and Rorc −/− ( n = 3) T cell receiving mice (D) were assessed by flow cytometry for the frequencies and absolute numbers of GM-CSF-, IFNγ- or interleukin 17a (IL-17a)-producing CD4 + donor T cells, respectively, employing intracellular cytokine staining techniques. Displayed data are derived from one representative of two independent experiments and were analyzed by Student’s t -test. * p

Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Transplantation Assay, Flow Cytometry, Cytometry, Staining, Derivative Assay

59) Product Images from "Combination of Gefitinib and DNA Methylation Inhibitor Decitabine Exerts Synergistic Anti-Cancer Activity in Colon Cancer Cells"

Article Title: Combination of Gefitinib and DNA Methylation Inhibitor Decitabine Exerts Synergistic Anti-Cancer Activity in Colon Cancer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0097719

XAF1 plays a critical role in cellular apoptosis triggered by decitabine combined with gefitinib. (A) and (B) SW1116 and LOVO cells were treated for 48 h with the indicated concentrations of decitabine (DAC) and gefitinib (GEF) either alone or in combination. The mRNA expression levels of XAF1 were determined by real-time quantitative PCR. Expression of β-actin served as control. The data are representative of three independent experiments. Columns, mean; bars, SD. **, P
Figure Legend Snippet: XAF1 plays a critical role in cellular apoptosis triggered by decitabine combined with gefitinib. (A) and (B) SW1116 and LOVO cells were treated for 48 h with the indicated concentrations of decitabine (DAC) and gefitinib (GEF) either alone or in combination. The mRNA expression levels of XAF1 were determined by real-time quantitative PCR. Expression of β-actin served as control. The data are representative of three independent experiments. Columns, mean; bars, SD. **, P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

60) Product Images from "Synthesis, Biological Evaluation, and Docking Studies of a Novel Sulfonamido-Based Gallate as Pro-Chondrogenic Agent for the Treatment of Cartilage"

Article Title: Synthesis, Biological Evaluation, and Docking Studies of a Novel Sulfonamido-Based Gallate as Pro-Chondrogenic Agent for the Treatment of Cartilage

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules22010003

Quantitative analysis of ECM-related gene expressions by qRT-PCR, ( A ) MMP-1; ( B ) MMP-3; and ( C ) TIMP-1. Chondrocytes were cultured with different concentrations of HAMDC, GA and SD-Na for 2, 4 and 6 days. They were then stimulated with IL-1β for another 24 h. Control: cells without any treatment; IL-1β: cells stimulated with IL-1β; C-1 to C-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL HAMDC, respectively; G-1 to G-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL GA, respectively; H-1 to H-3: cells pre-incubated with 2.344, 4.688, and 9.375 μg/mL SD-Na, respectively. The gene expressions of MMP-1, MMP-3, and TIMP-1 in cells treated with HAMDC, GA and SD-Na relative to control were analyzed by the 2 −ΔΔ C t method using GAPDH as the internal control. Each condition was repeated for three times, and data are presented as mean ± SD. ( p
Figure Legend Snippet: Quantitative analysis of ECM-related gene expressions by qRT-PCR, ( A ) MMP-1; ( B ) MMP-3; and ( C ) TIMP-1. Chondrocytes were cultured with different concentrations of HAMDC, GA and SD-Na for 2, 4 and 6 days. They were then stimulated with IL-1β for another 24 h. Control: cells without any treatment; IL-1β: cells stimulated with IL-1β; C-1 to C-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL HAMDC, respectively; G-1 to G-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL GA, respectively; H-1 to H-3: cells pre-incubated with 2.344, 4.688, and 9.375 μg/mL SD-Na, respectively. The gene expressions of MMP-1, MMP-3, and TIMP-1 in cells treated with HAMDC, GA and SD-Na relative to control were analyzed by the 2 −ΔΔ C t method using GAPDH as the internal control. Each condition was repeated for three times, and data are presented as mean ± SD. ( p

Techniques Used: Quantitative RT-PCR, Cell Culture, Incubation

Quantitative comparison of ECM-related gene expression by qRT-PCR: ( A – C ) aggrecan; ( D – F ) collagen II; ( G – I ) Sox9; and ( J – L ) collagen I. The chondrocytes were pre-incubated with different concentrations of HAMDC, GA and SD-Na for 2 days, 4 days and 6 days, and then stimulated by 10 ng/mL IL-1β. Control: cells without any treatment; IL-1β: cells stimulated with IL-1β; C-1 to C-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL HAMDC, respectively; G-1 to G-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL GA, respectively; H-1 to H-3: cells pre-incubated with 2.344, 4.688, and 9.375 μg/mL SD-Na, respectively. The gene expressions of aggrecan, collegen II, Sox9 and collagen I in cells treated with HAMDC, GA and SD-Na relative to control were analyzed by the 2 −ΔΔ C t method using GAPDH as the internal control. Each condition was repeated for three times, and data are presented as mean ± SD ( p
Figure Legend Snippet: Quantitative comparison of ECM-related gene expression by qRT-PCR: ( A – C ) aggrecan; ( D – F ) collagen II; ( G – I ) Sox9; and ( J – L ) collagen I. The chondrocytes were pre-incubated with different concentrations of HAMDC, GA and SD-Na for 2 days, 4 days and 6 days, and then stimulated by 10 ng/mL IL-1β. Control: cells without any treatment; IL-1β: cells stimulated with IL-1β; C-1 to C-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL HAMDC, respectively; G-1 to G-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL GA, respectively; H-1 to H-3: cells pre-incubated with 2.344, 4.688, and 9.375 μg/mL SD-Na, respectively. The gene expressions of aggrecan, collegen II, Sox9 and collagen I in cells treated with HAMDC, GA and SD-Na relative to control were analyzed by the 2 −ΔΔ C t method using GAPDH as the internal control. Each condition was repeated for three times, and data are presented as mean ± SD ( p

Techniques Used: Expressing, Quantitative RT-PCR, Incubation

61) Product Images from "Comparative transcriptome and metabolome analysis suggests bottlenecks that limit seed and oil yields in transgenic Camelina sativa expressing diacylglycerol acyltransferase 1 and glycerol-3-phosphate dehydrogenase"

Article Title: Comparative transcriptome and metabolome analysis suggests bottlenecks that limit seed and oil yields in transgenic Camelina sativa expressing diacylglycerol acyltransferase 1 and glycerol-3-phosphate dehydrogenase

Journal: Biotechnology for Biofuels

doi: 10.1186/s13068-018-1326-2

The gene expression analysis for the selected genes showing differential regulation in Camelina transgenic lines. Data are the fold changes (FC) in expression measured by using both RNA-Seq and qRT-PCR techniques ( a , b ) in both DGAT1 and GPD1, respectively, relative to WT. The fold change values used in the analysis are presented in Additional file 1 : Table S15. Data shown in c , d indicate the relative gene expression for the selected genes measured by qRT-PCR in both DGAT1 and GPD1 lines, respectively, relative to WT. The genes shown here are non-specific lipid transfer 4-like ( NSLT - L ), glycerol-3-phosphate sn -2-acyltransferase 1 ( GPAT1 ), oleosin 5 ( OLE5 ), 3-ketoacyl-synthase 18-like ( KCS18 ), TAG-lipase 2-like ( TAGL2 - L ), acyl CoA thioesterase 13-like ( ACOT13 - L ), cruciferin 3 ( CRU3 ), acyl-CoA:diacylglycerol acyltransferase 1 ( DGAT1 ), oleosin 1 ( OLE1 ), glycerol-3-phosphate acyltransferase 9 ( GPAT9 ), lysophosphatidyl acyltransferase 2 ( LPAT2 ), glycerol-3-phosphate transporter 1 ( GLPT1 ), lysophosphatidyl acyltransferase 5 ( LPAT5 ), glucose-6-phosphate l-epimerase ( G6Pe ), diacylglycerol kinase 3-like isoform X1 ( DAGK ), 3-keto acyl-synthase 6 ( KCS6 ), acyl-activating enzyme 7 ( Acylae7 ), glycerol-3-phosphate acyltransferase 5 ( GPAT5 )
Figure Legend Snippet: The gene expression analysis for the selected genes showing differential regulation in Camelina transgenic lines. Data are the fold changes (FC) in expression measured by using both RNA-Seq and qRT-PCR techniques ( a , b ) in both DGAT1 and GPD1, respectively, relative to WT. The fold change values used in the analysis are presented in Additional file 1 : Table S15. Data shown in c , d indicate the relative gene expression for the selected genes measured by qRT-PCR in both DGAT1 and GPD1 lines, respectively, relative to WT. The genes shown here are non-specific lipid transfer 4-like ( NSLT - L ), glycerol-3-phosphate sn -2-acyltransferase 1 ( GPAT1 ), oleosin 5 ( OLE5 ), 3-ketoacyl-synthase 18-like ( KCS18 ), TAG-lipase 2-like ( TAGL2 - L ), acyl CoA thioesterase 13-like ( ACOT13 - L ), cruciferin 3 ( CRU3 ), acyl-CoA:diacylglycerol acyltransferase 1 ( DGAT1 ), oleosin 1 ( OLE1 ), glycerol-3-phosphate acyltransferase 9 ( GPAT9 ), lysophosphatidyl acyltransferase 2 ( LPAT2 ), glycerol-3-phosphate transporter 1 ( GLPT1 ), lysophosphatidyl acyltransferase 5 ( LPAT5 ), glucose-6-phosphate l-epimerase ( G6Pe ), diacylglycerol kinase 3-like isoform X1 ( DAGK ), 3-keto acyl-synthase 6 ( KCS6 ), acyl-activating enzyme 7 ( Acylae7 ), glycerol-3-phosphate acyltransferase 5 ( GPAT5 )

Techniques Used: Expressing, Transgenic Assay, RNA Sequencing Assay, Quantitative RT-PCR

62) Product Images from "Ethanol inhibition of aspartyl-asparaginyl-?-hydroxylase in fetal alcohol spectrum disorder: Potential link to the impairments in central nervous system neuronal migration"

