Structured Review

Qiagen mrna expression
Schematic flow chart of the study design. Fecal and milk samples were collected from apparently healthy livestock and were subjected to PCR targeting H . pylori -specific 16s and ureA genes. The PCR positive samples were further subjected to genotyping of the virulence genes cagA and <t>vacA</t> and to bacteriological culture. The bacterial isolates were examined for cagA and vacA genotypes and for cytotoxicity using vacuolization assay. The fecal isolate with predominant cytotoxic genotype cagA + vacA s1a m1 i1 was inoculated in UHT milk and its survivability under different temperatures was determined. The UHT milk that contains the survived H . pylori strains were fed to healthy experimental mice groups for 40 days. The gastric mucosa was collected following scarification of the mice groups and was examined by real-time/quantitative polymerase chain reaction (qPCR) to estimate the load of infection. The gastric mucosa was further subjected to bacteriological culture and the <t>mRNA</t> expression of cagA and vacA was detected by reverse transcription PCR (RT-PCR).
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Images

1) Product Images from "Occurrence of cagA+vacA s1a m1 i1 Helicobacter pylori in farm animals in Egypt and ability to survive in experimentally contaminated UHT milk"

Article Title: Occurrence of cagA+vacA s1a m1 i1 Helicobacter pylori in farm animals in Egypt and ability to survive in experimentally contaminated UHT milk

Journal: Scientific Reports

doi: 10.1038/s41598-018-32671-0

Schematic flow chart of the study design. Fecal and milk samples were collected from apparently healthy livestock and were subjected to PCR targeting H . pylori -specific 16s and ureA genes. The PCR positive samples were further subjected to genotyping of the virulence genes cagA and vacA and to bacteriological culture. The bacterial isolates were examined for cagA and vacA genotypes and for cytotoxicity using vacuolization assay. The fecal isolate with predominant cytotoxic genotype cagA + vacA s1a m1 i1 was inoculated in UHT milk and its survivability under different temperatures was determined. The UHT milk that contains the survived H . pylori strains were fed to healthy experimental mice groups for 40 days. The gastric mucosa was collected following scarification of the mice groups and was examined by real-time/quantitative polymerase chain reaction (qPCR) to estimate the load of infection. The gastric mucosa was further subjected to bacteriological culture and the mRNA expression of cagA and vacA was detected by reverse transcription PCR (RT-PCR).
Figure Legend Snippet: Schematic flow chart of the study design. Fecal and milk samples were collected from apparently healthy livestock and were subjected to PCR targeting H . pylori -specific 16s and ureA genes. The PCR positive samples were further subjected to genotyping of the virulence genes cagA and vacA and to bacteriological culture. The bacterial isolates were examined for cagA and vacA genotypes and for cytotoxicity using vacuolization assay. The fecal isolate with predominant cytotoxic genotype cagA + vacA s1a m1 i1 was inoculated in UHT milk and its survivability under different temperatures was determined. The UHT milk that contains the survived H . pylori strains were fed to healthy experimental mice groups for 40 days. The gastric mucosa was collected following scarification of the mice groups and was examined by real-time/quantitative polymerase chain reaction (qPCR) to estimate the load of infection. The gastric mucosa was further subjected to bacteriological culture and the mRNA expression of cagA and vacA was detected by reverse transcription PCR (RT-PCR).

Techniques Used: Flow Cytometry, Polymerase Chain Reaction, Mouse Assay, Real-time Polymerase Chain Reaction, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction

2) Product Images from "Characterization of Transcriptome Transition Associates Long Noncoding RNAs with Glioma Progression"

Article Title: Characterization of Transcriptome Transition Associates Long Noncoding RNAs with Glioma Progression

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2018.10.009

CARD8-AS1 shRNA Lentivirus Suppresses Glioma Cell Proliferation and Migration and Induces Apoptosis In Vitro (A) CARD8-AS1 expression was quantified by qRT-PCR analysis. CARD8-AS1 shRNA lentivirus significantly reduced CARD8-AS1 expression, compared with the control groups. (B) Morphological alterations and accounted assay in the U251 and A172 cells upon CARD8-AS1 suppression, as assessed by phase contrast microscopy. (C and D) Representative images of in vitro Annexin V (C) and cell scratch assays (D) of U251 and A172 after transfection with CARD8-AS1 shRNA lentivirus or control lentivirus.
Figure Legend Snippet: CARD8-AS1 shRNA Lentivirus Suppresses Glioma Cell Proliferation and Migration and Induces Apoptosis In Vitro (A) CARD8-AS1 expression was quantified by qRT-PCR analysis. CARD8-AS1 shRNA lentivirus significantly reduced CARD8-AS1 expression, compared with the control groups. (B) Morphological alterations and accounted assay in the U251 and A172 cells upon CARD8-AS1 suppression, as assessed by phase contrast microscopy. (C and D) Representative images of in vitro Annexin V (C) and cell scratch assays (D) of U251 and A172 after transfection with CARD8-AS1 shRNA lentivirus or control lentivirus.

Techniques Used: shRNA, Migration, In Vitro, Expressing, Quantitative RT-PCR, Microscopy, Transfection

3) Product Images from "Characterization of the Interaction between Human Respiratory Syncytial Virus and the Cell Cycle in Continuous Cell Culture and Primary Human Airway Epithelial Cells ▿"

Article Title: Characterization of the Interaction between Human Respiratory Syncytial Virus and the Cell Cycle in Continuous Cell Culture and Primary Human Airway Epithelial Cells ▿

Journal: Journal of Virology

doi: 10.1128/JVI.05164-11

Model showing examples of cell cycle-regulatory proteins involved in progression whose abundances either increased (red) or decreased (green) in HRSV-infected cells. The model shows that although the abundances of different proteins were affected differently in HRSV-infected HBEpC or A549 cells, these changes will still result in G 0 /G 1 phase arrest. In addition, a reduction in the abundance of CDK1 is observed in HRSV-infected cells at 24 h postinfection, and this may act on G 2 /M phase progression.
Figure Legend Snippet: Model showing examples of cell cycle-regulatory proteins involved in progression whose abundances either increased (red) or decreased (green) in HRSV-infected cells. The model shows that although the abundances of different proteins were affected differently in HRSV-infected HBEpC or A549 cells, these changes will still result in G 0 /G 1 phase arrest. In addition, a reduction in the abundance of CDK1 is observed in HRSV-infected cells at 24 h postinfection, and this may act on G 2 /M phase progression.

Techniques Used: Infection, Activated Clotting Time Assay

(A) Ingenuity Pathway Analysis of the transcriptomic data sets, showing the relationship between cell cycle-regulatory factors whose mRNAs either were decreased in abundance more than 2-fold (green), remained unchanged (gray), or increased in abundance more than 2-fold (red) in HRSV-infected HBEpC cells. This diagram focuses on molecules involved in the G 1 /S phase transition. (B) Immunoblot analysis of the abundances of p21 WAF1/CIP1 (p21), CDK4, and CDK1 at 12 and 24 h postinfection in HBEpC and A549 cells that were either mock treated or infected with HRSV.
Figure Legend Snippet: (A) Ingenuity Pathway Analysis of the transcriptomic data sets, showing the relationship between cell cycle-regulatory factors whose mRNAs either were decreased in abundance more than 2-fold (green), remained unchanged (gray), or increased in abundance more than 2-fold (red) in HRSV-infected HBEpC cells. This diagram focuses on molecules involved in the G 1 /S phase transition. (B) Immunoblot analysis of the abundances of p21 WAF1/CIP1 (p21), CDK4, and CDK1 at 12 and 24 h postinfection in HBEpC and A549 cells that were either mock treated or infected with HRSV.

Techniques Used: Infection, Sublimation

Cell cycle profiles for HBEpC and A549 cells dually stained with EdU and DAPI. (Left) Bars in histograms represent the mean percentages of cells in the G 0 /G 1 , S, and G 2 /M phases of the cell cycle in mock-treated and HRSV-infected cells at 12 and 24 h postinfection. (Right) Representative cell cycle profiles of dually stained (EdU/DAPI) mock-treated and HRSV-infected (24-h) HBEpC and A549 cells are shown for each experimental condition.
Figure Legend Snippet: Cell cycle profiles for HBEpC and A549 cells dually stained with EdU and DAPI. (Left) Bars in histograms represent the mean percentages of cells in the G 0 /G 1 , S, and G 2 /M phases of the cell cycle in mock-treated and HRSV-infected cells at 12 and 24 h postinfection. (Right) Representative cell cycle profiles of dually stained (EdU/DAPI) mock-treated and HRSV-infected (24-h) HBEpC and A549 cells are shown for each experimental condition.

Techniques Used: Staining, Infection

(A) Histograms showing the proportion of cells in each stage of the cell cycle for HBEpC and A549 cells grown in the absence (−) or presence (+) of the specific inhibitor of CDK4/6. (B) Change in the amount of output virus from HRSV-infected HBEpC or A549 cells grown in the absence (−) or presence (+) of the specific inhibitor of CDK4/6, as determined by a plaque assay. These experiments were performed in triplicate and are representative of technical replicates.
Figure Legend Snippet: (A) Histograms showing the proportion of cells in each stage of the cell cycle for HBEpC and A549 cells grown in the absence (−) or presence (+) of the specific inhibitor of CDK4/6. (B) Change in the amount of output virus from HRSV-infected HBEpC or A549 cells grown in the absence (−) or presence (+) of the specific inhibitor of CDK4/6, as determined by a plaque assay. These experiments were performed in triplicate and are representative of technical replicates.

Techniques Used: Infection, Plaque Assay

4) Product Images from "Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans"

Article Title: Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans

Journal: Journal of Bacteriology

doi: 10.1128/JB.02144-12

An mlc mutant makes less leukotoxin protein and RNA. Samples were taken at log phase from aerobically (+) and anaerobically (−) grown cultures of the parent strain (JP2) and Δ mlc (AAM155) cells as well as the deletion strain with a plasmid carrying mlc + (the Δ mlc / mlc + strain). Cells were harvested by centrifugation, resuspended in SDS-PAGE loading buffer, boiled, and centrifuged, and the soluble material was electrophoresed on duplicate gels. (A) Gel stained with Coomassie brilliant blue. (B) Western blot analysis of total cell protein from the indicated strains using rabbit antileukotoxin antibody as the primary antibody. The arrows mark the position of leukotoxin. The numbers show the positions of molecular size markers (not shown) in kDa. (C) For each sample, total cell RNA was prepared from anaerobically grown log phase cells, and cDNA was synthesized with random hexamer primers and then used in quantitative RT-PCR. The level of ltxA mRNA in each sample is normalized to the level of pdxY mRNA in the same sample. The data are the average of the expression ratios from three biological replicates. The error bars are standard error, and the difference between the two samples is statistically significant ( P ≤ 0.05, by Student's t test).
Figure Legend Snippet: An mlc mutant makes less leukotoxin protein and RNA. Samples were taken at log phase from aerobically (+) and anaerobically (−) grown cultures of the parent strain (JP2) and Δ mlc (AAM155) cells as well as the deletion strain with a plasmid carrying mlc + (the Δ mlc / mlc + strain). Cells were harvested by centrifugation, resuspended in SDS-PAGE loading buffer, boiled, and centrifuged, and the soluble material was electrophoresed on duplicate gels. (A) Gel stained with Coomassie brilliant blue. (B) Western blot analysis of total cell protein from the indicated strains using rabbit antileukotoxin antibody as the primary antibody. The arrows mark the position of leukotoxin. The numbers show the positions of molecular size markers (not shown) in kDa. (C) For each sample, total cell RNA was prepared from anaerobically grown log phase cells, and cDNA was synthesized with random hexamer primers and then used in quantitative RT-PCR. The level of ltxA mRNA in each sample is normalized to the level of pdxY mRNA in the same sample. The data are the average of the expression ratios from three biological replicates. The error bars are standard error, and the difference between the two samples is statistically significant ( P ≤ 0.05, by Student's t test).