Article Title: Ethanol inhibition of aspartyl-asparaginyl-?-hydroxylase in fetal alcohol spectrum disorder: Potential link to the impairments in central nervous system neuronal migration

Journal: Alcohol (Fayetteville, N.Y.)

doi: 10.1016/j.alcohol.2008.09.009

Effects of chronic gestational exposure to ethanol on aspartyl (asparaginyl)-β-hydroxylase (AAH) expression. Protein homogenates of cerebella from P1 rat pups exposed to 0, 8, 18, 26, or 37% ethanol via pregnant dams’ liquid diets (see Experimental procedures) were used to measure AAH protein expression. (A) Example Western blot analysis using the A85G6 mAb. The blots were stripped and re-probed with polyclonal antibodies to the p85 subunit of PI3 kinase as a loading control. (B) Digital image quantification of A85G6 Western blot signals (~86 kD band-arrow), including three samples per ethanol dose. Levels of immunoreactivity were measured in arbitrary densitometry units (DU). (C) A85G6 immunoreactivity measured by enzyme-linked immunosorbent assay (eight samples/group) using HRP-conjugated secondary antibody, Amplex Red fluorophore, and an M5 microplate reader (Ex 530 nm/Em 590 nm). (D) Western blot analysis was also performed using the A85E6 mAb (blot not shown). Graph depicts results of digital image quantified signals reflecting A85E6 immunoreactivity (~52 kD band), including three samples per ethanol dose. (E) AAH mRNA and (F) 18S rRNA levels were measured by qRT-PCR. AAH mRNA levels were normalized to 18S measured in corresponding samples. All graphs depict mean ±standard error of the mean of results. Statistical comparisons were made using one-way analysis of variance and the post hoc Tukey—Kramer test for significance. Significant P values relative to control (0% ethanol) are indicated over the bars. * P
Figure Legend Snippet: Effects of chronic gestational exposure to ethanol on aspartyl (asparaginyl)-β-hydroxylase (AAH) expression. Protein homogenates of cerebella from P1 rat pups exposed to 0, 8, 18, 26, or 37% ethanol via pregnant dams’ liquid diets (see Experimental procedures) were used to measure AAH protein expression. (A) Example Western blot analysis using the A85G6 mAb. The blots were stripped and re-probed with polyclonal antibodies to the p85 subunit of PI3 kinase as a loading control. (B) Digital image quantification of A85G6 Western blot signals (~86 kD band-arrow), including three samples per ethanol dose. Levels of immunoreactivity were measured in arbitrary densitometry units (DU). (C) A85G6 immunoreactivity measured by enzyme-linked immunosorbent assay (eight samples/group) using HRP-conjugated secondary antibody, Amplex Red fluorophore, and an M5 microplate reader (Ex 530 nm/Em 590 nm). (D) Western blot analysis was also performed using the A85E6 mAb (blot not shown). Graph depicts results of digital image quantified signals reflecting A85E6 immunoreactivity (~52 kD band), including three samples per ethanol dose. (E) AAH mRNA and (F) 18S rRNA levels were measured by qRT-PCR. AAH mRNA levels were normalized to 18S measured in corresponding samples. All graphs depict mean ±standard error of the mean of results. Statistical comparisons were made using one-way analysis of variance and the post hoc Tukey—Kramer test for significance. Significant P values relative to control (0% ethanol) are indicated over the bars. * P

Techniques Used: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

63) Product Images from "Two Trichothecene Mycotoxins from Myrotheciumroridum Induce Apoptosis of HepG-2 Cells via Caspase Activation and Disruption of Mitochondrial Membrane Potential"

Article Title: Two Trichothecene Mycotoxins from Myrotheciumroridum Induce Apoptosis of HepG-2 Cells via Caspase Activation and Disruption of Mitochondrial Membrane Potential

Journal: Molecules

doi: 10.3390/molecules21060781

Western blot analysis of the activation of caspases and the expression of Bax and Bcl-2 proteins in HepG-2 cells treated with mytoxin B and epiroridin acid for 24 h, respectively: ( A ) detection of caspase-3 and caspase-9: ( 1 ) control; ( 2 ) mytoxin B-treated HepG-2 cells; ( 3 ) epiroridin acid-treated HepG-2 cells; ( B ) detection of Bcl-2 protein: ( 1 ) epiroridin acid-treated HepG-2 cells; ( 2 ) mytoxin B-treated HepG-2 cells; ( 3 ) control; ( C ) detection of Bax protein: ( 1 ) epiroridin acid-treated HepG-2 cells; ( 2 ) mytoxin B-treated HepG-2 cells; ( 3 ) control.
Figure Legend Snippet: Western blot analysis of the activation of caspases and the expression of Bax and Bcl-2 proteins in HepG-2 cells treated with mytoxin B and epiroridin acid for 24 h, respectively: ( A ) detection of caspase-3 and caspase-9: ( 1 ) control; ( 2 ) mytoxin B-treated HepG-2 cells; ( 3 ) epiroridin acid-treated HepG-2 cells; ( B ) detection of Bcl-2 protein: ( 1 ) epiroridin acid-treated HepG-2 cells; ( 2 ) mytoxin B-treated HepG-2 cells; ( 3 ) control; ( C ) detection of Bax protein: ( 1 ) epiroridin acid-treated HepG-2 cells; ( 2 ) mytoxin B-treated HepG-2 cells; ( 3 ) control.

Techniques Used: Western Blot, Activation Assay, Expressing

qRT-PCR analysis of the expression levels of bcl-2 , bax , and Fasl in HepG-2 cells treated with mytoxin B and epiroridin acid for 24 h, respectively: ( A ) the expression level of bcl-2 gene; ( B ) the expression level of bax gene; ( C ) the expression level of Fasl gene.
Figure Legend Snippet: qRT-PCR analysis of the expression levels of bcl-2 , bax , and Fasl in HepG-2 cells treated with mytoxin B and epiroridin acid for 24 h, respectively: ( A ) the expression level of bcl-2 gene; ( B ) the expression level of bax gene; ( C ) the expression level of Fasl gene.

Techniques Used: Quantitative RT-PCR, Expressing

64) Product Images from "Synthesis, Biological Evaluation, and Docking Studies of a Novel Sulfonamido-Based Gallate as Pro-Chondrogenic Agent for the Treatment of Cartilage"

Article Title: Synthesis, Biological Evaluation, and Docking Studies of a Novel Sulfonamido-Based Gallate as Pro-Chondrogenic Agent for the Treatment of Cartilage

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules22010003

Quantitative analysis of ECM-related gene expressions by qRT-PCR, ( A ) MMP-1; ( B ) MMP-3; and ( C ) TIMP-1. Chondrocytes were cultured with different concentrations of HAMDC, GA and SD-Na for 2, 4 and 6 days. They were then stimulated with IL-1β for another 24 h. Control: cells without any treatment; IL-1β: cells stimulated with IL-1β; C-1 to C-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL HAMDC, respectively; G-1 to G-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL GA, respectively; H-1 to H-3: cells pre-incubated with 2.344, 4.688, and 9.375 μg/mL SD-Na, respectively. The gene expressions of MMP-1, MMP-3, and TIMP-1 in cells treated with HAMDC, GA and SD-Na relative to control were analyzed by the 2 −ΔΔ C t method using GAPDH as the internal control. Each condition was repeated for three times, and data are presented as mean ± SD. ( p
Figure Legend Snippet: Quantitative analysis of ECM-related gene expressions by qRT-PCR, ( A ) MMP-1; ( B ) MMP-3; and ( C ) TIMP-1. Chondrocytes were cultured with different concentrations of HAMDC, GA and SD-Na for 2, 4 and 6 days. They were then stimulated with IL-1β for another 24 h. Control: cells without any treatment; IL-1β: cells stimulated with IL-1β; C-1 to C-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL HAMDC, respectively; G-1 to G-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL GA, respectively; H-1 to H-3: cells pre-incubated with 2.344, 4.688, and 9.375 μg/mL SD-Na, respectively. The gene expressions of MMP-1, MMP-3, and TIMP-1 in cells treated with HAMDC, GA and SD-Na relative to control were analyzed by the 2 −ΔΔ C t method using GAPDH as the internal control. Each condition was repeated for three times, and data are presented as mean ± SD. ( p

Techniques Used: Quantitative RT-PCR, Cell Culture, Incubation

Quantitative comparison of ECM-related gene expression by qRT-PCR: ( A – C ) aggrecan; ( D – F ) collagen II; ( G – I ) Sox9; and ( J – L ) collagen I. The chondrocytes were pre-incubated with different concentrations of HAMDC, GA and SD-Na for 2 days, 4 days and 6 days, and then stimulated by 10 ng/mL IL-1β. Control: cells without any treatment; IL-1β: cells stimulated with IL-1β; C-1 to C-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL HAMDC, respectively; G-1 to G-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL GA, respectively; H-1 to H-3: cells pre-incubated with 2.344, 4.688, and 9.375 μg/mL SD-Na, respectively. The gene expressions of aggrecan, collegen II, Sox9 and collagen I in cells treated with HAMDC, GA and SD-Na relative to control were analyzed by the 2 −ΔΔ C t method using GAPDH as the internal control. Each condition was repeated for three times, and data are presented as mean ± SD ( p
Figure Legend Snippet: Quantitative comparison of ECM-related gene expression by qRT-PCR: ( A – C ) aggrecan; ( D – F ) collagen II; ( G – I ) Sox9; and ( J – L ) collagen I. The chondrocytes were pre-incubated with different concentrations of HAMDC, GA and SD-Na for 2 days, 4 days and 6 days, and then stimulated by 10 ng/mL IL-1β. Control: cells without any treatment; IL-1β: cells stimulated with IL-1β; C-1 to C-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL HAMDC, respectively; G-1 to G-3: cells pre-incubated with 3.125, 6.250, and 12.500 μg/mL GA, respectively; H-1 to H-3: cells pre-incubated with 2.344, 4.688, and 9.375 μg/mL SD-Na, respectively. The gene expressions of aggrecan, collegen II, Sox9 and collagen I in cells treated with HAMDC, GA and SD-Na relative to control were analyzed by the 2 −ΔΔ C t method using GAPDH as the internal control. Each condition was repeated for three times, and data are presented as mean ± SD ( p

Techniques Used: Expressing, Quantitative RT-PCR, Incubation

65) Product Images from "SPARC-induced increase in glioma matrix and decrease in vascularity are associated with reduced VEGF expression and secretion"

Article Title: SPARC-induced increase in glioma matrix and decrease in vascularity are associated with reduced VEGF expression and secretion

Journal: International Journal of Cancer. Journal International du Cancer

doi: 10.1002/ijc.23450

Immunohistochemical, Western blot and RT-PCR analysis of VEGF protein and transcripts in control- (−SPARC) and SPARC- (+SPARC) transfected clones. ( a ) Representative tumor sections immunohistochemically stained for VEGF expression (×40). Note the decreased VEGF expression in the SPARC-expressing tumors. ( b ) Control and SPARC-expressing spheroids were assessed for VEGF and SPARC expression and secretion by Western blot analysis. L: lysate, M: medium. The same blot was used for all lysate analyses. Actin detection was used as a loading control. Note that increased SPARC expression correlated with decreased VEGF expression and secretion. ( c ) RT-PCR was performed to detect all 4 major VEGF isoforms as indicated. ( b and c ) (∧) Indicates that the signal is on the same gel but moved closer. ( d ) Real-time RT-PCR analysis of the VEGF165 isoform. Note that enhanced SPARC expression is associated with decreased VEGF165 transcript abundance. P-parental clonal U87MG-derived cell line, VC1- and VC1-vector control cell lines, S1- and S2- SPARC-transfected cell lines.
Figure Legend Snippet: Immunohistochemical, Western blot and RT-PCR analysis of VEGF protein and transcripts in control- (−SPARC) and SPARC- (+SPARC) transfected clones. ( a ) Representative tumor sections immunohistochemically stained for VEGF expression (×40). Note the decreased VEGF expression in the SPARC-expressing tumors. ( b ) Control and SPARC-expressing spheroids were assessed for VEGF and SPARC expression and secretion by Western blot analysis. L: lysate, M: medium. The same blot was used for all lysate analyses. Actin detection was used as a loading control. Note that increased SPARC expression correlated with decreased VEGF expression and secretion. ( c ) RT-PCR was performed to detect all 4 major VEGF isoforms as indicated. ( b and c ) (∧) Indicates that the signal is on the same gel but moved closer. ( d ) Real-time RT-PCR analysis of the VEGF165 isoform. Note that enhanced SPARC expression is associated with decreased VEGF165 transcript abundance. P-parental clonal U87MG-derived cell line, VC1- and VC1-vector control cell lines, S1- and S2- SPARC-transfected cell lines.

Techniques Used: Immunohistochemistry, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Clone Assay, Staining, Expressing, Quantitative RT-PCR, Derivative Assay, Plasmid Preparation

66) Product Images from "Generation of retinal pigmented epithelium from iPSCs derived from the conjunctiva of donors with and without age related macular degeneration"

Article Title: Generation of retinal pigmented epithelium from iPSCs derived from the conjunctiva of donors with and without age related macular degeneration

Journal: PLoS ONE

doi: 10.1371/journal.pone.0173575

Derivation of RPE from conjunctival iPSCs using fully defined culture conditions. A Comparison of qRT-PCR analysis of key markers of initial RPE phenotype induction from iPSC line UMN AMD1-2B6 differentiated on three different substrates, undefined Matrigel or defined substrates vitronectin or Synthemax ) compatible with requirements for cGMP-compliant RPE cell manufacture. Following the initial induction iPSC-derived RPE cultures were maintained and expanded on the different substrates for three passages. B Comparison of qRT PCR analysis of gene expression of RPE made from iPSC line UMN AMD1-2B6 maintained on the three different substrates.
Figure Legend Snippet: Derivation of RPE from conjunctival iPSCs using fully defined culture conditions. A Comparison of qRT-PCR analysis of key markers of initial RPE phenotype induction from iPSC line UMN AMD1-2B6 differentiated on three different substrates, undefined Matrigel or defined substrates vitronectin or Synthemax ) compatible with requirements for cGMP-compliant RPE cell manufacture. Following the initial induction iPSC-derived RPE cultures were maintained and expanded on the different substrates for three passages. B Comparison of qRT PCR analysis of gene expression of RPE made from iPSC line UMN AMD1-2B6 maintained on the three different substrates.

Techniques Used: Quantitative RT-PCR, Derivative Assay, Expressing

Characterization of the initial induction of RPE from donors with and without AMD. iPSC lines derived from donors with and without AMD were differentiated into RPE using the rapid 14 day induction protocol. A Immunohistochemistry detected the expression of proteins indicating the rapid establishment of early eye field and RPE identity at 6 and 14 days after the start of directed differentiation. Protein expression is shown from the differentiation of 2 iPSC lines from the same donor (UMN AMD1-1D3, UMN AMD1-2B6 –MGS1 no disease) and from iPSC line UMN-AMD2-2A4 (MGS2—AMD disease). (Scale bar 50μm) (B) qRT-PCR analysis comparing the time course of eye field and early RPE gene expression during RPE differentiation in multiple iPSC lines derived from three donors with and without AMD.
Figure Legend Snippet: Characterization of the initial induction of RPE from donors with and without AMD. iPSC lines derived from donors with and without AMD were differentiated into RPE using the rapid 14 day induction protocol. A Immunohistochemistry detected the expression of proteins indicating the rapid establishment of early eye field and RPE identity at 6 and 14 days after the start of directed differentiation. Protein expression is shown from the differentiation of 2 iPSC lines from the same donor (UMN AMD1-1D3, UMN AMD1-2B6 –MGS1 no disease) and from iPSC line UMN-AMD2-2A4 (MGS2—AMD disease). (Scale bar 50μm) (B) qRT-PCR analysis comparing the time course of eye field and early RPE gene expression during RPE differentiation in multiple iPSC lines derived from three donors with and without AMD.

Techniques Used: Derivative Assay, Immunohistochemistry, Expressing, Quantitative RT-PCR

67) Product Images from "In vitro model for the analysis of synovial fibroblast-mediated degradation of intact cartilage"

Article Title: In vitro model for the analysis of synovial fibroblast-mediated degradation of intact cartilage

Journal: Arthritis Research & Therapy

doi: 10.1186/ar2618

Expression of bovine MMP-1 and MMP-3 mRNA. Cartilage from (a, b) monoculture (n = 5, with two replicates each) and (c, d) human mRNA/protein in synovial fibroblasts (SFB) after co-culture with cartilage (n = 5, with two replicates each) with/without stimulation with TNF-α, IL-1β or TNF-α/IL-1β (14 days) were used.gene expression values (means ± standard error of the mean (SEM)), as determined by quantitiative PCR, are expressed as percentage of the values in non-stimulated samples (100%). In addition, (e, f) the values for human MMP-1 and MMP-3 protein secreted by SFB into the supernatant of co-cultures are shown. The protein levels, as measured in the supernatant by ELISA, are expressed as means +/- SEM. § p ≤ 0.05 Mann-Whitney U Test compared with non-stimulated control; * p ≤ 0.05 Mann-Whitney U Test compared with stimulation with TNF-α.
Figure Legend Snippet: Expression of bovine MMP-1 and MMP-3 mRNA. Cartilage from (a, b) monoculture (n = 5, with two replicates each) and (c, d) human mRNA/protein in synovial fibroblasts (SFB) after co-culture with cartilage (n = 5, with two replicates each) with/without stimulation with TNF-α, IL-1β or TNF-α/IL-1β (14 days) were used.gene expression values (means ± standard error of the mean (SEM)), as determined by quantitiative PCR, are expressed as percentage of the values in non-stimulated samples (100%). In addition, (e, f) the values for human MMP-1 and MMP-3 protein secreted by SFB into the supernatant of co-cultures are shown. The protein levels, as measured in the supernatant by ELISA, are expressed as means +/- SEM. § p ≤ 0.05 Mann-Whitney U Test compared with non-stimulated control; * p ≤ 0.05 Mann-Whitney U Test compared with stimulation with TNF-α.

Techniques Used: Expressing, Co-Culture Assay, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

68) Product Images from "Specific siRNA Targeting Receptor for Advanced Glycation End Products (RAGE) Decreases Proliferation in Human Breast Cancer Cell Lines"

Article Title: Specific siRNA Targeting Receptor for Advanced Glycation End Products (RAGE) Decreases Proliferation in Human Breast Cancer Cell Lines

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms14047959

RAGE siRNA silencing efficiency by ( a ) qRT-PCR ( * , ** , # p
Figure Legend Snippet: RAGE siRNA silencing efficiency by ( a ) qRT-PCR ( * , ** , # p

Techniques Used: Quantitative RT-PCR

RAGE mRNA and protein expression (normalized to β-actin expression) in MCF-7, SK-Br-3, MDA-MB-231 cell lines analyzed by ( A ) qRT-PCR and ( B ) Western Blot. The results indicate that RAGE expression in MDA-MB-231 was significantly higher than in MCF-7 and SK-Br-3; (# p
Figure Legend Snippet: RAGE mRNA and protein expression (normalized to β-actin expression) in MCF-7, SK-Br-3, MDA-MB-231 cell lines analyzed by ( A ) qRT-PCR and ( B ) Western Blot. The results indicate that RAGE expression in MDA-MB-231 was significantly higher than in MCF-7 and SK-Br-3; (# p

Techniques Used: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR, Western Blot

There was decreased expression of PCNA mRNA after transfection with RAGE siRNA as shown by qRT-PCR in MDA-MB-231 ( a ), SK-Br-3 ( b ) and MCF-7 ( c ) cell lines respectively ( * , ** , # p
Figure Legend Snippet: There was decreased expression of PCNA mRNA after transfection with RAGE siRNA as shown by qRT-PCR in MDA-MB-231 ( a ), SK-Br-3 ( b ) and MCF-7 ( c ) cell lines respectively ( * , ** , # p