Techniques Used: Mutagenesis, Plasmid Preparation, Centrifugation, SDS Page, Staining, Western Blot, Synthesized, Random Hexamer Labeling, Quantitative RT-PCR, Expressing

The genetic interactions of IHF, CRP, and Mlc in altering leukotoxin transcription. After each sample was grown to log phase, total cell RNA was isolated, and cDNA was prepared with random hexamer primers and then used in quantitative RT-PCR. The level of ltxA mRNA in each sample is normalized to the level of pdxY mRNA in the same sample. The data are the average of the expression ratios from three biological replicates. The error bars are standard error. A single asterisk indicates samples for which the ratio of ltxA mRNA to pdxY mRNA is statistically significantly ( P
Figure Legend Snippet: The genetic interactions of IHF, CRP, and Mlc in altering leukotoxin transcription. After each sample was grown to log phase, total cell RNA was isolated, and cDNA was prepared with random hexamer primers and then used in quantitative RT-PCR. The level of ltxA mRNA in each sample is normalized to the level of pdxY mRNA in the same sample. The data are the average of the expression ratios from three biological replicates. The error bars are standard error. A single asterisk indicates samples for which the ratio of ltxA mRNA to pdxY mRNA is statistically significantly ( P

Techniques Used: Immunohistofluorescence, Isolation, Random Hexamer Labeling, Quantitative RT-PCR, Expressing

Quantitative RT-PCR of BamHI mutations define key regulatory elements within the leukotoxin promoter. Each of the indicated strains was grown anaerobically (A) or aerobically (B) to log phase, total RNA was isolated, and cDNA was prepared with random hexamer primers and then used in quantitative RT-PCR with ltxA - and pdxY -specific primers. The levels of ltxA mRNA in a single sample were normalized to pdxY (internal control) levels in the same sample. The value for a given strain is the average of the ratios of the ltxA mRNA to pdxY mRNA levels from three biological replicates (samples) of that strain. The error bars are the sample standard deviation. A single asterisk indicates a sample for which the ratio of ltxA mRNA to pdxY mRNA is statistically significantly ( P
Figure Legend Snippet: Quantitative RT-PCR of BamHI mutations define key regulatory elements within the leukotoxin promoter. Each of the indicated strains was grown anaerobically (A) or aerobically (B) to log phase, total RNA was isolated, and cDNA was prepared with random hexamer primers and then used in quantitative RT-PCR with ltxA - and pdxY -specific primers. The levels of ltxA mRNA in a single sample were normalized to pdxY (internal control) levels in the same sample. The value for a given strain is the average of the ratios of the ltxA mRNA to pdxY mRNA levels from three biological replicates (samples) of that strain. The error bars are the sample standard deviation. A single asterisk indicates a sample for which the ratio of ltxA mRNA to pdxY mRNA is statistically significantly ( P

Techniques Used: Quantitative RT-PCR, Isolation, Random Hexamer Labeling, Standard Deviation

5) Product Images from "A Novel Glycerophosphodiester Phosphodiesterase, GDE5, Controls Skeletal Muscle Development via a Non-enzymatic Mechanism *"

Article Title: A Novel Glycerophosphodiester Phosphodiesterase, GDE5, Controls Skeletal Muscle Development via a Non-enzymatic Mechanism *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.106708

Retroviruses harboring GFP, GDE5, or GDE5ΔC471 were used to infect L6 myoblasts. A , total protein extracts (10 μg/lane) from infected L6 myoblasts were subjected to SDS-PAGE followed by Western blotting using an anti-GDE5 antibody. B , the morphology of each cell line cultured in the myogenic medium for 10 days was observed under light microscopy. Magnification, ×100. C , to quantitatively evaluate myogenesis, elongated cells (per 5000 cells) were counted in each dish. D , total RNA was extracted and subjected to RT-PCR analysis to measure the expression of myogenin and creatine kinase ( CK ) mRNAs. The level of β-actin transcript was used as a control. E and F , quantitative RT-PCR was performed to estimate the mRNA abundance of myogenin and creatine kinase. **, p
Figure Legend Snippet: Retroviruses harboring GFP, GDE5, or GDE5ΔC471 were used to infect L6 myoblasts. A , total protein extracts (10 μg/lane) from infected L6 myoblasts were subjected to SDS-PAGE followed by Western blotting using an anti-GDE5 antibody. B , the morphology of each cell line cultured in the myogenic medium for 10 days was observed under light microscopy. Magnification, ×100. C , to quantitatively evaluate myogenesis, elongated cells (per 5000 cells) were counted in each dish. D , total RNA was extracted and subjected to RT-PCR analysis to measure the expression of myogenin and creatine kinase ( CK ) mRNAs. The level of β-actin transcript was used as a control. E and F , quantitative RT-PCR was performed to estimate the mRNA abundance of myogenin and creatine kinase. **, p

Techniques Used: Infection, SDS Page, Western Blot, Cell Culture, Light Microscopy, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

6) Product Images from "Surface Proteome Analysis and Characterization of Surface Cell Antigen (Sca) or Autotransporter Family of Rickettsia typhi"

Article Title: Surface Proteome Analysis and Characterization of Surface Cell Antigen (Sca) or Autotransporter Family of Rickettsia typhi

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002856

Expression analysis by real-time RT-PCR of Sca genes during L929 cell infection and immunofluorescence assays of Sca protein expression in C. felis and rat spleens. RNA was extracted from R. typhi infected L929 cells at different time points as described and analyzed by multiplex real-time qPCR for expression of Sca , rpsL and GAPDH genes. A) Transcript abundance of each Sca gene was normalized to rpsL transcript abundance and B) rickettsial burden ( rpsL : GAPDH ) confirmed increased infection over time. The results represent data from 3 experiments with each gene analyzed in duplicate. C) Cat fleas were housed in an artificial dog and fed uninfected or R. typhi infected whole sheep's blood for 48 h.; they were then fed uninfected blood for 8 days. Capsules were placed at −20°C to immobilize fleas before placing them in 3% PFA overnight. Fleas were embedded in OCT media, frozen and cryosectioned. Sections were labeled with anti- R. typhi rat immune serum (Alexa488-conjugated anti-rat secondary Ab – green), anti-serum to the sca protein indicated on the left (Alexa594-conjugated anti-rabbit secondary Ab – red) and mounted in VectaShield medium with DAPI (blue) to counterstain DNA. Arrowheads point to examples of positively stained rickettsiae. Bar = 5 µm. D) Spleens were harvested from 9 day infected female Sprague-Dawley rats and fixed and embedded as described. Sections (5 µm) were stained with AlexaFluor 532-labeled anti- R. typhi rat immune serum (red), AlexaFluor 350-labeled anti-serum to the sca protein indicated on the left (blue) and propidium iodide (green) to counterstain DNA (not shown). Sections were mounted in VectaShield medium. Arrowheads indicate examples of positively labeled rickettsiae.
Figure Legend Snippet: Expression analysis by real-time RT-PCR of Sca genes during L929 cell infection and immunofluorescence assays of Sca protein expression in C. felis and rat spleens. RNA was extracted from R. typhi infected L929 cells at different time points as described and analyzed by multiplex real-time qPCR for expression of Sca , rpsL and GAPDH genes. A) Transcript abundance of each Sca gene was normalized to rpsL transcript abundance and B) rickettsial burden ( rpsL : GAPDH ) confirmed increased infection over time. The results represent data from 3 experiments with each gene analyzed in duplicate. C) Cat fleas were housed in an artificial dog and fed uninfected or R. typhi infected whole sheep's blood for 48 h.; they were then fed uninfected blood for 8 days. Capsules were placed at −20°C to immobilize fleas before placing them in 3% PFA overnight. Fleas were embedded in OCT media, frozen and cryosectioned. Sections were labeled with anti- R. typhi rat immune serum (Alexa488-conjugated anti-rat secondary Ab – green), anti-serum to the sca protein indicated on the left (Alexa594-conjugated anti-rabbit secondary Ab – red) and mounted in VectaShield medium with DAPI (blue) to counterstain DNA. Arrowheads point to examples of positively stained rickettsiae. Bar = 5 µm. D) Spleens were harvested from 9 day infected female Sprague-Dawley rats and fixed and embedded as described. Sections (5 µm) were stained with AlexaFluor 532-labeled anti- R. typhi rat immune serum (red), AlexaFluor 350-labeled anti-serum to the sca protein indicated on the left (blue) and propidium iodide (green) to counterstain DNA (not shown). Sections were mounted in VectaShield medium. Arrowheads indicate examples of positively labeled rickettsiae.

Techniques Used: Expressing, Quantitative RT-PCR, Infection, Immunofluorescence, Multiplex Assay, Real-time Polymerase Chain Reaction, Labeling, Staining

Transcriptional analysis of putative sca operons by RT-PCR. RNA was extracted from 72 h-infected L929 fibroblasts and RT-PCR with primers designed to amplify the regions indicated in (A). A) Schematic of putative sca operons. Blue bars indicate regions successfully amplified, red bars indicate regions that could not be amplified. B) RT-PCR amplification of regions between ORFs in putative sca operons. Left panel – lanes 2 thru 4 – RT-PCR amplification of the 400 bp region within the encoded passenger domain of sca3 used in time course analyses ; lanes 5 thru 7 – amplification of Lon-sca3 co-transcript. Right panel: lanes 2 thru 4 – amplification of sca4 region used in time course analyses; lanes 5 thru 7 – amplification of tlcD-sca4 transcript. C) Analysis of sca5 operon; labels indicate genes spanned by primers. RT – reverse transcriptase reaction, NRT – no reverse transcriptase, D – DNA control.
Figure Legend Snippet: Transcriptional analysis of putative sca operons by RT-PCR. RNA was extracted from 72 h-infected L929 fibroblasts and RT-PCR with primers designed to amplify the regions indicated in (A). A) Schematic of putative sca operons. Blue bars indicate regions successfully amplified, red bars indicate regions that could not be amplified. B) RT-PCR amplification of regions between ORFs in putative sca operons. Left panel – lanes 2 thru 4 – RT-PCR amplification of the 400 bp region within the encoded passenger domain of sca3 used in time course analyses ; lanes 5 thru 7 – amplification of Lon-sca3 co-transcript. Right panel: lanes 2 thru 4 – amplification of sca4 region used in time course analyses; lanes 5 thru 7 – amplification of tlcD-sca4 transcript. C) Analysis of sca5 operon; labels indicate genes spanned by primers. RT – reverse transcriptase reaction, NRT – no reverse transcriptase, D – DNA control.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Amplification

7) Product Images from "Insertion mutants in Drosophila melanogaster Hsc20 halt larval growth and lead to reduced iron-sulfur cluster enzyme activities and impaired iron homeostasis"

Article Title: Insertion mutants in Drosophila melanogaster Hsc20 halt larval growth and lead to reduced iron-sulfur cluster enzyme activities and impaired iron homeostasis

Journal: Journal of Biological Inorganic Chemistry

doi: 10.1007/s00775-013-0988-2

The Drosophila Hsc20 gene is broadly expressed at low levels and encodes a mitochondrial protein. a The information presented was retrieved and adapted from the FlyBase and modENCODE databases, where Hsc20 is listed as l(3)72Do or CG34246 . The gene is composed of four exons and three introns. The open reading frame is shown in beige and the noncoding region of the messenger RNA in gray . The two piggyBac insertions used in this study disrupt the first exon. The gene shows ubiquitous low level of expression throughout development and into adulthood. b Confirmation of the presence and small size of introns 2 and 3 by reverse transcription PCR ( RT-PCR ; the location of primers used shown is in a ) and localization of an Hsc20–red fluorescent protein ( RFP ) fusion protein in mitochondria of transfected HeLa cells
Figure Legend Snippet: The Drosophila Hsc20 gene is broadly expressed at low levels and encodes a mitochondrial protein. a The information presented was retrieved and adapted from the FlyBase and modENCODE databases, where Hsc20 is listed as l(3)72Do or CG34246 . The gene is composed of four exons and three introns. The open reading frame is shown in beige and the noncoding region of the messenger RNA in gray . The two piggyBac insertions used in this study disrupt the first exon. The gene shows ubiquitous low level of expression throughout development and into adulthood. b Confirmation of the presence and small size of introns 2 and 3 by reverse transcription PCR ( RT-PCR ; the location of primers used shown is in a ) and localization of an Hsc20–red fluorescent protein ( RFP ) fusion protein in mitochondria of transfected HeLa cells

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

8) Product Images from "The Alkaloid Compound Harmane Increases the Lifespan of Caenorhabditis elegans during Bacterial Infection, by Modulating the Nematode's Innate Immune Response"

Article Title: The Alkaloid Compound Harmane Increases the Lifespan of Caenorhabditis elegans during Bacterial Infection, by Modulating the Nematode's Innate Immune Response

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060519

Harmane induces the immune response gene F35E12.5 . (A) Fluorescence microscopy pictures of F35E12.5::gfp transgenic C. elegans , exposed to DMSO or Harmane for 20 hours, at 15°C (feeding on E. coli OP50). The fluorescence was always seen at the tail end of the animals. (B) Individual nematodes in fluorescence pictures were quantified, and data normalized to the level of the DMSO treated nematodes. There was a significant difference between the two samples [ P
Figure Legend Snippet: Harmane induces the immune response gene F35E12.5 . (A) Fluorescence microscopy pictures of F35E12.5::gfp transgenic C. elegans , exposed to DMSO or Harmane for 20 hours, at 15°C (feeding on E. coli OP50). The fluorescence was always seen at the tail end of the animals. (B) Individual nematodes in fluorescence pictures were quantified, and data normalized to the level of the DMSO treated nematodes. There was a significant difference between the two samples [ P

Techniques Used: Fluorescence, Microscopy, Transgenic Assay

9) Product Images from "Statins Reduce Melanoma Development and Metastasis through MICA Overexpression"

Article Title: Statins Reduce Melanoma Development and Metastasis through MICA Overexpression