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Multiple Displacement Amplification

There was decreased expression of CyclinD1 mRNA after transfection with RAGE siRNA as shown by qRT-PCR in MDA-MB-231 ( a ), SK-Br-3 ( b ) and MCF-7 ( c ) cell lines respectively ( * , ** , # p
Figure Legend Snippet: There was decreased expression of CyclinD1 mRNA after transfection with RAGE siRNA as shown by qRT-PCR in MDA-MB-231 ( a ), SK-Br-3 ( b ) and MCF-7 ( c ) cell lines respectively ( * , ** , # p

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Multiple Displacement Amplification

There was decreased expression of NF-κB p65 mRNA after transfection with RAGE siRNA as shown by qRT-PCR in MDA-MB-231 ( a ), SK-Br-3 ( b ) and MCF-7 ( c ) cell lines respectively ( * , ** , # p
Figure Legend Snippet: There was decreased expression of NF-κB p65 mRNA after transfection with RAGE siRNA as shown by qRT-PCR in MDA-MB-231 ( a ), SK-Br-3 ( b ) and MCF-7 ( c ) cell lines respectively ( * , ** , # p

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Multiple Displacement Amplification

69) Product Images from "Ectopic Expression of Aeluropus littoralis Plasma Membrane Protein Gene AlTMP1 Confers Abiotic Stress Tolerance in Transgenic Tobacco by Improving Water Status and Cation Homeostasis"

Article Title: Ectopic Expression of Aeluropus littoralis Plasma Membrane Protein Gene AlTMP1 Confers Abiotic Stress Tolerance in Transgenic Tobacco by Improving Water Status and Cation Homeostasis

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18040692

Molecular analysis of T2 transgenic tobacco lines (L1, L2, and L3) expressing AlTMP1 . ( A ) Schematic map of the T-DNA inserted into the binary vector pCAMBIA1390:: AlTMP1 , used for tobacco transformation; LB: left border T-DNA repeat; RB: right border T-DNA repeat; hpt : gene codes for hygromycin phosphotransferase which detoxifies the antibiotic hygromycin B; ( B ) PCR amplification products from T2 transgenic and non-transgenic (NT) tobacco plants. Genomic DNA of NT and transgenic tobacco lines was used to amplify AlTMP1 gene with specific primers; lane 1: ddH 2 O; lane 2: NT tobacco DNA; lanes 3–5: transgenic clones L1, L2, and L3 DNA; lane 6: pCAMBIA1390:: AlTMP1 binary vector DNA used as positive control; lane L: Lambda DNA Pst I Marker; ( C ) Expression pattern of the AlTMP1 gene in transgenic lines (L1, L2, and L3). The RT-PCR analysis was performed with AlTMP1 specific primers using the RNA isolated from the transgenic lines and NT plants. Actin amplification was used as an internal control.
Figure Legend Snippet: Molecular analysis of T2 transgenic tobacco lines (L1, L2, and L3) expressing AlTMP1 . ( A ) Schematic map of the T-DNA inserted into the binary vector pCAMBIA1390:: AlTMP1 , used for tobacco transformation; LB: left border T-DNA repeat; RB: right border T-DNA repeat; hpt : gene codes for hygromycin phosphotransferase which detoxifies the antibiotic hygromycin B; ( B ) PCR amplification products from T2 transgenic and non-transgenic (NT) tobacco plants. Genomic DNA of NT and transgenic tobacco lines was used to amplify AlTMP1 gene with specific primers; lane 1: ddH 2 O; lane 2: NT tobacco DNA; lanes 3–5: transgenic clones L1, L2, and L3 DNA; lane 6: pCAMBIA1390:: AlTMP1 binary vector DNA used as positive control; lane L: Lambda DNA Pst I Marker; ( C ) Expression pattern of the AlTMP1 gene in transgenic lines (L1, L2, and L3). The RT-PCR analysis was performed with AlTMP1 specific primers using the RNA isolated from the transgenic lines and NT plants. Actin amplification was used as an internal control.

Techniques Used: Transgenic Assay, Expressing, Plasmid Preparation, Transformation Assay, Polymerase Chain Reaction, Amplification, Clone Assay, Positive Control, Lambda DNA Preparation, Marker, Reverse Transcription Polymerase Chain Reaction, Isolation

qRT-PCR analysis of AlTMP1 transcripts in A. littoralis plants challenged with various stresses. Two-month-old A. littoralis plants were treated with 300 mM NaCl, 4 °C, 20% PEG 8000 and 100 µM abscisic acid (ABA) for 2, 4, 6, 24 and 48 h. The rRNA18s gene was used as an internal control. Data from qRT-PCR experiments were analyzed according to the 2 −∆∆ C t method. Vertical bars indicate standard deviation calculated from three replicates. Values are mean ± s.e. ( n = 3). Means denoted by the same letter did not differ significantly at p
Figure Legend Snippet: qRT-PCR analysis of AlTMP1 transcripts in A. littoralis plants challenged with various stresses. Two-month-old A. littoralis plants were treated with 300 mM NaCl, 4 °C, 20% PEG 8000 and 100 µM abscisic acid (ABA) for 2, 4, 6, 24 and 48 h. The rRNA18s gene was used as an internal control. Data from qRT-PCR experiments were analyzed according to the 2 −∆∆ C t method. Vertical bars indicate standard deviation calculated from three replicates. Values are mean ± s.e. ( n = 3). Means denoted by the same letter did not differ significantly at p

Techniques Used: Quantitative RT-PCR, Standard Deviation

70) Product Images from "Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens"

Article Title: Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens

Journal: Analytical chemistry

doi: 10.1021/acs.analchem.5b01594

Paper-based extraction of influenza A (H1N1) RNA. (a) Schematic of the paper RNA extraction method. Nasopharyngeal swab samples are lysed in a Glycoblue-containing lysis buffer and filtered through our paper extraction setup (scale bar = 10 mm). Co-precipitated RNA and Glycoblue result in a visible blue film (inset, scale bar = 1 mm). (b) Extraction set up. (c) Paper extractions of H1N1 RNA standards and centrifuge control extraction yields quantified via qRT-PCR. Error bars: standard deviation, n = 3. Percentage values indicate paper extraction yields compared to centrifuge control yields.
Figure Legend Snippet: Paper-based extraction of influenza A (H1N1) RNA. (a) Schematic of the paper RNA extraction method. Nasopharyngeal swab samples are lysed in a Glycoblue-containing lysis buffer and filtered through our paper extraction setup (scale bar = 10 mm). Co-precipitated RNA and Glycoblue result in a visible blue film (inset, scale bar = 1 mm). (b) Extraction set up. (c) Paper extractions of H1N1 RNA standards and centrifuge control extraction yields quantified via qRT-PCR. Error bars: standard deviation, n = 3. Percentage values indicate paper extraction yields compared to centrifuge control yields.

Techniques Used: RNA Extraction, Lysis, Quantitative RT-PCR, Standard Deviation

71) Product Images from "Comparative transcriptome profiling of Blumeria graminis f. sp. tritici during compatible and incompatible interactions with sister wheat lines carrying and lacking Pm40"

Article Title: Comparative transcriptome profiling of Blumeria graminis f. sp. tritici during compatible and incompatible interactions with sister wheat lines carrying and lacking Pm40

Journal: PLoS ONE

doi: 10.1371/journal.pone.0198891

qRT-PCR of cDNA from a time-course of L958 interactions and L658 interactions of five target genes. (A), (C), (E), (G), and (I) represent TID_seq197, TID_seq273, TID_seq1665, TID_seq1719, and TID_seq3742 in L658 interactions, respectively; (B), (D), (F), (H), and (J) represent TID_seq197, TID_seq273, TID_seq1665, TID_seq1719, and TID_seq3742 in L958 interactions, respectively. Elongation factor 1-alpha (EF-1α) was used as a reference gene. The bar chart represents the relative expression levels validated by qRT-PCR, and the line chart represents the FPKM of RNA-seq. Different letters indicate statistically significant difference in relative expression of each gene among different time-points. The capital letter represents p
Figure Legend Snippet: qRT-PCR of cDNA from a time-course of L958 interactions and L658 interactions of five target genes. (A), (C), (E), (G), and (I) represent TID_seq197, TID_seq273, TID_seq1665, TID_seq1719, and TID_seq3742 in L658 interactions, respectively; (B), (D), (F), (H), and (J) represent TID_seq197, TID_seq273, TID_seq1665, TID_seq1719, and TID_seq3742 in L958 interactions, respectively. Elongation factor 1-alpha (EF-1α) was used as a reference gene. The bar chart represents the relative expression levels validated by qRT-PCR, and the line chart represents the FPKM of RNA-seq. Different letters indicate statistically significant difference in relative expression of each gene among different time-points. The capital letter represents p

Techniques Used: Quantitative RT-PCR, Expressing, RNA Sequencing Assay

72) Product Images from "Microparticles Mediate the Intercellular Regulation of microRNA-503 and Proline-Rich Tyrosine Kinase 2 to Alter the Migration and Invasion Capacity of Breast Cancer Cells"

Article Title: Microparticles Mediate the Intercellular Regulation of microRNA-503 and Proline-Rich Tyrosine Kinase 2 to Alter the Migration and Invasion Capacity of Breast Cancer Cells