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2013.00062

Inhibition of Rho and Ras GTPases proteins do not induce MICA membrane overexpression . LB1319-MEL cells were untreated or treated with TatC3 exoenzyme (TatC3) 10 μg/mL for 24 h (A,B) . LB1319-MEL cells were transfected with control siRNA (siCtrl) (C–G) , two RhoA-specific siRNAs (siRhoA1, siRhoA2) (C) , two RhoB-specific siRNAs (siRhoB1, siRhoB2) (D) , two RhoC-specific siRNAs (siRhoC1, siRhoC2) (E) , two Rac1-specific siRNAs (siRac1.1, siRac1.2) (F) , or two Cdc42-specific siRNAs (siCdc42.1, siCdc42.2) (G) . LB1319-MEL cells were treated or not with a Ras inhibitor, FTS, 50–100 μM for 24 h (H,I) . MICA membrane expression was analyzed by flow cytometry 72 h after transfection or 24 h after treatment. Representative illustrations of untreated cells (black) and of treated cells (filled in gray) with Tat-C3 exoenzyme (A) or FTS (H) are shown. To evaluate membrane antigen expression, the index of specific fluorescence (ISF) was calculated as indicated in the Section “Materials and Methods.” Results are expressed as mean ± SD (error bars, n = 3 experiments). * P
Figure Legend Snippet: Inhibition of Rho and Ras GTPases proteins do not induce MICA membrane overexpression . LB1319-MEL cells were untreated or treated with TatC3 exoenzyme (TatC3) 10 μg/mL for 24 h (A,B) . LB1319-MEL cells were transfected with control siRNA (siCtrl) (C–G) , two RhoA-specific siRNAs (siRhoA1, siRhoA2) (C) , two RhoB-specific siRNAs (siRhoB1, siRhoB2) (D) , two RhoC-specific siRNAs (siRhoC1, siRhoC2) (E) , two Rac1-specific siRNAs (siRac1.1, siRac1.2) (F) , or two Cdc42-specific siRNAs (siCdc42.1, siCdc42.2) (G) . LB1319-MEL cells were treated or not with a Ras inhibitor, FTS, 50–100 μM for 24 h (H,I) . MICA membrane expression was analyzed by flow cytometry 72 h after transfection or 24 h after treatment. Representative illustrations of untreated cells (black) and of treated cells (filled in gray) with Tat-C3 exoenzyme (A) or FTS (H) are shown. To evaluate membrane antigen expression, the index of specific fluorescence (ISF) was calculated as indicated in the Section “Materials and Methods.” Results are expressed as mean ± SD (error bars, n = 3 experiments). * P

Techniques Used: Inhibition, Over Expression, Transfection, Expressing, Flow Cytometry, Cytometry, Fluorescence

Atorvastatin treatment inhibits melanoma in vivo development and metastasis . LB1319-MEL cells untreated (H 2 O) or pretreated with atorvastatin 5 μM for 48 h (Ator) were injected subcutaneously into the flank of six nude mice. Tumor growth was measured every 2–3 days (A) . *** P
Figure Legend Snippet: Atorvastatin treatment inhibits melanoma in vivo development and metastasis . LB1319-MEL cells untreated (H 2 O) or pretreated with atorvastatin 5 μM for 48 h (Ator) were injected subcutaneously into the flank of six nude mice. Tumor growth was measured every 2–3 days (A) . *** P

Techniques Used: In Vivo, Injection, Mouse Assay

PPARγ and SAPK/JNK are involved in atorvastatin-induced MICA overexpression . LB1319-MEL cells were treated with DMSO or a PPARγ inhibitor, T0070907 25 nM for 24 h (A,B) . LB1319-MEL cells were transfected with an empty plasmid (Ctrl) or a plasmid encoding a PPARγ dominant negative mutant (PPARγ DN) (C,D) . MICA membrane expression was analyzed 24 h after treatment or transfection. Representative illustrations of controlled cells (black) and treated cells (filled in gray) with T0070907 (A) or PPARγ DN (C) are shown. The ISF were calculated (B,D) . LB1319-MEL cells were transfected with Firefly Luc control plasmids (pGL4.23-FLuc) or plasmid reporting the PPARγ PPRE activity (pGL4.3xPPRE-FLuc) and these cells were untreated (H 2 O) or treated with atorvastatin 5 μM (Ator 5 μM). Twenty-four hours later, luciferase assays were performed (E) . LB1319-MEL cells were untreated (H 2 O) or treated with atorvastatin 5 μM for 48 h and a Proteome Profiler Array was performed to compare kinases phosphorylation (F,G) . One, two, and three correspond respectively to a reference spot (REF), panJNK T182/Y185, T221/Y223 (panJNK), and c-Jun S63 (c-Jun) phosphorylations (F) . The densities of the signals were analyzed with ImageJ software and the ratios of atorvastatin on untreated conditions were calculated (G) . Results are expressed as mean ± SD (error bars, n = 3 experiments). * P
Figure Legend Snippet: PPARγ and SAPK/JNK are involved in atorvastatin-induced MICA overexpression . LB1319-MEL cells were treated with DMSO or a PPARγ inhibitor, T0070907 25 nM for 24 h (A,B) . LB1319-MEL cells were transfected with an empty plasmid (Ctrl) or a plasmid encoding a PPARγ dominant negative mutant (PPARγ DN) (C,D) . MICA membrane expression was analyzed 24 h after treatment or transfection. Representative illustrations of controlled cells (black) and treated cells (filled in gray) with T0070907 (A) or PPARγ DN (C) are shown. The ISF were calculated (B,D) . LB1319-MEL cells were transfected with Firefly Luc control plasmids (pGL4.23-FLuc) or plasmid reporting the PPARγ PPRE activity (pGL4.3xPPRE-FLuc) and these cells were untreated (H 2 O) or treated with atorvastatin 5 μM (Ator 5 μM). Twenty-four hours later, luciferase assays were performed (E) . LB1319-MEL cells were untreated (H 2 O) or treated with atorvastatin 5 μM for 48 h and a Proteome Profiler Array was performed to compare kinases phosphorylation (F,G) . One, two, and three correspond respectively to a reference spot (REF), panJNK T182/Y185, T221/Y223 (panJNK), and c-Jun S63 (c-Jun) phosphorylations (F) . The densities of the signals were analyzed with ImageJ software and the ratios of atorvastatin on untreated conditions were calculated (G) . Results are expressed as mean ± SD (error bars, n = 3 experiments). * P

Techniques Used: Over Expression, Transfection, Plasmid Preparation, Dominant Negative Mutation, Expressing, Activity Assay, Luciferase, Software

Statin treatment induces MICA overexpression and increases NK-dependent cytotoxicity . LB1319-MEL cells were treated with 5 μM atorvastatin for 48 h (Ator) or untreated (H 2 O). The atorvastatin treatment efficiency was controlled by the analysis of Rap1A unprenylation (Rap1A u.p) compared to the total protein (Rap1A total) (A) . MICA total expression was analyzed by western blot (A) , membrane expression by flow cytometry (B,C) , and cleavage by ELISA (D) ( n = 3). To evaluate membrane antigen expression, the index of specific fluorescence (ISF) was calculated as indicated in the Section “Materials and Methods” (C) . Results are expressed as mean ± SD [error bars, n = 5 experiments (C) or 3 experiments (D) ]. * P
Figure Legend Snippet: Statin treatment induces MICA overexpression and increases NK-dependent cytotoxicity . LB1319-MEL cells were treated with 5 μM atorvastatin for 48 h (Ator) or untreated (H 2 O). The atorvastatin treatment efficiency was controlled by the analysis of Rap1A unprenylation (Rap1A u.p) compared to the total protein (Rap1A total) (A) . MICA total expression was analyzed by western blot (A) , membrane expression by flow cytometry (B,C) , and cleavage by ELISA (D) ( n = 3). To evaluate membrane antigen expression, the index of specific fluorescence (ISF) was calculated as indicated in the Section “Materials and Methods” (C) . Results are expressed as mean ± SD [error bars, n = 5 experiments (C) or 3 experiments (D) ]. * P

Techniques Used: Over Expression, Expressing, Western Blot, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Fluorescence

10) Product Images from "The Ca2+/Mn2+ ion-pump PMR1 links elevation of cytosolic Ca2+ levels to α-synuclein toxicity in Parkinson's disease models"

Article Title: The Ca2+/Mn2+ ion-pump PMR1 links elevation of cytosolic Ca2+ levels to α-synuclein toxicity in Parkinson's disease models

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2012.142

Expression of α Syn causes a slight upregulation of PMR1 and CCH1 mRNA levels. ( a ) Representative micrographs of yeast cells expressing endogenously GFP-tagged Pmr1p in combination with α Syn or corresponding vector control at indicated time points after induction of α Syn expression. ( b ) Western blot analysis of cells described in ( a ) at indicated time points after induction of α Syn expression. Blots were probed with antibodies against GFP to detect Pmr1p-GFP fusion protein, against FLAG-epitope to detect FLAG-tagged α Syn and against glyceraldehyd-3-phosphate dehydrogenase (GAPDH) as loading control. ( c and d ) Q-PCR-based quantification of PMR1 mRNA levels ( c ) and of CCH1 and MID1 mRNA levels ( d ) in WT cells expressing α Syn or harbouring the empty vector control for 14 h or 24 h, respectively, normalized to actin mRNA levels. Means±S.E.M., n =6–9; * P
Figure Legend Snippet: Expression of α Syn causes a slight upregulation of PMR1 and CCH1 mRNA levels. ( a ) Representative micrographs of yeast cells expressing endogenously GFP-tagged Pmr1p in combination with α Syn or corresponding vector control at indicated time points after induction of α Syn expression. ( b ) Western blot analysis of cells described in ( a ) at indicated time points after induction of α Syn expression. Blots were probed with antibodies against GFP to detect Pmr1p-GFP fusion protein, against FLAG-epitope to detect FLAG-tagged α Syn and against glyceraldehyd-3-phosphate dehydrogenase (GAPDH) as loading control. ( c and d ) Q-PCR-based quantification of PMR1 mRNA levels ( c ) and of CCH1 and MID1 mRNA levels ( d ) in WT cells expressing α Syn or harbouring the empty vector control for 14 h or 24 h, respectively, normalized to actin mRNA levels. Means±S.E.M., n =6–9; * P

Techniques Used: Expressing, Plasmid Preparation, Western Blot, FLAG-tag, Polymerase Chain Reaction

11) Product Images from "Tgf-?1 Inhibits Cftr Biogenesis and Prevents Functional Rescue of ?F508-Cftr in Primary Differentiated Human Bronchial Epithelial Cells"

Article Title: Tgf-?1 Inhibits Cftr Biogenesis and Prevents Functional Rescue of ?F508-Cftr in Primary Differentiated Human Bronchial Epithelial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063167

Summary of qRT-PCR experiments demonstrating that TGF-β1 decreases ΔF508-CFTR mRNA levels in CF-HBE cells. TGF-β1 (15 ng/ml) or vehicle control (CTRL) was added to the basolateral medium and cells were incubated for 24 h. Raw data were analyzed using the ΔΔC t method. Changes in the ΔF508-CFTR mRNA were normalized to GAPDH. Data are expressed as fold change in ΔF508-CFTR mRNA vs. CTRL. All experiments were performed in triplicates in cell obtained from 3 donors. *, p
Figure Legend Snippet: Summary of qRT-PCR experiments demonstrating that TGF-β1 decreases ΔF508-CFTR mRNA levels in CF-HBE cells. TGF-β1 (15 ng/ml) or vehicle control (CTRL) was added to the basolateral medium and cells were incubated for 24 h. Raw data were analyzed using the ΔΔC t method. Changes in the ΔF508-CFTR mRNA were normalized to GAPDH. Data are expressed as fold change in ΔF508-CFTR mRNA vs. CTRL. All experiments were performed in triplicates in cell obtained from 3 donors. *, p

Techniques Used: Quantitative RT-PCR, Incubation

qRT-PCR and western blot experiments examining effects of TGF-β1 on the epithelial phenotype in HBE cells. To examine whether the 24 h treatment with TGF-β1 alters the epithelial phenotype of HBE cells we examined the mRNA levels ( A ) and protein abundance ( B ) of E- and N-cadherin, markers of epithelial and mesenchymal phenotype, respectively. TGF-β1 (15 ng/ml) or vehicle control (CTRL) was added to the basolateral medium for 6, 12, or 24 h. ( A ) qRT-PCR experiments. Raw data were analyzed using the ΔΔC t method. Changes in the E- and N-cadherin mRNA were normalized to GAPDH. Data are expressed as fold change in E- or N-cadherin mRNA vs. CTRL. The 24 h treatment with TGF-β1 increased levels of N-cadherin mRNA without reducing the E-cadherin mRNA. All experiments were performed in triplicate in cells obtained from 3 donors. *, p
Figure Legend Snippet: qRT-PCR and western blot experiments examining effects of TGF-β1 on the epithelial phenotype in HBE cells. To examine whether the 24 h treatment with TGF-β1 alters the epithelial phenotype of HBE cells we examined the mRNA levels ( A ) and protein abundance ( B ) of E- and N-cadherin, markers of epithelial and mesenchymal phenotype, respectively. TGF-β1 (15 ng/ml) or vehicle control (CTRL) was added to the basolateral medium for 6, 12, or 24 h. ( A ) qRT-PCR experiments. Raw data were analyzed using the ΔΔC t method. Changes in the E- and N-cadherin mRNA were normalized to GAPDH. Data are expressed as fold change in E- or N-cadherin mRNA vs. CTRL. The 24 h treatment with TGF-β1 increased levels of N-cadherin mRNA without reducing the E-cadherin mRNA. All experiments were performed in triplicate in cells obtained from 3 donors. *, p

Techniques Used: Quantitative RT-PCR, Western Blot

12) Product Images from "Nucleoprotein Nanostructures Combined with Adjuvants Adapted to the Neonatal Immune Context: A Candidate Mucosal RSV Vaccine"

Article Title: Nucleoprotein Nanostructures Combined with Adjuvants Adapted to the Neonatal Immune Context: A Candidate Mucosal RSV Vaccine

Journal: PLoS ONE

doi: 10.1371/journal.pone.0037722

Neonatal nasal vaccination with N+LT conferred viral protection but exacerbated airway disease upon RSV challenge. Male and female pups (5–7 day-old) were vaccinated by intranasal instillation of 10 µL saline containing or not 3 µg N and 2 µg LT as indicated. At 5 weeks of age, mice were challenged by intranasal instillation of 50 µL (5.10 6 pfu) hRSV A2. Controls included unvaccinated infected (C+) and uninfected (C−) littermates. Animals were killed 5 days post challenge (d5 p.i.). (A) Individual viral load assessed by qRT-PCR: R.Q. of N transcripts, normalized to HPRT, are expressed as % of the unvaccinated infected control group (C+) (R.Q. = 100×2 −ΔΔCt ). Four independent experiments combining different treatment groups are shown with ≥5 mice per group (C−: n = 7, C+: n = 10, LT: n = 5, N: n = 16, N+LT: n = 20). Mann-Whitney U-test was used for comparison between treatments (* p
Figure Legend Snippet: Neonatal nasal vaccination with N+LT conferred viral protection but exacerbated airway disease upon RSV challenge. Male and female pups (5–7 day-old) were vaccinated by intranasal instillation of 10 µL saline containing or not 3 µg N and 2 µg LT as indicated. At 5 weeks of age, mice were challenged by intranasal instillation of 50 µL (5.10 6 pfu) hRSV A2. Controls included unvaccinated infected (C+) and uninfected (C−) littermates. Animals were killed 5 days post challenge (d5 p.i.). (A) Individual viral load assessed by qRT-PCR: R.Q. of N transcripts, normalized to HPRT, are expressed as % of the unvaccinated infected control group (C+) (R.Q. = 100×2 −ΔΔCt ). Four independent experiments combining different treatment groups are shown with ≥5 mice per group (C−: n = 7, C+: n = 10, LT: n = 5, N: n = 16, N+LT: n = 20). Mann-Whitney U-test was used for comparison between treatments (* p

Techniques Used: Mouse Assay, Infection, Quantitative RT-PCR, MANN-WHITNEY

CpG added to the neonatal vaccine decreased pulmonary CCL11 mRNA expression upon RSV challenge. The level of expression of genes encoding mCCL2 (A), mCCL3 (C), mCCL5 (D) and mCCL11 (B) was monitored by quantitative real time PCR of RNAs extracted from individual lungs collected at d5 p.i. Data were normalized to the mHPRT and expressed relative to the unvaccinated infected control group (C+) (R.Q. = 100×2 −ΔΔCt ). Data are mean±SEM from n≥4 mice per group (Mann Whitney test, * p
Figure Legend Snippet: CpG added to the neonatal vaccine decreased pulmonary CCL11 mRNA expression upon RSV challenge. The level of expression of genes encoding mCCL2 (A), mCCL3 (C), mCCL5 (D) and mCCL11 (B) was monitored by quantitative real time PCR of RNAs extracted from individual lungs collected at d5 p.i. Data were normalized to the mHPRT and expressed relative to the unvaccinated infected control group (C+) (R.Q. = 100×2 −ΔΔCt ). Data are mean±SEM from n≥4 mice per group (Mann Whitney test, * p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Infection, Mouse Assay, MANN-WHITNEY

13) Product Images from "A Comparison of the Rest Complex Binding Patterns in Embryonic Stem Cells and Epiblast Stem Cells"

Article Title: A Comparison of the Rest Complex Binding Patterns in Embryonic Stem Cells and Epiblast Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0095374

Rest binding sites that affect transcription. (A) Rest knock-down analysis. We show the indicated gene mRNA expression levels measured using RT-qPCR and the Rest results from two independent qPCR primers. (B) The ES-unique (left) and common (right) Rest binding sites distributions that show the indicated fold change in transcription. Populations with greater than a 2-fold increase in transcription are indicated with arrows. (C) The Gabrb3 Rest binding and transcript levels in ES and EpiS cells are compared. Gabrb3 induction through Rest knock-down and the Gabrb3 regulatory network model are also shown. (D) Rest and Sin3A ChIP Seq tags in the indicated cells are shown. Signal intensities of the active chromatin modifications (H3K4me3, RNA Pol2, and H3ac) surrounding Gabrb3 in ES and EpiS cells. (E) The Igfbp6 and Gli1 Rest binding and transcript levels in ES and EpiS cells are compared. The Rest binding sites around Igfbp6 and Gli1 are located in 50 kbp upstream and 80 kbp downstream of each genes, respectively. The Igfbp6 and Gli1 expression change due to Rest knock-down and the Igf2 regulatory network model are also shown.
Figure Legend Snippet: Rest binding sites that affect transcription. (A) Rest knock-down analysis. We show the indicated gene mRNA expression levels measured using RT-qPCR and the Rest results from two independent qPCR primers. (B) The ES-unique (left) and common (right) Rest binding sites distributions that show the indicated fold change in transcription. Populations with greater than a 2-fold increase in transcription are indicated with arrows. (C) The Gabrb3 Rest binding and transcript levels in ES and EpiS cells are compared. Gabrb3 induction through Rest knock-down and the Gabrb3 regulatory network model are also shown. (D) Rest and Sin3A ChIP Seq tags in the indicated cells are shown. Signal intensities of the active chromatin modifications (H3K4me3, RNA Pol2, and H3ac) surrounding Gabrb3 in ES and EpiS cells. (E) The Igfbp6 and Gli1 Rest binding and transcript levels in ES and EpiS cells are compared. The Rest binding sites around Igfbp6 and Gli1 are located in 50 kbp upstream and 80 kbp downstream of each genes, respectively. The Igfbp6 and Gli1 expression change due to Rest knock-down and the Igf2 regulatory network model are also shown.

Techniques Used: Binding Assay, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

14) Product Images from "Chronic Respiratory Aeroallergen Exposure in Mice Induces Epithelial-Mesenchymal Transition in the Large Airways"

Article Title: Chronic Respiratory Aeroallergen Exposure in Mice Induces Epithelial-Mesenchymal Transition in the Large Airways

Journal: PLoS ONE

doi: 10.1371/journal.pone.0016175

Prolonged respiratory HDM exposure induces epithelial-to-mesenchymal transition. Lung sections (15 µm thick) were prepared from control mice and mice exposed to HDM for 5, 10 or 15 weeks and immunofluorescent staining for the co-expression of the LacZ reporter in airway epithelial cells with α-SMA and occludin (A–D) and with vimentin and E-cadherin (E–H) was performed. Scale bars 10 µm. Quantification of lung fibrosis was performed by morphometric analysis of lung sections stained for α-SMA (I) or LacZ and vimentin (J). * p
Figure Legend Snippet: Prolonged respiratory HDM exposure induces epithelial-to-mesenchymal transition. Lung sections (15 µm thick) were prepared from control mice and mice exposed to HDM for 5, 10 or 15 weeks and immunofluorescent staining for the co-expression of the LacZ reporter in airway epithelial cells with α-SMA and occludin (A–D) and with vimentin and E-cadherin (E–H) was performed. Scale bars 10 µm. Quantification of lung fibrosis was performed by morphometric analysis of lung sections stained for α-SMA (I) or LacZ and vimentin (J). * p

Techniques Used: Mouse Assay, Staining, Expressing

Induction of EMT in A549 cells. Progression through EMT was evaluated in the adenocarcinoma-derived human lung epithelial cell line A549 72h after the addition of TGF-β1 (10 ng/mL) and EGF (50 ng/mL) to the culture medium. The epithelial junction proteins CAR and E-cadherin (A, D), the tight junction protein occludin and the mesenchymal marker vimentin (B, E) as well as the EMT-associated transcription factors pSmad3 and Snail1 (C, F) were assessed by immunofluorescent staining. Scale bar 10 µm. Quantification of the relative mRNA expression of CAR (G), occludin (H), E-cadherin (I), vimentin (J) ), α-SMA (K) and Snail1 (L) was assessed by qPCR.* p
Figure Legend Snippet: Induction of EMT in A549 cells. Progression through EMT was evaluated in the adenocarcinoma-derived human lung epithelial cell line A549 72h after the addition of TGF-β1 (10 ng/mL) and EGF (50 ng/mL) to the culture medium. The epithelial junction proteins CAR and E-cadherin (A, D), the tight junction protein occludin and the mesenchymal marker vimentin (B, E) as well as the EMT-associated transcription factors pSmad3 and Snail1 (C, F) were assessed by immunofluorescent staining. Scale bar 10 µm. Quantification of the relative mRNA expression of CAR (G), occludin (H), E-cadherin (I), vimentin (J) ), α-SMA (K) and Snail1 (L) was assessed by qPCR.* p

Techniques Used: Derivative Assay, Marker, Staining, Expressing, Real-time Polymerase Chain Reaction

Partial induction of EMT in 16HBE14o- cells. (A–I) Progression through EMT was evaluated in the phenotypically normal human lung epithelial cell line 16HBE14o- 72 h after the addition of TGF-β1 (10 ng/mL) and EGF (50 ng/mL) to the culture medium. The epithelial junction proteins CAR and E-cadherin (A, D), the tight junction protein occludin and the mesenchymal marker vimentin (B, E) as well as the EMT-associated transcription factors pSmad3 and Snail1 (C, F) were assessed by immunofluorescent staining. Scale bar 10 µm. Quantification of the relative mRNA expression of CAR (G), occludin (H), E-cadherin (I), vimentin (J), α-SMA (K) and Snail1 (L) was assessed by qPCR. * p
Figure Legend Snippet: Partial induction of EMT in 16HBE14o- cells. (A–I) Progression through EMT was evaluated in the phenotypically normal human lung epithelial cell line 16HBE14o- 72 h after the addition of TGF-β1 (10 ng/mL) and EGF (50 ng/mL) to the culture medium. The epithelial junction proteins CAR and E-cadherin (A, D), the tight junction protein occludin and the mesenchymal marker vimentin (B, E) as well as the EMT-associated transcription factors pSmad3 and Snail1 (C, F) were assessed by immunofluorescent staining. Scale bar 10 µm. Quantification of the relative mRNA expression of CAR (G), occludin (H), E-cadherin (I), vimentin (J), α-SMA (K) and Snail1 (L) was assessed by qPCR. * p

Techniques Used: Marker, Staining, Expressing, Real-time Polymerase Chain Reaction

15) Product Images from "A Novel Secretion Pathway of Salmonella enterica Acts as an Antivirulence Modulator during Salmonellosis"

Article Title: A Novel Secretion Pathway of Salmonella enterica Acts as an Antivirulence Modulator during Salmonellosis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000036

The ZirTS pathway is negatively regulated by zinc and OxyR. A. OxyR negatively regulates the expression of zirTS . Wild-type S. Typhimurium SL1344 (wild-type) and an isogenic strain harboring a mutation in oxyR ( oxyR ) expressing zirTS::lacZ were grown in M9 minimal medium to late stationary phase. β-galactosidase assay was performed to evaluate the expression of zirTS::lacZ in each strain. The presented values represent the average of at least 7 independent cultures with a standard deviation shown by the error bars. B. Expression of zirT and zirS in the oxyR background vs. the wild-type strain as determined by q-RT-PCR. RNA was harvested from SL1344 and the oxyR mutant strains grown in M9; reverse-transcribed and the expression of zirT and zirS was examined by quantitative real-time PCR. The fold change in the expression of zirT and zirS in the oxyR background, relative to their expression in the wild-type strain is presented. Expression was normalized using the housekeeping rpoD gene as a control. The results represent the average of 4 independent experiments (each included 3–5 replicates) with a standard deviation shown by the error bars. C. Expression of zirTS in the presence of metals. S. Typhimurium SL1344 expressing zirTS::lacZ was grown in M9 medium (M9) or M9 supplemented with the following metal salts: 0.1 mM MgSO 4 (Mg), 0.1 mM FeCl 3 (Fe 3+ ), 0.1 mM FeSO 4 (Fe 2+ ), and ZnSO 4 (Zn) in the indicated concentrations (0.01, 0.1, or 0.5 mM). Cultures were grown to a late stationary phase and β-galactosidase assay was performed to evaluate the expression levels of zirTS::lacZ under each condition. The presented values represent the average of at least 8 independent cultures with a standard deviation shown by the error bars. D. Expression in the presence of metal chelators. S. Typhimurium SL1344 expressing zirTS::lacZ was grown in LB broth (LB) or LB supplemented with the following compounds: 1 mM ZnSO 4 (LB+Zn), 0.1 mM metal chelators (DTPA, EDDA, or TPEN), metals chelators with 1 mM ZnSO 4 (Zn), or metal chelators with 1 mM FeSO 4 (Fe). Cultures were grown to a late stationary phase following by β-galactosidase assay. The presented values represent the average of at least 8 independent cultures with a standard deviation shown by the error bars. E. Expression of zirT and zirS in the presence and absence of zinc and metal chelators as determined by q-RT-PCR. RNA was harvested from wild-type Salmonella cultures that were grown either in M9 medium in the presence or absence of 0.5 mM ZnSO 4 ; or in LB supplemented with or lacking DTPA (0.1 mM) or TPEN (0.05 mM). RNA was reverse-transcribed and the expression of zirT and zirS was examined by real-time PCR. The fold change in the expression of zirT and zirS in cultures that were grown in the presence of zinc (M9+Zn) or metal chelators (LB+DTPA/TPEN) relative to their expression in the appropriate unsupplemented media is presented. Expression was normalized using the housekeeping rpoD gene as a control. The results represent the average of 3 independent experiments (each included 3–5 replicates) with a standard deviation shown by the error bars.
Figure Legend Snippet: The ZirTS pathway is negatively regulated by zinc and OxyR. A. OxyR negatively regulates the expression of zirTS . Wild-type S. Typhimurium SL1344 (wild-type) and an isogenic strain harboring a mutation in oxyR ( oxyR ) expressing zirTS::lacZ were grown in M9 minimal medium to late stationary phase. β-galactosidase assay was performed to evaluate the expression of zirTS::lacZ in each strain. The presented values represent the average of at least 7 independent cultures with a standard deviation shown by the error bars. B. Expression of zirT and zirS in the oxyR background vs. the wild-type strain as determined by q-RT-PCR. RNA was harvested from SL1344 and the oxyR mutant strains grown in M9; reverse-transcribed and the expression of zirT and zirS was examined by quantitative real-time PCR. The fold change in the expression of zirT and zirS in the oxyR background, relative to their expression in the wild-type strain is presented. Expression was normalized using the housekeeping rpoD gene as a control. The results represent the average of 4 independent experiments (each included 3–5 replicates) with a standard deviation shown by the error bars. C. Expression of zirTS in the presence of metals. S. Typhimurium SL1344 expressing zirTS::lacZ was grown in M9 medium (M9) or M9 supplemented with the following metal salts: 0.1 mM MgSO 4 (Mg), 0.1 mM FeCl 3 (Fe 3+ ), 0.1 mM FeSO 4 (Fe 2+ ), and ZnSO 4 (Zn) in the indicated concentrations (0.01, 0.1, or 0.5 mM). Cultures were grown to a late stationary phase and β-galactosidase assay was performed to evaluate the expression levels of zirTS::lacZ under each condition. The presented values represent the average of at least 8 independent cultures with a standard deviation shown by the error bars. D. Expression in the presence of metal chelators. S. Typhimurium SL1344 expressing zirTS::lacZ was grown in LB broth (LB) or LB supplemented with the following compounds: 1 mM ZnSO 4 (LB+Zn), 0.1 mM metal chelators (DTPA, EDDA, or TPEN), metals chelators with 1 mM ZnSO 4 (Zn), or metal chelators with 1 mM FeSO 4 (Fe). Cultures were grown to a late stationary phase following by β-galactosidase assay. The presented values represent the average of at least 8 independent cultures with a standard deviation shown by the error bars. E. Expression of zirT and zirS in the presence and absence of zinc and metal chelators as determined by q-RT-PCR. RNA was harvested from wild-type Salmonella cultures that were grown either in M9 medium in the presence or absence of 0.5 mM ZnSO 4 ; or in LB supplemented with or lacking DTPA (0.1 mM) or TPEN (0.05 mM). RNA was reverse-transcribed and the expression of zirT and zirS was examined by real-time PCR. The fold change in the expression of zirT and zirS in cultures that were grown in the presence of zinc (M9+Zn) or metal chelators (LB+DTPA/TPEN) relative to their expression in the appropriate unsupplemented media is presented. Expression was normalized using the housekeeping rpoD gene as a control. The results represent the average of 3 independent experiments (each included 3–5 replicates) with a standard deviation shown by the error bars.