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2014.00220

Upregulation of PYK2 protein and transcript following co-culture with DxMPs . (A) PYK2 protein expression as determined by Western blotting. Total lysates of MCF-7 cells (lane 1), MCF-7 cells co-cultured with DxMPs (lanes 2, 3, and 4), MCF-7/Dx cells (lane 5), and DxMPs (lane 7) were examined for PYK2 expression by Western blotting using the PYK2 (clone 5E2) mAb. β-actin was used as an internal control. Data was representative of at least three independent experiments. (B) Pyk2 gene transcript expression as determined by qRT-PCR. Pyk2 gene transcript in ( ): MCF-7 cells, ( ): MCF-7/Dx cells, ( ): DxMPs, ( ): MCF-7 cells co-cultured with MCFMPs, and ( ): MCF-7 cells co-cultured with DxMPs. Values are expressed as the fold difference relative to MCF-7 cells using GAPDH as the endogenous control. Data represent the mean ± SEM of at least three independent experiments. * p
Figure Legend Snippet: Upregulation of PYK2 protein and transcript following co-culture with DxMPs . (A) PYK2 protein expression as determined by Western blotting. Total lysates of MCF-7 cells (lane 1), MCF-7 cells co-cultured with DxMPs (lanes 2, 3, and 4), MCF-7/Dx cells (lane 5), and DxMPs (lane 7) were examined for PYK2 expression by Western blotting using the PYK2 (clone 5E2) mAb. β-actin was used as an internal control. Data was representative of at least three independent experiments. (B) Pyk2 gene transcript expression as determined by qRT-PCR. Pyk2 gene transcript in ( ): MCF-7 cells, ( ): MCF-7/Dx cells, ( ): DxMPs, ( ): MCF-7 cells co-cultured with MCFMPs, and ( ): MCF-7 cells co-cultured with DxMPs. Values are expressed as the fold difference relative to MCF-7 cells using GAPDH as the endogenous control. Data represent the mean ± SEM of at least three independent experiments. * p

Techniques Used: Co-Culture Assay, Expressing, Western Blot, Cell Culture, Quantitative RT-PCR

73) Product Images from "Endocytosis-Mediated Vacuolar Accumulation of the Human ApoE Apolipoprotein-Derived ApoEdpL-W Antimicrobial Peptide Contributes to Its Antifungal Activity in Candida albicans ▿ ▿ †"

Article Title: Endocytosis-Mediated Vacuolar Accumulation of the Human ApoE Apolipoprotein-Derived ApoEdpL-W Antimicrobial Peptide Contributes to Its Antifungal Activity in Candida albicans ▿ ▿ †

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.00319-11

C. albicans amino acid transporter genes are upregulated upon exposure to ApoEdpL-W. Shown are qRT-PCR results for genes identified as upregulated by ApoEdpL-W by microarray experiments, exposed to 2.5 μM ApoEdpL-W, and sampled at 10, 30, and 60 min of exposure. Fold changes were determined by the ΔΔ CT method, using CEF3 transcript levels for normalization.
Figure Legend Snippet: C. albicans amino acid transporter genes are upregulated upon exposure to ApoEdpL-W. Shown are qRT-PCR results for genes identified as upregulated by ApoEdpL-W by microarray experiments, exposed to 2.5 μM ApoEdpL-W, and sampled at 10, 30, and 60 min of exposure. Fold changes were determined by the ΔΔ CT method, using CEF3 transcript levels for normalization.

Techniques Used: Quantitative RT-PCR, Microarray

74) Product Images from "Adiponectin Agonist ADP355 Attenuates CCl4-Induced Liver Fibrosis in Mice"

Article Title: Adiponectin Agonist ADP355 Attenuates CCl4-Induced Liver Fibrosis in Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0110405

Effect of ADP355 on CCl 4 -induced liver fibrosis in mice. A ) Representative immunohistochemical (α-SMA) stained liver sections obtained for various treatments outlined (left panel); antibody control were presented in right panel. The original magnification was 4X. B ) Representative Western blot analysis and densitometry for α-SMA liver lysates obtained from saline, CCl 4 , CCl 4 –CTN and CCl 4 –ADN treated mice. C ) Hepatic α-SMA mRNA expression was assessed by qRT-PCR compared to housekeeping gene 18 s. *p
Figure Legend Snippet: Effect of ADP355 on CCl 4 -induced liver fibrosis in mice. A ) Representative immunohistochemical (α-SMA) stained liver sections obtained for various treatments outlined (left panel); antibody control were presented in right panel. The original magnification was 4X. B ) Representative Western blot analysis and densitometry for α-SMA liver lysates obtained from saline, CCl 4 , CCl 4 –CTN and CCl 4 –ADN treated mice. C ) Hepatic α-SMA mRNA expression was assessed by qRT-PCR compared to housekeeping gene 18 s. *p

Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Western Blot, Expressing, Quantitative RT-PCR

75) Product Images from "A Crp-Dependent Two-Component System Regulates Nitrate and Nitrite Respiration in Shewanella oneidensis"

Article Title: A Crp-Dependent Two-Component System Regulates Nitrate and Nitrite Respiration in Shewanella oneidensis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0051643

Expression analysis of nrfA during aerobic growth of S. oneidensis . A. A lacZ -based reporter analysis of the nap and nrfA promoters. Presented in columns is expression of nap and nrfA in the cells cultured aerobically in the absence of nitrite (/−) and in the presence of either nitrate (/NO 3 − ) or nitrite (/NO 2 − ). The napA mutant instead of the wild type was used here to keep nitrate unreduced. The nrfA mRNAs in the samples growing with NaNO 2 were also analyzed by qRT-PCR and presented (dash line, abundance relative to 16 S rRNA). Error bars represent the standard deviation (SD) ( n = 3). B. Western analysis of the cell samples used in A. Wild type cells grown in the absence or presence of nitrite at indicated time points were assayed, respectively. The complemented Δ nrfA (Δ nrfA c , carrying P nrfA -nrfA ) exhibited over-production of NrfA and Δ nrfA was used as the negative control.
Figure Legend Snippet: Expression analysis of nrfA during aerobic growth of S. oneidensis . A. A lacZ -based reporter analysis of the nap and nrfA promoters. Presented in columns is expression of nap and nrfA in the cells cultured aerobically in the absence of nitrite (/−) and in the presence of either nitrate (/NO 3 − ) or nitrite (/NO 2 − ). The napA mutant instead of the wild type was used here to keep nitrate unreduced. The nrfA mRNAs in the samples growing with NaNO 2 were also analyzed by qRT-PCR and presented (dash line, abundance relative to 16 S rRNA). Error bars represent the standard deviation (SD) ( n = 3). B. Western analysis of the cell samples used in A. Wild type cells grown in the absence or presence of nitrite at indicated time points were assayed, respectively. The complemented Δ nrfA (Δ nrfA c , carrying P nrfA -nrfA ) exhibited over-production of NrfA and Δ nrfA was used as the negative control.

Techniques Used: Expressing, Cell Culture, Mutagenesis, Quantitative RT-PCR, Standard Deviation, Western Blot, Negative Control

Binding analysis of NarP to nap and nrfA promoters. A. EMSA assay with carbamoyl phosphate (lanes 2–6) and NarQ 51–585 (lanes 7–11) treated NarP. NarP # represents NarP carrying a D57N mutation. Binding assays with NarP # treated with NarQ 51–585 and ATP are shown in lanes 12–13. All of the binding assays were performed with 2 ng of nrfA upstream fragments in the presence of 2 µg non-specific competitor DNA poly(dI · dC). Lanes 6 and 11 contain 10 µM of unlabeled nrfA upstream fragments as competitor DNA. The concentration of NarP or NarP # is indicated in the figure (µM). B. The EMSA assay was performed with 2 µM phosphorylated NarP and various amounts of 33 P end-labeled nrfA (−50 to −200 relative to the translation start codon) and nap (−50 to −200) upstream fragments. Non-specific competitor DNA, 2 µg poly(dI·dC), was added in all lanes. C. Western blotting analysis. Upper panel, analysis of NrfA in Δ narP . Cells grown in the presence of nitrite at the indicated time points were assayed. Lower panel, analysis of NarP. Cells grown in the presence of nitrate and/or nitrite at the indicated time points were assayed. In both panels, Δ narP c represents Δ narP containing pHG102- narP (P arcA -narP ), in which narP is over-expressed. D. qRT-PCR analysis of the narQ-narP operon. The wild type cells grown with nitrate or nitrate aerobically were collected at the indicated time points and assayed. Abundance is given relative to 16S rRNA. Error bars represent the standard deviation (SD) ( n = 3).
Figure Legend Snippet: Binding analysis of NarP to nap and nrfA promoters. A. EMSA assay with carbamoyl phosphate (lanes 2–6) and NarQ 51–585 (lanes 7–11) treated NarP. NarP # represents NarP carrying a D57N mutation. Binding assays with NarP # treated with NarQ 51–585 and ATP are shown in lanes 12–13. All of the binding assays were performed with 2 ng of nrfA upstream fragments in the presence of 2 µg non-specific competitor DNA poly(dI · dC). Lanes 6 and 11 contain 10 µM of unlabeled nrfA upstream fragments as competitor DNA. The concentration of NarP or NarP # is indicated in the figure (µM). B. The EMSA assay was performed with 2 µM phosphorylated NarP and various amounts of 33 P end-labeled nrfA (−50 to −200 relative to the translation start codon) and nap (−50 to −200) upstream fragments. Non-specific competitor DNA, 2 µg poly(dI·dC), was added in all lanes. C. Western blotting analysis. Upper panel, analysis of NrfA in Δ narP . Cells grown in the presence of nitrite at the indicated time points were assayed. Lower panel, analysis of NarP. Cells grown in the presence of nitrate and/or nitrite at the indicated time points were assayed. In both panels, Δ narP c represents Δ narP containing pHG102- narP (P arcA -narP ), in which narP is over-expressed. D. qRT-PCR analysis of the narQ-narP operon. The wild type cells grown with nitrate or nitrate aerobically were collected at the indicated time points and assayed. Abundance is given relative to 16S rRNA. Error bars represent the standard deviation (SD) ( n = 3).