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

16) Product Images from "The molecular signature of therapeutic mesenchymal stem cells exposes the architecture of the hematopoietic stem cell niche synapse"

Article Title: The molecular signature of therapeutic mesenchymal stem cells exposes the architecture of the hematopoietic stem cell niche synapse

Journal: BMC Genomics

doi: 10.1186/1471-2164-8-65

Therapeutic MSCs show a unique gene expression signature . ( a ) Principal component analysis of samples based on the expression of 500 genes. ( b ) Hierarchical clustering of genes and averaged samples utilizing smooth correlation metric with indication of the germinal layer of origin. MSCs: mesenchymal stem cells; MEFs: mouse embryonic fibroblasts; ESCs: embryonic stem cells; NSCs: neural stem cells; HSCs: hematopoietic stem cells; DCs: dendritic cells.
Figure Legend Snippet: Therapeutic MSCs show a unique gene expression signature . ( a ) Principal component analysis of samples based on the expression of 500 genes. ( b ) Hierarchical clustering of genes and averaged samples utilizing smooth correlation metric with indication of the germinal layer of origin. MSCs: mesenchymal stem cells; MEFs: mouse embryonic fibroblasts; ESCs: embryonic stem cells; NSCs: neural stem cells; HSCs: hematopoietic stem cells; DCs: dendritic cells.

Techniques Used: Expressing

Validation of microarray results . (a) Microarray levels (grey bars) represent the signal intensity of probe-sets corresponding to the same gene (averaged when more than one present). qRT-PCR data (black bars) are obtained utilizing a GAPDH standard curve. Relative expression levels with respect to those of MSCs are shown. ( b ) Correlation between microarray (X axis) and qRT-PCR (Y axis) expression values (fold-differences versus MSCs). triangles = Spp1 , circles = Fn1 , diamonds = Thbs1 , squares = Thbs2 , violet = Heart, red = Brain, yellow = Lung, rose = Kidney, orange = DCs, green = MEFs, tan = NSCs, grey = T cells, blue = ESCs, gold = Muscle, turqoise = Liver.
Figure Legend Snippet: Validation of microarray results . (a) Microarray levels (grey bars) represent the signal intensity of probe-sets corresponding to the same gene (averaged when more than one present). qRT-PCR data (black bars) are obtained utilizing a GAPDH standard curve. Relative expression levels with respect to those of MSCs are shown. ( b ) Correlation between microarray (X axis) and qRT-PCR (Y axis) expression values (fold-differences versus MSCs). triangles = Spp1 , circles = Fn1 , diamonds = Thbs1 , squares = Thbs2 , violet = Heart, red = Brain, yellow = Lung, rose = Kidney, orange = DCs, green = MEFs, tan = NSCs, grey = T cells, blue = ESCs, gold = Muscle, turqoise = Liver.

Techniques Used: Microarray, Quantitative RT-PCR, Expressing

17) Product Images from "Tetra-O-methyl nordihydroguaiaretic acid (Terameprocol) inhibits the NF-?B-dependent transcription of TNF-? and MCP-1/CCL2 genes by preventing RelA from binding its cognate sites on DNA"

Article Title: Tetra-O-methyl nordihydroguaiaretic acid (Terameprocol) inhibits the NF-?B-dependent transcription of TNF-? and MCP-1/CCL2 genes by preventing RelA from binding its cognate sites on DNA

Journal: Journal of Inflammation (London, England)

doi: 10.1186/1476-9255-7-59

TMP inhibits TNF-α and MCP-1/CCL-2 protein and mRNA . RAW 264.7 cells were either stimulated with 1 μg/ml of LPS or 1 μg/ml of LPS and 25 μM TMP. Following 24 h of treatment, supernatants were collected and levels of TNF-α (A) and MCP-1/CCL2 (B) were determined by ELISA. To assess the effects of TMP on the transcription of TNF-α (C) and MCP-1/CCL2 (D) genes, RNA was prepared from RAW 264.7 cells stimulated with 1 μg/ml of LPS or 1 μg/ml of LPS and 25 μM TMP for the indicated time periods. Quantitative RT-PCR was used to analyze the levels of TNF-α and MCP-1/CCL2 mRNA. Asterisks indicate significant differences between treatments with LPS and LPS and TMP (p
Figure Legend Snippet: TMP inhibits TNF-α and MCP-1/CCL-2 protein and mRNA . RAW 264.7 cells were either stimulated with 1 μg/ml of LPS or 1 μg/ml of LPS and 25 μM TMP. Following 24 h of treatment, supernatants were collected and levels of TNF-α (A) and MCP-1/CCL2 (B) were determined by ELISA. To assess the effects of TMP on the transcription of TNF-α (C) and MCP-1/CCL2 (D) genes, RNA was prepared from RAW 264.7 cells stimulated with 1 μg/ml of LPS or 1 μg/ml of LPS and 25 μM TMP for the indicated time periods. Quantitative RT-PCR was used to analyze the levels of TNF-α and MCP-1/CCL2 mRNA. Asterisks indicate significant differences between treatments with LPS and LPS and TMP (p

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

18) Product Images from "Uridine Metabolism in HIV-1-Infected Patients: Effect of Infection, of Antiretroviral Therapy and of HIV-1/ART-Associated Lipodystrophy Syndrome"

Article Title: Uridine Metabolism in HIV-1-Infected Patients: Effect of Infection, of Antiretroviral Therapy and of HIV-1/ART-Associated Lipodystrophy Syndrome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013896

Expression of genes encoding enzymes involved in uridine metabolism and concentrative nucleoside transporters (CNT). Data from subcutaneous adipose tissue of uninfected controls and HIV-1 infected patients. Figure shows means + SEM for each specific mRNA concentration (expressed as ratio relative to HPRT mRNA) from 6-8 patients/group. P values for statistical comparisons between groups are shown when
Figure Legend Snippet: Expression of genes encoding enzymes involved in uridine metabolism and concentrative nucleoside transporters (CNT). Data from subcutaneous adipose tissue of uninfected controls and HIV-1 infected patients. Figure shows means + SEM for each specific mRNA concentration (expressed as ratio relative to HPRT mRNA) from 6-8 patients/group. P values for statistical comparisons between groups are shown when

Techniques Used: Expressing, Infection, Concentration Assay

19) Product Images from "Folliculostellate Cells Are Required for Laminin Release from Gonadotrophs in Rat Anterior Pituitary"

Article Title: Folliculostellate Cells Are Required for Laminin Release from Gonadotrophs in Rat Anterior Pituitary

Journal: Acta Histochemica et Cytochemica

doi: 10.1267/ahc.14036

Relative mRNA concentration of laminin α1 chain ( Lama1 ), evaluated by quantitative real-time PCR. The white and gray bars represent cell aggregates without FS cells (0%) and with FS cells (5%, 10%, and 20%), respectively (n=4–5, mean±SEM). The concentrations were normalized with β-actin mRNA concentration. The expression level of Lama1 mRNA did not significantly differ in relation to the presence or absence of FS cells.
Figure Legend Snippet: Relative mRNA concentration of laminin α1 chain ( Lama1 ), evaluated by quantitative real-time PCR. The white and gray bars represent cell aggregates without FS cells (0%) and with FS cells (5%, 10%, and 20%), respectively (n=4–5, mean±SEM). The concentrations were normalized with β-actin mRNA concentration. The expression level of Lama1 mRNA did not significantly differ in relation to the presence or absence of FS cells.

Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Expressing

20) Product Images from "Folliculostellate Cells Are Required for Laminin Release from Gonadotrophs in Rat Anterior Pituitary"

Article Title: Folliculostellate Cells Are Required for Laminin Release from Gonadotrophs in Rat Anterior Pituitary

Journal: Acta Histochemica et Cytochemica

doi: 10.1267/ahc.14036

Immunofluorescence of laminin in 3D cell aggregates with different proportions of FS cells (0%–20%). Cell aggregates were fixed 5 days after plating and stained with laminin antibody. The top panels are phase-contrast images of cell aggregates superimposed on fluorescence images of FS cells ( a–d ). FS cells expressing GFP are shown in green. During the 5-day culture, amorphous aggregates were observed in the absence of FS cells ( a ). In the presence of FS cells, the cells formed round or oval aggregates with a smooth outer layer ( b–d ). The confocal images of FS cells and laminin immunofluorescence are shown in e–h and i–l , respectively. The bottom panels show merged images ( m–p ; FS cells, green; laminin, red; DAPI, blue). Extracellular laminin deposition was observed as the number of FS cells increased ( k, l, o, p ), while laminin-immunopositive cells (arrowheads) decreased as the number of FS cells increased ( i, j, m, n ). Bars=100 μm ( a–d ) and 10 μm ( e–l ).
Figure Legend Snippet: Immunofluorescence of laminin in 3D cell aggregates with different proportions of FS cells (0%–20%). Cell aggregates were fixed 5 days after plating and stained with laminin antibody. The top panels are phase-contrast images of cell aggregates superimposed on fluorescence images of FS cells ( a–d ). FS cells expressing GFP are shown in green. During the 5-day culture, amorphous aggregates were observed in the absence of FS cells ( a ). In the presence of FS cells, the cells formed round or oval aggregates with a smooth outer layer ( b–d ). The confocal images of FS cells and laminin immunofluorescence are shown in e–h and i–l , respectively. The bottom panels show merged images ( m–p ; FS cells, green; laminin, red; DAPI, blue). Extracellular laminin deposition was observed as the number of FS cells increased ( k, l, o, p ), while laminin-immunopositive cells (arrowheads) decreased as the number of FS cells increased ( i, j, m, n ). Bars=100 μm ( a–d ) and 10 μm ( e–l ).

Techniques Used: Immunofluorescence, Staining, Fluorescence, Expressing

21) Product Images from "Targeting Bone Remodeling by Isoflavone and 3,3?-Diindolylmethane in the Context of Prostate Cancer Bone Metastasis"

Article Title: Targeting Bone Remodeling by Isoflavone and 3,3?-Diindolylmethane in the Context of Prostate Cancer Bone Metastasis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0033011

Gene expressions validated by RT-PCR and Western Blot analysis. BR-DIM and isoflavone regulated the expression of MITF, p38, Akt, NKX3-1, p27, cyclin D, CREB and PSA at mRNA and protein levels tested by real-time PCR (A, B) and Western Blot analysis (C, D). C4-2B (A, C) and PC-3 (B, D) cells were treated with 25 µM G2535 or 25 µM BR-DIM for 24 hours (for RNA extraction) or 48 hours (for protein extraction). (BD: BR-DIM).
Figure Legend Snippet: Gene expressions validated by RT-PCR and Western Blot analysis. BR-DIM and isoflavone regulated the expression of MITF, p38, Akt, NKX3-1, p27, cyclin D, CREB and PSA at mRNA and protein levels tested by real-time PCR (A, B) and Western Blot analysis (C, D). C4-2B (A, C) and PC-3 (B, D) cells were treated with 25 µM G2535 or 25 µM BR-DIM for 24 hours (for RNA extraction) or 48 hours (for protein extraction). (BD: BR-DIM).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Real-time Polymerase Chain Reaction, RNA Extraction, Protein Extraction

22) Product Images from "Dynamics of Protein Phosphatase Gene Expression in Corbicula fluminea Exposed to Microcystin-LR and to Toxic Microcystis aeruginosa Cells"

Article Title: Dynamics of Protein Phosphatase Gene Expression in Corbicula fluminea Exposed to Microcystin-LR and to Toxic Microcystis aeruginosa Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms12129172

Projection of the normalized gene expression values of PPP1, PPP2 and PPP4 in C. fluminea visceral mass after exposure to 5 μg L −1 of MC-LR during 96 h, in relation to the studied independent variables, according to the multiple linear regression analysis (Control groups-dashed line; Exposure groups-continuous line). The regression models describing the functions represented in the figure include only the significant regression variables (PPP1: none; PPP2: Treatment; PPP4: none).
Figure Legend Snippet: Projection of the normalized gene expression values of PPP1, PPP2 and PPP4 in C. fluminea visceral mass after exposure to 5 μg L −1 of MC-LR during 96 h, in relation to the studied independent variables, according to the multiple linear regression analysis (Control groups-dashed line; Exposure groups-continuous line). The regression models describing the functions represented in the figure include only the significant regression variables (PPP1: none; PPP2: Treatment; PPP4: none).

Techniques Used: Expressing

Tissue content (μg MC-LR g −1 DW) of unbound MC-LR in the visceral mass during exposure of C. fluminea to 5 μg L −1 MC-LR for 96 h. Values represent average of three replicates and bars represent confidence interval for mean level (95%).
Figure Legend Snippet: Tissue content (μg MC-LR g −1 DW) of unbound MC-LR in the visceral mass during exposure of C. fluminea to 5 μg L −1 MC-LR for 96 h. Values represent average of three replicates and bars represent confidence interval for mean level (95%).

Techniques Used:

Projection of the normalized gene expression values of PPP2 in C. fluminea visceral mass after exposure to 1 × 10 5 cells cm −3 of a M. aeruginosa toxic strain during 96 h, in relation to the studied independent variables, according to the multiple linear regression analysis (Control groups-dashed line; Exposure groups-continuous line). The regression models describing the functions represented in the figure include only the significant regression variables (Treatment, Exposure time, Exposure time squared , Treatment × Exposure time, Treatment × Exposure time squared , Treatment × Exposure time cubed ).
Figure Legend Snippet: Projection of the normalized gene expression values of PPP2 in C. fluminea visceral mass after exposure to 1 × 10 5 cells cm −3 of a M. aeruginosa toxic strain during 96 h, in relation to the studied independent variables, according to the multiple linear regression analysis (Control groups-dashed line; Exposure groups-continuous line). The regression models describing the functions represented in the figure include only the significant regression variables (Treatment, Exposure time, Exposure time squared , Treatment × Exposure time, Treatment × Exposure time squared , Treatment × Exposure time cubed ).

Techniques Used: Expressing

Tissue content (μg MC-LReq. g −1 DW) of unbound MC-LReq. in the visceral mass during exposure of C. fluminea to 1 × 10 5 cells cm −3 of a M. aeruginosa toxic strain for 96 h. Values represent average of three replicates and bars represent confidence interval for mean level (95%).
Figure Legend Snippet: Tissue content (μg MC-LReq. g −1 DW) of unbound MC-LReq. in the visceral mass during exposure of C. fluminea to 1 × 10 5 cells cm −3 of a M. aeruginosa toxic strain for 96 h. Values represent average of three replicates and bars represent confidence interval for mean level (95%).

Techniques Used:

Projection of enzyme activity values of PPP2 in C. fluminea visceral mass after exposure to 1 × 10 5 cells mL −1 of a M. aeruginosa toxic strain during 96 h, in relation to the studied independent variables, according to the multiple linear regression analysis (Control groups-dashed line; Exposure groups-continuous line). The regression models describing the functions represented in the figure include only the significant regression variables (Treatment, Exposure time, Treatment × Exposure time, Treatment × Exposure time squared , Treatment × Exposure time cubed ).
Figure Legend Snippet: Projection of enzyme activity values of PPP2 in C. fluminea visceral mass after exposure to 1 × 10 5 cells mL −1 of a M. aeruginosa toxic strain during 96 h, in relation to the studied independent variables, according to the multiple linear regression analysis (Control groups-dashed line; Exposure groups-continuous line). The regression models describing the functions represented in the figure include only the significant regression variables (Treatment, Exposure time, Treatment × Exposure time, Treatment × Exposure time squared , Treatment × Exposure time cubed ).

Techniques Used: Activity Assay

23) Product Images from "No Dopamine Cell Loss or Changes in Cytoskeleton Function in Transgenic Mice Expressing Physiological Levels of Wild Type or G2019S Mutant LRRK2 and in Human Fibroblasts"

Article Title: No Dopamine Cell Loss or Changes in Cytoskeleton Function in Transgenic Mice Expressing Physiological Levels of Wild Type or G2019S Mutant LRRK2 and in Human Fibroblasts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0118947

Generation of human wild type and G2019S mutant LRRK2 transgenic mice. A: Human full length wild type or G2019S mutant LRRK2 cDNA were cloned into the murine Thy-1.2 promoter element driving neuronal-specific transgene expression. B: Western blot analysis of LRRK2 protein expression in brain lysates from LRRK2, GS-LRRK2 line 1, GS-LRRK2 line 2 and non-tg littermates using MID antibody which recognizes human and murine LRRK2 protein. C: Densitometry quantification revealed approximately twice the amount of total LRRK2 protein in all transgenic lines compared to endogenous Lrrk2 levels in non-tg controls. Data represent means ± SEM; n = 2 for each transgenic line.
Figure Legend Snippet: Generation of human wild type and G2019S mutant LRRK2 transgenic mice. A: Human full length wild type or G2019S mutant LRRK2 cDNA were cloned into the murine Thy-1.2 promoter element driving neuronal-specific transgene expression. B: Western blot analysis of LRRK2 protein expression in brain lysates from LRRK2, GS-LRRK2 line 1, GS-LRRK2 line 2 and non-tg littermates using MID antibody which recognizes human and murine LRRK2 protein. C: Densitometry quantification revealed approximately twice the amount of total LRRK2 protein in all transgenic lines compared to endogenous Lrrk2 levels in non-tg controls. Data represent means ± SEM; n = 2 for each transgenic line.

Techniques Used: Mutagenesis, Transgenic Assay, Mouse Assay, Clone Assay, Expressing, Western Blot

LRRK2 mRNA and protein expression in brain regions in the three transgenic mouse lines. A: RT-PCR semi-quantification of LRRK2 expression in whole brains at different embryonic and postnatal stages indicates robust transgene expression at postnatal day 2 (P2) in all three lines. Data represents means ± SEM; n = 3–5 animals per group. B: In-situ hybridization of coronal brain sections at the level of posterior hippocampus and midbrain with two human specific LRRK2 probes showed comparable transgene expression levels in hippocampus and cortex of 11-month-old LRRK2 and GS-LRRK2 lines 1 and 2. C: Western blot analysis of LRRK2 protein showed robust expression levels of LRRK2 in hippocampus (HC) and cortex (CTX) of 10-month-old animals with the human-specific LRRK2 antibody MJFF5; n = 3 animals per genotype.
Figure Legend Snippet: LRRK2 mRNA and protein expression in brain regions in the three transgenic mouse lines. A: RT-PCR semi-quantification of LRRK2 expression in whole brains at different embryonic and postnatal stages indicates robust transgene expression at postnatal day 2 (P2) in all three lines. Data represents means ± SEM; n = 3–5 animals per group. B: In-situ hybridization of coronal brain sections at the level of posterior hippocampus and midbrain with two human specific LRRK2 probes showed comparable transgene expression levels in hippocampus and cortex of 11-month-old LRRK2 and GS-LRRK2 lines 1 and 2. C: Western blot analysis of LRRK2 protein showed robust expression levels of LRRK2 in hippocampus (HC) and cortex (CTX) of 10-month-old animals with the human-specific LRRK2 antibody MJFF5; n = 3 animals per genotype.

Techniques Used: Expressing, Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Western Blot

Analysis of neurite outgrowth and branching complexity of primary hippocampal neurons. A-B: Neurite parameters of neuronal cultures from LRRK2, GS-LRRK2 (line 2) and their respective non-tg littermate, which were treated with vehicle-control or LRRK2-IN-1 (0.1 M) for seven days (DIV7). Comparison of parameters describing neurite branching (A) included number of branches, number of neurite trees and number of segments. Comparison of neurite length parameters (B) included total neurite length, average neurite length and maximal neurite length. Data represent mean ± SEM and were analyzed with two-way ANOVA. No significant difference was detected. Number of neurons analyzed for cultures obtained from LRRK2 transgenic mice: non-tg = 1339, non-tg + LRRK2-IN-1 = 1609; LRRK2 = 1697, LRRK2 + LRRK2-IN-1 = 1542, n = 4 independent experiments; Number of neurons analyzed for cultures obtained from GS-LRRK2 transgenic mice: non-tg = 1268; non-tg + LRRK2-IN-1 = 1522; GS-LRRK2 = 1526; GS-LRRK2 + LRRK2-IN-1 = 1844, n = 4 independent experiments; C-H: Representative pictures of ß-Tubulin III stained neurons on DIV7 derived from wild type, GS- LRRK2 (line 2), their non-transgenic littermates. Pictures were obtained with the BD Pathway 855 high content Bioimager. C1-H2: Total neurite length (C1-H1) and number of branches (C2-H2) segmentation corresponding to ß-tubulin III staining images (C-H) obtained from Attovision Software.
Figure Legend Snippet: Analysis of neurite outgrowth and branching complexity of primary hippocampal neurons. A-B: Neurite parameters of neuronal cultures from LRRK2, GS-LRRK2 (line 2) and their respective non-tg littermate, which were treated with vehicle-control or LRRK2-IN-1 (0.1 M) for seven days (DIV7). Comparison of parameters describing neurite branching (A) included number of branches, number of neurite trees and number of segments. Comparison of neurite length parameters (B) included total neurite length, average neurite length and maximal neurite length. Data represent mean ± SEM and were analyzed with two-way ANOVA. No significant difference was detected. Number of neurons analyzed for cultures obtained from LRRK2 transgenic mice: non-tg = 1339, non-tg + LRRK2-IN-1 = 1609; LRRK2 = 1697, LRRK2 + LRRK2-IN-1 = 1542, n = 4 independent experiments; Number of neurons analyzed for cultures obtained from GS-LRRK2 transgenic mice: non-tg = 1268; non-tg + LRRK2-IN-1 = 1522; GS-LRRK2 = 1526; GS-LRRK2 + LRRK2-IN-1 = 1844, n = 4 independent experiments; C-H: Representative pictures of ß-Tubulin III stained neurons on DIV7 derived from wild type, GS- LRRK2 (line 2), their non-transgenic littermates. Pictures were obtained with the BD Pathway 855 high content Bioimager. C1-H2: Total neurite length (C1-H1) and number of branches (C2-H2) segmentation corresponding to ß-tubulin III staining images (C-H) obtained from Attovision Software.

Techniques Used: Transgenic Assay, Mouse Assay, Staining, Derivative Assay, Software

No neuronal loss or neurodegeneration was detected in SNpc in transgenic mice. A: Representative coronal section of midbrain sections from 12- to 13-month-old non-tg, LRRK2 and GS-LRRK2 (line 2) transgenic mice immunostained for TH. (Scale bars: 100μm). B: Cell counts of TH+ and Nissl+ neurons in SNpc from non-tg, LRRK2 and GS-LRRK2 transgenic mice. Data represent mean ± SEM; transgenic mice n = 2, non-tg mice n = 3. n . s = not significant, (one-way ANOVA, Tukey’s post hoc analysis).
Figure Legend Snippet: No neuronal loss or neurodegeneration was detected in SNpc in transgenic mice. A: Representative coronal section of midbrain sections from 12- to 13-month-old non-tg, LRRK2 and GS-LRRK2 (line 2) transgenic mice immunostained for TH. (Scale bars: 100μm). B: Cell counts of TH+ and Nissl+ neurons in SNpc from non-tg, LRRK2 and GS-LRRK2 transgenic mice. Data represent mean ± SEM; transgenic mice n = 2, non-tg mice n = 3. n . s = not significant, (one-way ANOVA, Tukey’s post hoc analysis).