Techniques Used: Binding Assay, Mutagenesis, Concentration Assay, Labeling, Western Blot, Quantitative RT-PCR, Standard Deviation

Crp and Fnr in aerobic nitrate/nitrite respiration of S. oneidensis . A. qRT-PCR analysis of crp and fnr . The wild type cells grown in the presence or absence of nitrate or nitrate aerobically were collected at the indicated time points and assayed. Expression level of each gene was presented under three conditions: –, no addition of nitrate or nitrite, NO 3 − , nitrate added, and NO 2 − , nitrite added. Abundance is given relative to 16 S rRNA. B. Western blotting analysis of Crp. Upper panel, the wild type cells cultured in the absence of nitrate or presence of nitrate at 4, 8, and 12 h were assayed. Lower panel, the wild type cells cultured in the absence of nitrite or presence of nitrite at 8, 12, and 16 h were assayed. Δ crp was used as the negative control. C. qRT-PCR analysis of nap , nrfA , and narP in cells grown with nitrate. The wild type, Δ crp , and Δ fnr mutant strains were assayed at indicated time points. Abundance is given relative to 16 S rRNA. D. Nitrate/nitrite assay. Cells of tested strains grown aerobically in the presence of nitrate or nitrate were collected at the indicated times. Concentrations of nitrate and nitrite (8 h and after) remaining in cultures were measured. Δ crp c represented the mutant containing a copy of crp on the complementation plasmid. Solid and dash lines represent cells grown in the presence of nitrate and nitrite, respectively. The wild type and Δ fnr were indistinguishable from each other and thus data for the wild type and Error bars (SD, n = 4) were omitted for clarity.
Figure Legend Snippet: Crp and Fnr in aerobic nitrate/nitrite respiration of S. oneidensis . A. qRT-PCR analysis of crp and fnr . The wild type cells grown in the presence or absence of nitrate or nitrate aerobically were collected at the indicated time points and assayed. Expression level of each gene was presented under three conditions: –, no addition of nitrate or nitrite, NO 3 − , nitrate added, and NO 2 − , nitrite added. Abundance is given relative to 16 S rRNA. B. Western blotting analysis of Crp. Upper panel, the wild type cells cultured in the absence of nitrate or presence of nitrate at 4, 8, and 12 h were assayed. Lower panel, the wild type cells cultured in the absence of nitrite or presence of nitrite at 8, 12, and 16 h were assayed. Δ crp was used as the negative control. C. qRT-PCR analysis of nap , nrfA , and narP in cells grown with nitrate. The wild type, Δ crp , and Δ fnr mutant strains were assayed at indicated time points. Abundance is given relative to 16 S rRNA. D. Nitrate/nitrite assay. Cells of tested strains grown aerobically in the presence of nitrate or nitrate were collected at the indicated times. Concentrations of nitrate and nitrite (8 h and after) remaining in cultures were measured. Δ crp c represented the mutant containing a copy of crp on the complementation plasmid. Solid and dash lines represent cells grown in the presence of nitrate and nitrite, respectively. The wild type and Δ fnr were indistinguishable from each other and thus data for the wild type and Error bars (SD, n = 4) were omitted for clarity.

Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Cell Culture, Negative Control, Mutagenesis, Nitration, Plasmid Preparation

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Amplification:

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Synthesized:

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Quantitative RT-PCR:

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Real-time Polymerase Chain Reaction:

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Incubation:

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Activity Assay:

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Expressing:

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Countercurrent Chromatography:

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Immunoprecipitation:

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Serial Dilution:

Article Title: Threonine-deficient diets induced changes in hepatic bioenergetics
Article Snippet: Real-time PCR reactions were performed on the Eppendorf MasterCycler real-time detection system using SYBR Green. .. Each assay included (in triplicates) a standard curve of five serial dilution points of control cDNA (ranging from 100 ng to 100 pg), a no template control, and 25–50 ng of each sample cDNA.

Inhibition:

Article Title: Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species – A proof-of-concept using Alatospora pulchella
Article Snippet: Each qPCR reaction was performed in a total volume of 25 μL, containing 5 μL template DNA, 8.7 μL sterile ddH2 O, 1.3 μL primer-probe-mix (primers: 18 μM and probe: 5 μM in the stock), and 10 μL TaqMan Environmental Master Mix (Applied Biosystems, Foster City, California) avoiding PCR inhibition [ ]. .. Eppendorf real-time PCR tube strips covered with Masterclear® cap strips (Eppendorf, Hamburg, Germany) were used as reaction vessels for amplification and detection.

Polymerase Chain Reaction:

Article Title: Transcriptional Profiling of mRNA Expression in the Mouse Distal Colon
Article Snippet: Paragraph title: Quantitative Reverse-Transcriptase Polymerase Chain Reaction ... Real-time PCR was performed on an Eppendorf real-time PCR machine (Eppendorf North America, Westbury, NY).

Article Title: Insights Into an Unexplored Component of the Mosquito Repeatome: Distribution and Variability of Viral Sequences Integrated Into the Genome of the Arboviral Vector Aedes albopictus
Article Snippet: Reaction mixtures were prepared containing 10 μL QuantiNova Sybr Green PCR Master Mix (Qiagen, Hilden, Germany), 1 μL of each 10 μM primer, and template DNA diluted in distilled H2 O up to 20 μL total reaction volume. .. Real-time PCR reactions were performed in a two-step amplification protocol consisting of 2 min at 95°C, followed by 40 cycles of 95°C for 5 s and 60°C for 10 s. Reactions were run on a RealPlex Real-Time PCR Detection System (Eppendorf, Hamburg, Germany).

Article Title: Myosin XI Is Essential for Tip Growth in Physcomitrella patens [W]
Article Snippet: The PCR conditions were as follows: 95°C for 10 min, followed by 50 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 40 s. The primer sets were designed to have similar amplification efficiencies and are listed in Supplemental Table 1 online. .. Real-time PCR reactions were performed in an Eppendorf Mastercycler ep Realplex2 thermal cycler, and data were analyzed with the Realplex software 2.2.

Article Title: Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species – A proof-of-concept using Alatospora pulchella
Article Snippet: Each qPCR reaction was performed in a total volume of 25 μL, containing 5 μL template DNA, 8.7 μL sterile ddH2 O, 1.3 μL primer-probe-mix (primers: 18 μM and probe: 5 μM in the stock), and 10 μL TaqMan Environmental Master Mix (Applied Biosystems, Foster City, California) avoiding PCR inhibition [ ]. .. Eppendorf real-time PCR tube strips covered with Masterclear® cap strips (Eppendorf, Hamburg, Germany) were used as reaction vessels for amplification and detection.

Article Title: Vildagliptin improves high glucose‐induced endothelial mitochondrial dysfunction via inhibiting mitochondrial fission, et al. Vildagliptin improves high glucose‐induced endothelial mitochondrial dysfunction via inhibiting mitochondrial fission
Article Snippet: .. Briefly, DNA was extracted using a QIAamp DNA Micro kit (Qiagen, West Sussex, UK) and real‐time PCR reactions were performed on the Mastercycler EP realplex PCR System (Eppendorf, Wesseling‐Berzdorf, Germany) according to manufacturer's protocol. .. The primers for mtDNA and β‐globin were as follows: mtDNA (sense: 5′‐CCC CAC AAA CCC CAT TAC TAA ACC CA‐3′; antisense: 5′‐TTT CAT CAT GCG GAG ATG TTG GAT GG‐3′); β‐globin (sense: 5′‐CGA GTA AGA GAC CAT TGT GGC AG‐3′; antisense: 5′‐GCT GTT CTG TCA ATA AAT TTC CTTC‐3′).

Sonication:

Article Title: DNA Damage-induced Nuclear Export of Precursor MicroRNAs is Regulated by the ATM-AKT Pathway
Article Snippet: Briefly, cells were crosslinked for 20 min with 1% formaldehyde, and cell pellets were resuspended in buffer B (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1], 1x protease inhibitor, 50 U/ml RNase inhibitor) and disrupted by sonication. .. Quantitative PCR reactions were then preformed on real-time PCR machine (Realplex2, Eppendorf).

Article Title: The ATM Kinase Induces MicroRNA Biogenesis in the DNA Damage Response
Article Snippet: Incubated 10 min in ice, the pellets were disrupted by sonication and the lysates were cleared and subjected to immunoprecipitation with anti-FLAG, anti-KSRP, anti-Drosha, or control antibody, followed by stringent washing, elution and reversal of crosslinking. .. Quantitative PCR reactions were then performed on real-time PCR machine (Realplex2, Eppendorf).

Isolation:

Article Title: Threonine-deficient diets induced changes in hepatic bioenergetics
Article Snippet: Total RNA was isolated from liver with TRIzol (Invitrogen) and treated with RNase-free DNase (Ambion) to remove any contaminating genomic DNA. .. Real-time PCR reactions were performed on the Eppendorf MasterCycler real-time detection system using SYBR Green.

Article Title: Long noncoding RNA SNHG15 serves as an oncogene and predicts poor prognosis in epithelial ovarian cancer
Article Snippet: qRT-PCR assay Total RNA was isolated from EOC cells and human tissues using TRIzol reagent (Invitrogen). .. Quantitative PCR was performed under standard conditions using an Eppendorf real-time PCR machine and an SYBR Green I Master Kit (Roche Diagnostics).

Size-exclusion Chromatography:

Article Title: Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species – A proof-of-concept using Alatospora pulchella
Article Snippet: Eppendorf real-time PCR tube strips covered with Masterclear® cap strips (Eppendorf, Hamburg, Germany) were used as reaction vessels for amplification and detection. .. The qPCR temperature profile entailed the following steps: initial denaturation (2 min at 50°C) for optimal Uracil-N-Glycosylase enzyme activity and successive activation of the DNA polymerase (10 min at 95°C), followed by 50 cycles of 15 sec at 95°C and 1 min at 60°C.

Chloramphenicol Acetyltransferase Assay:

Article Title: Vildagliptin improves high glucose‐induced endothelial mitochondrial dysfunction via inhibiting mitochondrial fission, et al. Vildagliptin improves high glucose‐induced endothelial mitochondrial dysfunction via inhibiting mitochondrial fission
Article Snippet: Briefly, DNA was extracted using a QIAamp DNA Micro kit (Qiagen, West Sussex, UK) and real‐time PCR reactions were performed on the Mastercycler EP realplex PCR System (Eppendorf, Wesseling‐Berzdorf, Germany) according to manufacturer's protocol. .. The primers for mtDNA and β‐globin were as follows: mtDNA (sense: 5′‐CCC CAC AAA CCC CAT TAC TAA ACC CA‐3′; antisense: 5′‐TTT CAT CAT GCG GAG ATG TTG GAT GG‐3′); β‐globin (sense: 5′‐CGA GTA AGA GAC CAT TGT GGC AG‐3′; antisense: 5′‐GCT GTT CTG TCA ATA AAT TTC CTTC‐3′).