Techniques Used: Transgenic Assay, Mouse Assay

24) Product Images from "Probiotic Escherichia coli Nissle 1917 Inhibits Leaky Gut by Enhancing Mucosal Integrity"

Article Title: Probiotic Escherichia coli Nissle 1917 Inhibits Leaky Gut by Enhancing Mucosal Integrity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0001308

Increased ZO-1 mRNA expression in mice treated with DSS and EcN. IECs were isolated from indicated mice and relative levels of ZO-1 mRNA were normalized with respect to the expression level of IECs from DSS treated mice (fold change = 1). Data are presented as mean of four independent experiments (n = 3/group). * p
Figure Legend Snippet: Increased ZO-1 mRNA expression in mice treated with DSS and EcN. IECs were isolated from indicated mice and relative levels of ZO-1 mRNA were normalized with respect to the expression level of IECs from DSS treated mice (fold change = 1). Data are presented as mean of four independent experiments (n = 3/group). * p

Techniques Used: Expressing, Mouse Assay, Isolation

Isolation of IECs from gnotobiotic mice colonized with EcN or E. coli MG1655 and ZO-1 expression analysis. Gnotobiotic BALB/c mice were colonized with E. coli MG1655 (K12) or EcN for 6 days, respectively. Application of PBS was used as control. (A) Whole intestinal cell populations were isolated from gnotobiotic mice treated either with PBS, K12 or EcN. For sorting of a pure intestinal epithelial cell population, cells were labeled with anti-CD45 antibody to exclude hematopoietic cells and further distinguished by cell granularity and size (SSC) (Pre sorting). IECs were FACS-sorted by negative selection. Re-analysis was performed to determine the purity of sorted IECs (Post sorting). (B) Quantitative ZO-1 mRNA expression in IECs. Relative mRNA amounts were normalized with respect to expression levels of IECs from control mice (fold change = 1). Data are presented as mean of three independent experiments (n = 3/group). (C) Quantitative ZO-2 mRNA expression in IECs. Relative mRNA amounts were normalized with respect to expression levels of IECs from control mice (fold change = 1). Data are presented as mean of three independent experiments (n = 3/group). * p
Figure Legend Snippet: Isolation of IECs from gnotobiotic mice colonized with EcN or E. coli MG1655 and ZO-1 expression analysis. Gnotobiotic BALB/c mice were colonized with E. coli MG1655 (K12) or EcN for 6 days, respectively. Application of PBS was used as control. (A) Whole intestinal cell populations were isolated from gnotobiotic mice treated either with PBS, K12 or EcN. For sorting of a pure intestinal epithelial cell population, cells were labeled with anti-CD45 antibody to exclude hematopoietic cells and further distinguished by cell granularity and size (SSC) (Pre sorting). IECs were FACS-sorted by negative selection. Re-analysis was performed to determine the purity of sorted IECs (Post sorting). (B) Quantitative ZO-1 mRNA expression in IECs. Relative mRNA amounts were normalized with respect to expression levels of IECs from control mice (fold change = 1). Data are presented as mean of three independent experiments (n = 3/group). (C) Quantitative ZO-2 mRNA expression in IECs. Relative mRNA amounts were normalized with respect to expression levels of IECs from control mice (fold change = 1). Data are presented as mean of three independent experiments (n = 3/group). * p

Techniques Used: Isolation, Mouse Assay, Expressing, Labeling, FACS, Selection

25) Product Images from "Demethylation of the Coding Region Triggers the Activation of the Human Testis-Specific PDHA2 Gene in Somatic Tissues"

Article Title: Demethylation of the Coding Region Triggers the Activation of the Human Testis-Specific PDHA2 Gene in Somatic Tissues

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038076

Recruitment of RNA pol II to the human PDHA2 gene. Chromatin from SH-SY5Y was prepared at 72 hours after treatment with 5 µM DAC and precipitated with antibodies directed against IgG, Sp1 and RNA pol II. After DNA recovery, the precipitates were evaluated by real-time PCR as described in Experimental Procedures. Results are expressed as fold change over IgG and represent means of at least three independent experiments ± SEM (* p
Figure Legend Snippet: Recruitment of RNA pol II to the human PDHA2 gene. Chromatin from SH-SY5Y was prepared at 72 hours after treatment with 5 µM DAC and precipitated with antibodies directed against IgG, Sp1 and RNA pol II. After DNA recovery, the precipitates were evaluated by real-time PCR as described in Experimental Procedures. Results are expressed as fold change over IgG and represent means of at least three independent experiments ± SEM (* p

Techniques Used: Real-time Polymerase Chain Reaction

26) Product Images from "Nitrogen regulation of protein-protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea"

Article Title: Nitrogen regulation of protein-protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea

Journal: MicrobiologyOpen

doi: 10.1002/mbo3.120

Northern blot analysis of the amtB-glnK genomic region of Hfx. mediterranei . (A) Four different DIG-labeled PCR products were used as probes. Primers (listed in Table S1 ) were amt1-mRNA-3F and amt1-mRNA-3R (probe A, 515 bp), glnK1-mRNA-1F and glnK1-mRNA-1R (probe B, 364 bp), amt2-mRNA-4F and amt2-mRNA-4R (probe C, 473), glnK2-mRNA-2F and glnK2-mRNA-2R (probe D, 337 bp). (B) 1 μg or 0.5 μg (‘samples) of RNA from Hfx. mediterranei wild-type cells grown with different nitrogen sources were probed with the four DIG-labeled PCR fragments. Lanes correspond to (1 and 1′) complex medium samples, (2 and 2′) 5 mmol/L NH 4 Cl samples, (3 and 3′) 5 mmol/L KNO 3 samples, (M) RiboRuler High Range RNA Ladder from Fermentas , (RNA) RNA sample stained with ethidium bromide and visualized using ultraviolet light.
Figure Legend Snippet: Northern blot analysis of the amtB-glnK genomic region of Hfx. mediterranei . (A) Four different DIG-labeled PCR products were used as probes. Primers (listed in Table S1 ) were amt1-mRNA-3F and amt1-mRNA-3R (probe A, 515 bp), glnK1-mRNA-1F and glnK1-mRNA-1R (probe B, 364 bp), amt2-mRNA-4F and amt2-mRNA-4R (probe C, 473), glnK2-mRNA-2F and glnK2-mRNA-2R (probe D, 337 bp). (B) 1 μg or 0.5 μg (‘samples) of RNA from Hfx. mediterranei wild-type cells grown with different nitrogen sources were probed with the four DIG-labeled PCR fragments. Lanes correspond to (1 and 1′) complex medium samples, (2 and 2′) 5 mmol/L NH 4 Cl samples, (3 and 3′) 5 mmol/L KNO 3 samples, (M) RiboRuler High Range RNA Ladder from Fermentas , (RNA) RNA sample stained with ethidium bromide and visualized using ultraviolet light.

Techniques Used: Northern Blot, Labeling, Polymerase Chain Reaction, Staining

RT-PCR analysis of amtB-glnK transcription in ΔglnK and flag:amtB mutant strains. (A) RT-PCR analysis of amtB-glnK transcription in ΔglnK strains. First panel: nitrate RNA samples amplified with amtB1 primers; second panel: ammonium RNA samples amplified with amtB1 primers; third panel: nitrate RNA samples amplified with amtB2 primers; fourth panel: ammonium RNA samples amplified with amtB2 primers. Lanes correspond to the following PCR products: (1) PCR negative control (water instead of template cDNA), (2) RT negative control (cDNA obtained from RT reactions without template RNA), (3) PCR positive control (genomic DNA amplification), (M) Quick-Load 100 bp DNA Ladder from New England Biolabs , (4 and 5) HM26 RNA sample and corresponding negative control (cDNA obtained from RT reaction with RNA but without retrotranscriptase), (6 and 7) HM26-K1 RNA sample and corresponding negative control, (8 and 9) HM26-K2 RNA sample and corresponding negative control, (10 and 11) HM26-K1K2 RNA sample and corresponding negative control. (B) RT-PCR analysis of the amtB transcription in flag:amtB strains. The four Flag-tagged strains and HM26 as control were grown in complex medium (OD 600 of 1) and nitrogen starved for 48 h prior to RNA isolation. Lanes correspond to (1) negative control for the PCR reaction performed in the absence of cDNA, (2) positive control with genomic DNA as PCR template, (M) Quick-Load 100 bp DNA Ladder from New England Biolabs , (3 and 4) HM26 RT-PCR reaction and negative control in the absence of retrotranscriptase to check for DNA contamination, respectively, the same for (5 and 6) HM26-F1, (7 and 8) HM26-F2, (9 and 10) HM26-F3, (11 and 12) HM26-F4. (A) and (B) RT of the RNA samples was performed with random hexamers and PCR amplification of cDNA with amtB1 and amtB2 specific primers (RT-Amt1For/RT-Amt1Rev and RT-Amt2For/RT-Amt2Rev, respectively). PCR amplification products of the cDNA separated by 1.8% (w/v) agarose gel electrophoresis and stained with ethidium bromide are presented.
Figure Legend Snippet: RT-PCR analysis of amtB-glnK transcription in ΔglnK and flag:amtB mutant strains. (A) RT-PCR analysis of amtB-glnK transcription in ΔglnK strains. First panel: nitrate RNA samples amplified with amtB1 primers; second panel: ammonium RNA samples amplified with amtB1 primers; third panel: nitrate RNA samples amplified with amtB2 primers; fourth panel: ammonium RNA samples amplified with amtB2 primers. Lanes correspond to the following PCR products: (1) PCR negative control (water instead of template cDNA), (2) RT negative control (cDNA obtained from RT reactions without template RNA), (3) PCR positive control (genomic DNA amplification), (M) Quick-Load 100 bp DNA Ladder from New England Biolabs , (4 and 5) HM26 RNA sample and corresponding negative control (cDNA obtained from RT reaction with RNA but without retrotranscriptase), (6 and 7) HM26-K1 RNA sample and corresponding negative control, (8 and 9) HM26-K2 RNA sample and corresponding negative control, (10 and 11) HM26-K1K2 RNA sample and corresponding negative control. (B) RT-PCR analysis of the amtB transcription in flag:amtB strains. The four Flag-tagged strains and HM26 as control were grown in complex medium (OD 600 of 1) and nitrogen starved for 48 h prior to RNA isolation. Lanes correspond to (1) negative control for the PCR reaction performed in the absence of cDNA, (2) positive control with genomic DNA as PCR template, (M) Quick-Load 100 bp DNA Ladder from New England Biolabs , (3 and 4) HM26 RT-PCR reaction and negative control in the absence of retrotranscriptase to check for DNA contamination, respectively, the same for (5 and 6) HM26-F1, (7 and 8) HM26-F2, (9 and 10) HM26-F3, (11 and 12) HM26-F4. (A) and (B) RT of the RNA samples was performed with random hexamers and PCR amplification of cDNA with amtB1 and amtB2 specific primers (RT-Amt1For/RT-Amt1Rev and RT-Amt2For/RT-Amt2Rev, respectively). PCR amplification products of the cDNA separated by 1.8% (w/v) agarose gel electrophoresis and stained with ethidium bromide are presented.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Amplification, Polymerase Chain Reaction, Negative Control, Positive Control, Isolation, Agarose Gel Electrophoresis, Staining

27) Product Images from "The Uptake of Apoptotic Cells Drives Coxiella burnetii Replication and Macrophage Polarization: A Model for Q Fever Endocarditis"

Article Title: The Uptake of Apoptotic Cells Drives Coxiella burnetii Replication and Macrophage Polarization: A Model for Q Fever Endocarditis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000066

Leukocyte apoptosis and Q fever evolution. Valvulopathy is associated with increased apoptosis of circulating leukocytes. In the model of Q fever endocarditis, the uptake of apoptotic cells by Mo and MDM increases bacterial replication through the polarization of Mo and MDM toward M2 profiles that are non-protective against most pathogens. In patients without valvulopathy or in immunocompetent patients that produce IFN-γ, Mo and MDM are polarized toward a M1 program and are able to kill C. burnetii .
Figure Legend Snippet: Leukocyte apoptosis and Q fever evolution. Valvulopathy is associated with increased apoptosis of circulating leukocytes. In the model of Q fever endocarditis, the uptake of apoptotic cells by Mo and MDM increases bacterial replication through the polarization of Mo and MDM toward M2 profiles that are non-protective against most pathogens. In patients without valvulopathy or in immunocompetent patients that produce IFN-γ, Mo and MDM are polarized toward a M1 program and are able to kill C. burnetii .