Software:

Article Title: Myosin XI Is Essential for Tip Growth in Physcomitrella patens [W]
Article Snippet: .. Real-time PCR reactions were performed in an Eppendorf Mastercycler ep Realplex2 thermal cycler, and data were analyzed with the Realplex software 2.2. .. To determine the number of nuclei per plant, cells were simultaneously stained with calcofluor (fluorescent brightener 28) and 4',6-diamidino-2-phenylindole (DAPI).

SYBR Green Assay:

Article Title: Insights Into an Unexplored Component of the Mosquito Repeatome: Distribution and Variability of Viral Sequences Integrated Into the Genome of the Arboviral Vector Aedes albopictus
Article Snippet: Reaction mixtures were prepared containing 10 μL QuantiNova Sybr Green PCR Master Mix (Qiagen, Hilden, Germany), 1 μL of each 10 μM primer, and template DNA diluted in distilled H2 O up to 20 μL total reaction volume. .. Real-time PCR reactions were performed in a two-step amplification protocol consisting of 2 min at 95°C, followed by 40 cycles of 95°C for 5 s and 60°C for 10 s. Reactions were run on a RealPlex Real-Time PCR Detection System (Eppendorf, Hamburg, Germany).

Article Title: Myosin XI Is Essential for Tip Growth in Physcomitrella patens [W]
Article Snippet: All real-time PCR reactions used 2 to 8 ng of cDNA template in a 25-μL reaction using the Brilliant II SYBR Green QPCR Master Mix (Stratagene). .. Real-time PCR reactions were performed in an Eppendorf Mastercycler ep Realplex2 thermal cycler, and data were analyzed with the Realplex software 2.2.

Article Title: Threonine-deficient diets induced changes in hepatic bioenergetics
Article Snippet: .. Real-time PCR reactions were performed on the Eppendorf MasterCycler real-time detection system using SYBR Green. .. Each assay included (in triplicates) a standard curve of five serial dilution points of control cDNA (ranging from 100 ng to 100 pg), a no template control, and 25–50 ng of each sample cDNA.

Article Title: Long noncoding RNA SNHG15 serves as an oncogene and predicts poor prognosis in epithelial ovarian cancer
Article Snippet: .. Quantitative PCR was performed under standard conditions using an Eppendorf real-time PCR machine and an SYBR Green I Master Kit (Roche Diagnostics). ..

Activation Assay:

Article Title: Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species – A proof-of-concept using Alatospora pulchella
Article Snippet: Eppendorf real-time PCR tube strips covered with Masterclear® cap strips (Eppendorf, Hamburg, Germany) were used as reaction vessels for amplification and detection. .. The qPCR temperature profile entailed the following steps: initial denaturation (2 min at 50°C) for optimal Uracil-N-Glycosylase enzyme activity and successive activation of the DNA polymerase (10 min at 95°C), followed by 50 cycles of 15 sec at 95°C and 1 min at 60°C.

Protease Inhibitor:

Article Title: DNA Damage-induced Nuclear Export of Precursor MicroRNAs is Regulated by the ATM-AKT Pathway
Article Snippet: Briefly, cells were crosslinked for 20 min with 1% formaldehyde, and cell pellets were resuspended in buffer B (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1], 1x protease inhibitor, 50 U/ml RNase inhibitor) and disrupted by sonication. .. Quantitative PCR reactions were then preformed on real-time PCR machine (Realplex2, Eppendorf).

Article Title: The ATM Kinase Induces MicroRNA Biogenesis in the DNA Damage Response
Article Snippet: Briefly, cells were crosslinked for 20 min with 1% formaldehyde, and cell pellets was resuspended in Buffer B (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH8.1, 1x protease inhibitor, 50U/ml RNase inhibitor). .. Quantitative PCR reactions were then performed on real-time PCR machine (Realplex2, Eppendorf).

CTG Assay:

Article Title: Vildagliptin improves high glucose‐induced endothelial mitochondrial dysfunction via inhibiting mitochondrial fission, et al. Vildagliptin improves high glucose‐induced endothelial mitochondrial dysfunction via inhibiting mitochondrial fission
Article Snippet: Briefly, DNA was extracted using a QIAamp DNA Micro kit (Qiagen, West Sussex, UK) and real‐time PCR reactions were performed on the Mastercycler EP realplex PCR System (Eppendorf, Wesseling‐Berzdorf, Germany) according to manufacturer's protocol. .. The primers for mtDNA and β‐globin were as follows: mtDNA (sense: 5′‐CCC CAC AAA CCC CAT TAC TAA ACC CA‐3′; antisense: 5′‐TTT CAT CAT GCG GAG ATG TTG GAT GG‐3′); β‐globin (sense: 5′‐CGA GTA AGA GAC CAT TGT GGC AG‐3′; antisense: 5′‐GCT GTT CTG TCA ATA AAT TTC CTTC‐3′).

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  • 94
    Eppendorf AG quantitative pcr assays
    Detection and quantification of Simian Foamy Virus (SFV) <t>DNA</t> in samples of PBMCs and saliva derived from 14 humans infected by a gorilla strain of SFV. Detection of SFV DNA: Presence (+) or absence (-) of SFV DNA in PBMCs and saliva samples of each individual are indicated, based on <t>nested-PCR</t> results. Quantification of SFV DNA: Blue and red bars represent mean SFV DNA levels from at least two independent assays run in duplicate in PBMCs and saliva samples, for each individual respectively. Viral loads were normalized to cell equivalents by q-PCR for albumin. Error bars represent the standard deviations from an individual participant. Mean of SFV DNA copies in PBMCs (blue) and saliva (red) of the 14 individuals are shown by the dotted lines.
    Quantitative Pcr Assays, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Eppendorf AG real time quantitative pcr 3d liver models
    Expression of hepatocyte functions in the human <t>3D-liver</t> model. (A) Schematic representation of the different set-ups used for the analyses. Standard 2D Huh-7 monocultures (2D hepatocytes), the COL-I sandwich with a Huh-7 monolayer (no LSEC) and the 3D-liver model with a Huh-7 and LSEC layer. (B) Human albumin secretion into the medium on top of the cultures measured by ELISA. Cultures were used 3d after seeding. Albumin secretion into fresh serum-free medium was determined after 1.5–6 h incubation. (C, D) Expression of hepatocyte markers by <t>Q-RT-PCR</t> analysis (see Figure S3 for complete datasets). (C) Amount of transcripts expressed in the 3D-liver model monitored over time of culture (3–14d). Changes in transcript levels were expressed as log2-fold changes in comparison to levels determined for the 3d cultures (set to 0). The graph represents the 5 markers whose expression was maintained over time. (D) Transcript levels in 3d cultures represented as log2-fold changes over levels in 2D Huh-7 cultures (set to 0). The graph shows the functions for which the levels were significantly increased. Note that for the 3D-liver model, normalization of Q-PCR data with GAPDH (expressed by both hepatocytes and LSEC) leads to an underestimation of the transcript level for the hepatocyte-specific markers.
    Real Time Quantitative Pcr 3d Liver Models, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Eppendorf AG real time qpcr measurements
    Isoliquiritigenin (ISL) reduced lipopolysaccharide (LPS)-induced inflammatory response and increased the expression of genes involved in cholesterol homeostasis in macrophages. The <t>mRNA</t> levels of inflammation-associated cytokines interleukin 6 ( IL-6 ) ( a ); tumor necrosis factor α ( TNF-α ) ( b ); monocyte chemotactic protein-1 ( MCP-1 ) ( c ); and lipid flux-related receptors liver X receptor α ( LXRα ) ( d ); ATP-binding cassette transporter A1 ( ABCA1 ) ( e ); and cluster of differentiation 36 ( CD36 ) ( f ) were determined using <t>qPCR.</t> Peritoneal macrophages from apolipoprotein E-deficient (apoE −/− ) mice were treated with LPS (50 ng/mL) and ISL (0.1 µg/mL) for 6 h. Total RNA was extracted and reverse transcribed into cDNA ( n = 3–4; * p
    Real Time Qpcr Measurements, supplied by Eppendorf AG, 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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    Eppendorf AG real time quantitative pcr rt qpcr amplification
    LY294002 impacts ProDH1 steady state transcript and proteins levels . Northern- and western blot analysis were performed on total RNA or proteins extracted, respectively, from 12-days-old seedlings treated with either 0.5× MS alone (control) or supplemented with 200 mM NaCl (NaCl) during 3 h or 24 h with 100 μM LY294002 (+) or DMSO (−) as described in the legend of Figure 1 . (A) Total RNA (10 μg) was loaded in each lane and northern blots were probed with DNA fragments specific for AtP5CS1 and AtProDH1 . Methylene blue staining of rRNAs is shown as a loading control. (B) Total proteins (20 μg) were separated on an 8% SDS-PAGE gel. Western blots were incubated with specific antibodies directed against AtP5CS and AtProDH proteins. Detection of the immunolabeled proteins was done by autoradiography using an ECL kit. Membranes were stained with Ponceau S Red as control for protein loading (Rubisco). (C) Relative expression levels of P5CS1 and ProDH1 genes measured by real-time quantitative <t>PCR</t> after 24 h treatment as described in (A) . Results expressed as percentage compared to APT1 as a reference gene are means ± SD of 3 replicates. Letters indicate statistical differences in P5CS1 (blue letter) or ProDH1 (red letter) gene expression depending on treatment conditions (Two-Way ANOVA test, P
    Real Time Quantitative Pcr Rt Qpcr Amplification, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Detection and quantification of Simian Foamy Virus (SFV) DNA in samples of PBMCs and saliva derived from 14 humans infected by a gorilla strain of SFV. Detection of SFV DNA: Presence (+) or absence (-) of SFV DNA in PBMCs and saliva samples of each individual are indicated, based on nested-PCR results. Quantification of SFV DNA: Blue and red bars represent mean SFV DNA levels from at least two independent assays run in duplicate in PBMCs and saliva samples, for each individual respectively. Viral loads were normalized to cell equivalents by q-PCR for albumin. Error bars represent the standard deviations from an individual participant. Mean of SFV DNA copies in PBMCs (blue) and saliva (red) of the 14 individuals are shown by the dotted lines.

    Journal: PLoS ONE

    Article Title: Viral Latency in Blood and Saliva of Simian Foamy Virus-Infected Humans

    doi: 10.1371/journal.pone.0077072

    Figure Lengend Snippet: Detection and quantification of Simian Foamy Virus (SFV) DNA in samples of PBMCs and saliva derived from 14 humans infected by a gorilla strain of SFV. Detection of SFV DNA: Presence (+) or absence (-) of SFV DNA in PBMCs and saliva samples of each individual are indicated, based on nested-PCR results. Quantification of SFV DNA: Blue and red bars represent mean SFV DNA levels from at least two independent assays run in duplicate in PBMCs and saliva samples, for each individual respectively. Viral loads were normalized to cell equivalents by q-PCR for albumin. Error bars represent the standard deviations from an individual participant. Mean of SFV DNA copies in PBMCs (blue) and saliva (red) of the 14 individuals are shown by the dotted lines.

    Article Snippet: Quantitative PCR assays for DNA (qPCR) were performed using the Eppendorf realplex master gradient detection system.

    Techniques: Derivative Assay, Infection, Nested PCR, Polymerase Chain Reaction

    Expression of hepatocyte functions in the human 3D-liver model. (A) Schematic representation of the different set-ups used for the analyses. Standard 2D Huh-7 monocultures (2D hepatocytes), the COL-I sandwich with a Huh-7 monolayer (no LSEC) and the 3D-liver model with a Huh-7 and LSEC layer. (B) Human albumin secretion into the medium on top of the cultures measured by ELISA. Cultures were used 3d after seeding. Albumin secretion into fresh serum-free medium was determined after 1.5–6 h incubation. (C, D) Expression of hepatocyte markers by Q-RT-PCR analysis (see Figure S3 for complete datasets). (C) Amount of transcripts expressed in the 3D-liver model monitored over time of culture (3–14d). Changes in transcript levels were expressed as log2-fold changes in comparison to levels determined for the 3d cultures (set to 0). The graph represents the 5 markers whose expression was maintained over time. (D) Transcript levels in 3d cultures represented as log2-fold changes over levels in 2D Huh-7 cultures (set to 0). The graph shows the functions for which the levels were significantly increased. Note that for the 3D-liver model, normalization of Q-PCR data with GAPDH (expressed by both hepatocytes and LSEC) leads to an underestimation of the transcript level for the hepatocyte-specific markers.

    Journal: PLoS Pathogens

    Article Title: A New Human 3D-Liver Model Unravels the Role of Galectins in Liver Infection by the Parasite Entamoeba histolytica

    doi: 10.1371/journal.ppat.1004381

    Figure Lengend Snippet: Expression of hepatocyte functions in the human 3D-liver model. (A) Schematic representation of the different set-ups used for the analyses. Standard 2D Huh-7 monocultures (2D hepatocytes), the COL-I sandwich with a Huh-7 monolayer (no LSEC) and the 3D-liver model with a Huh-7 and LSEC layer. (B) Human albumin secretion into the medium on top of the cultures measured by ELISA. Cultures were used 3d after seeding. Albumin secretion into fresh serum-free medium was determined after 1.5–6 h incubation. (C, D) Expression of hepatocyte markers by Q-RT-PCR analysis (see Figure S3 for complete datasets). (C) Amount of transcripts expressed in the 3D-liver model monitored over time of culture (3–14d). Changes in transcript levels were expressed as log2-fold changes in comparison to levels determined for the 3d cultures (set to 0). The graph represents the 5 markers whose expression was maintained over time. (D) Transcript levels in 3d cultures represented as log2-fold changes over levels in 2D Huh-7 cultures (set to 0). The graph shows the functions for which the levels were significantly increased. Note that for the 3D-liver model, normalization of Q-PCR data with GAPDH (expressed by both hepatocytes and LSEC) leads to an underestimation of the transcript level for the hepatocyte-specific markers.

    Article Snippet: Real-time quantitative PCR 3D-liver models, the setup without LSEC, and Huh-7 cell standard 2D cultures, cultivated for 3–14d, were independently transferred to Eppendorf tubes and centrifuged for 5 min at 16000 g. The pellets (containing cells and the COL-I matrix) were lysed with lysis buffer from the RNAeasy Plus Mini kit (Qiagen) and total RNA was prepared with the kit.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Isoliquiritigenin (ISL) reduced lipopolysaccharide (LPS)-induced inflammatory response and increased the expression of genes involved in cholesterol homeostasis in macrophages. The mRNA levels of inflammation-associated cytokines interleukin 6 ( IL-6 ) ( a ); tumor necrosis factor α ( TNF-α ) ( b ); monocyte chemotactic protein-1 ( MCP-1 ) ( c ); and lipid flux-related receptors liver X receptor α ( LXRα ) ( d ); ATP-binding cassette transporter A1 ( ABCA1 ) ( e ); and cluster of differentiation 36 ( CD36 ) ( f ) were determined using qPCR. Peritoneal macrophages from apolipoprotein E-deficient (apoE −/− ) mice were treated with LPS (50 ng/mL) and ISL (0.1 µg/mL) for 6 h. Total RNA was extracted and reverse transcribed into cDNA ( n = 3–4; * p

    Journal: International Journal of Molecular Sciences

    Article Title: Isoliquiritigenin Attenuates Atherogenesis in Apolipoprotein E-Deficient Mice

    doi: 10.3390/ijms17111932

    Figure Lengend Snippet: Isoliquiritigenin (ISL) reduced lipopolysaccharide (LPS)-induced inflammatory response and increased the expression of genes involved in cholesterol homeostasis in macrophages. The mRNA levels of inflammation-associated cytokines interleukin 6 ( IL-6 ) ( a ); tumor necrosis factor α ( TNF-α ) ( b ); monocyte chemotactic protein-1 ( MCP-1 ) ( c ); and lipid flux-related receptors liver X receptor α ( LXRα ) ( d ); ATP-binding cassette transporter A1 ( ABCA1 ) ( e ); and cluster of differentiation 36 ( CD36 ) ( f ) were determined using qPCR. Peritoneal macrophages from apolipoprotein E-deficient (apoE −/− ) mice were treated with LPS (50 ng/mL) and ISL (0.1 µg/mL) for 6 h. Total RNA was extracted and reverse transcribed into cDNA ( n = 3–4; * p

    Article Snippet: Real-time qPCR measurements of target mRNA levels were performed on an Eppendorf Realplex2 Mastercycler (Eppendorf, Hamburg, Germany) according to the manufacturer’s instructions.

    Techniques: Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Mouse Assay

    LY294002 impacts ProDH1 steady state transcript and proteins levels . Northern- and western blot analysis were performed on total RNA or proteins extracted, respectively, from 12-days-old seedlings treated with either 0.5× MS alone (control) or supplemented with 200 mM NaCl (NaCl) during 3 h or 24 h with 100 μM LY294002 (+) or DMSO (−) as described in the legend of Figure 1 . (A) Total RNA (10 μg) was loaded in each lane and northern blots were probed with DNA fragments specific for AtP5CS1 and AtProDH1 . Methylene blue staining of rRNAs is shown as a loading control. (B) Total proteins (20 μg) were separated on an 8% SDS-PAGE gel. Western blots were incubated with specific antibodies directed against AtP5CS and AtProDH proteins. Detection of the immunolabeled proteins was done by autoradiography using an ECL kit. Membranes were stained with Ponceau S Red as control for protein loading (Rubisco). (C) Relative expression levels of P5CS1 and ProDH1 genes measured by real-time quantitative PCR after 24 h treatment as described in (A) . Results expressed as percentage compared to APT1 as a reference gene are means ± SD of 3 replicates. Letters indicate statistical differences in P5CS1 (blue letter) or ProDH1 (red letter) gene expression depending on treatment conditions (Two-Way ANOVA test, P

    Journal: Frontiers in Plant Science

    Article Title: Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana

    doi: 10.3389/fpls.2014.00772

    Figure Lengend Snippet: LY294002 impacts ProDH1 steady state transcript and proteins levels . Northern- and western blot analysis were performed on total RNA or proteins extracted, respectively, from 12-days-old seedlings treated with either 0.5× MS alone (control) or supplemented with 200 mM NaCl (NaCl) during 3 h or 24 h with 100 μM LY294002 (+) or DMSO (−) as described in the legend of Figure 1 . (A) Total RNA (10 μg) was loaded in each lane and northern blots were probed with DNA fragments specific for AtP5CS1 and AtProDH1 . Methylene blue staining of rRNAs is shown as a loading control. (B) Total proteins (20 μg) were separated on an 8% SDS-PAGE gel. Western blots were incubated with specific antibodies directed against AtP5CS and AtProDH proteins. Detection of the immunolabeled proteins was done by autoradiography using an ECL kit. Membranes were stained with Ponceau S Red as control for protein loading (Rubisco). (C) Relative expression levels of P5CS1 and ProDH1 genes measured by real-time quantitative PCR after 24 h treatment as described in (A) . Results expressed as percentage compared to APT1 as a reference gene are means ± SD of 3 replicates. Letters indicate statistical differences in P5CS1 (blue letter) or ProDH1 (red letter) gene expression depending on treatment conditions (Two-Way ANOVA test, P

    Article Snippet: The resulting first-strand cDNA was 20-fold diluted and used as the template for real-time quantitative PCR (RT-qPCR) amplification performed on a MasterCycler®ep realplex thermocycler (Eppendorf) with Maxima® SYBR Green/ROX qPCR Master Mix (Thermo Scientific) following the manufacturer protocol.

    Techniques: Northern Blot, Western Blot, Mass Spectrometry, Staining, SDS Page, Incubation, Immunolabeling, Autoradiography, Expressing, Real-time Polymerase Chain Reaction