Techniques Used:

Effect of AL binding on the maturation of C. burnetii -phagosomes. MDM (A), AL-MDM (B) or NL-MDM (C) were stimulated with C. burnetii for 4 h, washed and then cultured for 24 h. Cells were labeled with anti- C. burnetii (Alexa 546), anti-Lamp-1 (Alexa 488) and anti-cathepsin D (Alexa 647) Abs and analyzed under a confocal microscope. A–C, Representative micrographs are shown with expanded images (white rectangle). D, Percentage of C. burnetii phagosomes that colocalized with Lamp-1 and cathepsin D. The results represent the mean±SEM of 4 independent experiments. * p
Figure Legend Snippet: Effect of AL binding on the maturation of C. burnetii -phagosomes. MDM (A), AL-MDM (B) or NL-MDM (C) were stimulated with C. burnetii for 4 h, washed and then cultured for 24 h. Cells were labeled with anti- C. burnetii (Alexa 546), anti-Lamp-1 (Alexa 488) and anti-cathepsin D (Alexa 647) Abs and analyzed under a confocal microscope. A–C, Representative micrographs are shown with expanded images (white rectangle). D, Percentage of C. burnetii phagosomes that colocalized with Lamp-1 and cathepsin D. The results represent the mean±SEM of 4 independent experiments. * p

Techniques Used: Binding Assay, Cell Culture, Labeling, Microscopy

Polarization of C. burnetii -stimulated Mo and MDM after AL uptake. A, Transcriptional responses of C. burnetii -stimulated Mo and MDM that had bound AL or NL were analyzed by DNA microarrays. Modulated genes (fold-change ≥2) were compared by hierarchical clustering analysis. APC, antigen presenting cells and TF, transcription factors. B and C, Cytokine release by C. burnetii -stimulated Mo (B) and MDM (C) after AL or NL uptake. The results are expressed in pg/ml and represent the mean±SEM of 5 experiments. D and E, Membrane expression of CD14 and MR on C. burnetii -stimulated Mo (D) and MDM (E) after AL or NL binding. The results are expressed as the percentages of positive cells and represent the mean±SEM of 5 experiments. F and G, Mo and MDM were incubated with AL for 2 h and then infected with C. burnetii for 4 h. Cells were then cultured for 9 days in the presence of 10 µg/ml of monoclonal anti-IL-10 or anti-TGF-β1 Abs. The number of bacterial DNA copies was determined by qPCR. The results represent the mean±SEM of 3 experiments. * p
Figure Legend Snippet: Polarization of C. burnetii -stimulated Mo and MDM after AL uptake. A, Transcriptional responses of C. burnetii -stimulated Mo and MDM that had bound AL or NL were analyzed by DNA microarrays. Modulated genes (fold-change ≥2) were compared by hierarchical clustering analysis. APC, antigen presenting cells and TF, transcription factors. B and C, Cytokine release by C. burnetii -stimulated Mo (B) and MDM (C) after AL or NL uptake. The results are expressed in pg/ml and represent the mean±SEM of 5 experiments. D and E, Membrane expression of CD14 and MR on C. burnetii -stimulated Mo (D) and MDM (E) after AL or NL binding. The results are expressed as the percentages of positive cells and represent the mean±SEM of 5 experiments. F and G, Mo and MDM were incubated with AL for 2 h and then infected with C. burnetii for 4 h. Cells were then cultured for 9 days in the presence of 10 µg/ml of monoclonal anti-IL-10 or anti-TGF-β1 Abs. The number of bacterial DNA copies was determined by qPCR. The results represent the mean±SEM of 3 experiments. * p

Techniques Used: Expressing, Binding Assay, Incubation, Infection, Cell Culture, Real-time Polymerase Chain Reaction

Effect of AL binding on the intracellular survival of C. burnetii . A–D, Mo (A, C) and MDM (B, D) were incubated with AL or NL for 2 h, infected with C. burnetii for 4 h (insets) and cultured for 9 days. The number of bacterial DNA copies was determined by qPCR in Mo (A) and MDM (B). The percentages of Mo (C) and MDM (D) that bound more than 5 bacteria were determined by indirect immunofluorescence. The results represent the mean±SEM of 5 experiments. E and F, Mo (E) and MDM (F) were incubated with AL and NL in separate chambers for 2 h, then infected with C. burnetii for 4 h, and cultured for 9 days. The number of bacterial DNA copies was determined by qPCR at day 9. The results represent the mean±SEM of 3 experiments. * p
Figure Legend Snippet: Effect of AL binding on the intracellular survival of C. burnetii . A–D, Mo (A, C) and MDM (B, D) were incubated with AL or NL for 2 h, infected with C. burnetii for 4 h (insets) and cultured for 9 days. The number of bacterial DNA copies was determined by qPCR in Mo (A) and MDM (B). The percentages of Mo (C) and MDM (D) that bound more than 5 bacteria were determined by indirect immunofluorescence. The results represent the mean±SEM of 5 experiments. E and F, Mo (E) and MDM (F) were incubated with AL and NL in separate chambers for 2 h, then infected with C. burnetii for 4 h, and cultured for 9 days. The number of bacterial DNA copies was determined by qPCR at day 9. The results represent the mean±SEM of 3 experiments. * p

Techniques Used: Binding Assay, Incubation, Infection, Cell Culture, Real-time Polymerase Chain Reaction, Immunofluorescence

Effect of IFN-γ on AL-Mo and AL-MDM responses. Mo and MDM that had or not ingested AL were infected with C. burnetii for 4 h in the presence of 1000 U/ml of IFN-γ. A and B, After washing, Mo and AL-Mo (A) or MDM and AL-MDM (B) were cultured for 9 days in the presence of IFN-γ. C. burnetii replication was determined by qPCR. The results represent the mean±SEM of 3 experiments. C–E, After washing, MDM and AL-MDM were cultured for 24 h in the presence of IFN-γ. Cells were labeled with anti- C. burnetii (Alexa 546), anti-Lamp-1 (Alexa 488) and anti-cathepsin D (Alexa 647) Abs and analyzed under a confocal microscope. Representative micrographs of IFN-γ-treated MDM (C) and AL-MDM (D) are shown with expanded images (white rectangle). In E, the results are expressed as the percentages of C. burnetii phagosomes that colocalised with Lamp-1 and/or cathepsin D. F, The transcriptional response of AL-Mo and AL-MDM stimulated with C. burnetii for 4 h in the presence or the absence of IFN-γ was analyzed by qRT-PCR. The results are expressed as the Log2 fold change and analyzed by hierarchical clustering. G, AL-Mo and AL-MDM were stimulated with heat-killed C. burnetii for 24 h in the presence or the absence of IFN-γ. The cytokine release was determined by immunoassays and expressed in pg/ml. The results represent the mean±SEM of 3 experiments. * p
Figure Legend Snippet: Effect of IFN-γ on AL-Mo and AL-MDM responses. Mo and MDM that had or not ingested AL were infected with C. burnetii for 4 h in the presence of 1000 U/ml of IFN-γ. A and B, After washing, Mo and AL-Mo (A) or MDM and AL-MDM (B) were cultured for 9 days in the presence of IFN-γ. C. burnetii replication was determined by qPCR. The results represent the mean±SEM of 3 experiments. C–E, After washing, MDM and AL-MDM were cultured for 24 h in the presence of IFN-γ. Cells were labeled with anti- C. burnetii (Alexa 546), anti-Lamp-1 (Alexa 488) and anti-cathepsin D (Alexa 647) Abs and analyzed under a confocal microscope. Representative micrographs of IFN-γ-treated MDM (C) and AL-MDM (D) are shown with expanded images (white rectangle). In E, the results are expressed as the percentages of C. burnetii phagosomes that colocalised with Lamp-1 and/or cathepsin D. F, The transcriptional response of AL-Mo and AL-MDM stimulated with C. burnetii for 4 h in the presence or the absence of IFN-γ was analyzed by qRT-PCR. The results are expressed as the Log2 fold change and analyzed by hierarchical clustering. G, AL-Mo and AL-MDM were stimulated with heat-killed C. burnetii for 24 h in the presence or the absence of IFN-γ. The cytokine release was determined by immunoassays and expressed in pg/ml. The results represent the mean±SEM of 3 experiments. * p

Techniques Used: Infection, Cell Culture, Real-time Polymerase Chain Reaction, Labeling, Microscopy, Quantitative RT-PCR

28) Product Images from "Podocytic PKC-Alpha Is Regulated in Murine and Human Diabetes and Mediates Nephrin Endocytosis"

Article Title: Podocytic PKC-Alpha Is Regulated in Murine and Human Diabetes and Mediates Nephrin Endocytosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0010185

Inducible interaction of transiently overexpressed and endogenous nephrin with PKCα and PICK1. (A) HEKT293T cells were transiently transfected with mNephrin-flag, PKCα-GFP and PICK1-myc and stimulated with PMA for 0, 5 and 15 min. Nephrin-flag was precipitated with agarose labelled with an anti-Flag-antibody. The probes were blotted and analyzed for GFP and Myc. (B) Endogenous nephrin and PICK1 were precipitated (IP) using an anti-nephrin and anti-PICK1 antibody from whole cell lysates of PKCα+/+ and PKCα−/− podocytes which were treated for 0, 5 and 15 min with PMA. The probes were blotted and analyzed for PKCα, nephrin and PICK1.
Figure Legend Snippet: Inducible interaction of transiently overexpressed and endogenous nephrin with PKCα and PICK1. (A) HEKT293T cells were transiently transfected with mNephrin-flag, PKCα-GFP and PICK1-myc and stimulated with PMA for 0, 5 and 15 min. Nephrin-flag was precipitated with agarose labelled with an anti-Flag-antibody. The probes were blotted and analyzed for GFP and Myc. (B) Endogenous nephrin and PICK1 were precipitated (IP) using an anti-nephrin and anti-PICK1 antibody from whole cell lysates of PKCα+/+ and PKCα−/− podocytes which were treated for 0, 5 and 15 min with PMA. The probes were blotted and analyzed for PKCα, nephrin and PICK1.

Techniques Used: Transfection

PKCα promotes nephrin endocytosis. (A) PKCα+/+ (a, b, c) and PKCα−/− (d, e, f) podocytes were double labelled with Cholera toxin-B (green) and an ectodomain anti-nephrin antibody detected with an Cy3-labeled secondary antibody (red). The cells are labelled at 4°C and incubated at 4°C (a, d) or shifted to 37°C (b, c, e, f) for 20 min to induce internalization in the absence (b, e) or presence of the PKCα-inhibitor GÖ6976 (20 µM) (c, f) (scale bars 10 µm). (B, C) Nephrin expressed at the surface of murine PKCα+/+ and PKCα−/− and human podocytes is labelled with an anti-nephrin antibody at 4°C. (B) For temperature shift experiments cells were left at 4°C or incubated at 37°C for 20 min in the absence or presence of the PKCα-Inhibitor GÖ6976 (20 µM) to induce internalization. (C) For glucose experiments murine PKCα+/+ and PKCα−/− and human podocytes are stimulated for 1 hour with high glucose (30 mM) or Mannitol. Endocytosis of nephrin is stopped by cooling down at 4°C and labelled with an anti-nephrin antibody. Extinction was measured at 405 nm. Bar graphs represent the graphical summary of three independent experiments. *p
Figure Legend Snippet: PKCα promotes nephrin endocytosis. (A) PKCα+/+ (a, b, c) and PKCα−/− (d, e, f) podocytes were double labelled with Cholera toxin-B (green) and an ectodomain anti-nephrin antibody detected with an Cy3-labeled secondary antibody (red). The cells are labelled at 4°C and incubated at 4°C (a, d) or shifted to 37°C (b, c, e, f) for 20 min to induce internalization in the absence (b, e) or presence of the PKCα-inhibitor GÖ6976 (20 µM) (c, f) (scale bars 10 µm). (B, C) Nephrin expressed at the surface of murine PKCα+/+ and PKCα−/− and human podocytes is labelled with an anti-nephrin antibody at 4°C. (B) For temperature shift experiments cells were left at 4°C or incubated at 37°C for 20 min in the absence or presence of the PKCα-Inhibitor GÖ6976 (20 µM) to induce internalization. (C) For glucose experiments murine PKCα+/+ and PKCα−/− and human podocytes are stimulated for 1 hour with high glucose (30 mM) or Mannitol. Endocytosis of nephrin is stopped by cooling down at 4°C and labelled with an anti-nephrin antibody. Extinction was measured at 405 nm. Bar graphs represent the graphical summary of three independent experiments. *p

Techniques Used: Labeling, Incubation

Glomerular PKCα is upregulated in mice and patients with diabetic nephropathy. (A) Immunofluorescence using an anti-PKCα (green) and anti-podocalyxin (red) antibody on frozen kidney cortex sections of control and diabetic mice. White arrowheads in the merged panel depict colocalization of PKCα with podocalyxin. Picture is representative for the majority of glomeruli in control and diabetic mice (n = 5) in each group). (B) Immunohistochemistry using an anti-PKCα antibody shows expression of PKCα in human biopsy samples of healthy control individual (a, b) and a patient with diabetic nephropathy (c, d). Black arrowheads depict expression of PKCα in the glomerular endothelium and open arrowheads depict podocytes in normal and diabetic individual (panel b, d). Bar graphs (e) summarize semiquantitative score of all glomeruli (n = 74 for patients with DN and n = 150 for control cases), indicating 1 for absent or moderate or 2 for strong expression of PKCα in podocytes and mRNA expression of PKCα isolated from control (n = 300) and diabetic (n = 298) human glomeruli in relation to Wt-1 mRNA expression, *p
Figure Legend Snippet: Glomerular PKCα is upregulated in mice and patients with diabetic nephropathy. (A) Immunofluorescence using an anti-PKCα (green) and anti-podocalyxin (red) antibody on frozen kidney cortex sections of control and diabetic mice. White arrowheads in the merged panel depict colocalization of PKCα with podocalyxin. Picture is representative for the majority of glomeruli in control and diabetic mice (n = 5) in each group). (B) Immunohistochemistry using an anti-PKCα antibody shows expression of PKCα in human biopsy samples of healthy control individual (a, b) and a patient with diabetic nephropathy (c, d). Black arrowheads depict expression of PKCα in the glomerular endothelium and open arrowheads depict podocytes in normal and diabetic individual (panel b, d). Bar graphs (e) summarize semiquantitative score of all glomeruli (n = 74 for patients with DN and n = 150 for control cases), indicating 1 for absent or moderate or 2 for strong expression of PKCα in podocytes and mRNA expression of PKCα isolated from control (n = 300) and diabetic (n = 298) human glomeruli in relation to Wt-1 mRNA expression, *p

Techniques Used: Mouse Assay, Immunofluorescence, Immunohistochemistry, Expressing, Isolation

PKCα expression is upregulated by high glucose in murine and human podocytes. Quantitative PCR (a) and Western blot analysis (b) for PKCα in murine and human podocytes depicts induction of protein in a time course experiment after stimulation with high glucose (30 mM) and mannitol as osmotic control for up to 24 hrs (results are representative for 3 independent experiments). Scale bars of quantitative PCR summarizes means of high glucose (white bars) and Mannitol (black bars) of n = 3 independent experiments. (*p
Figure Legend Snippet: PKCα expression is upregulated by high glucose in murine and human podocytes. Quantitative PCR (a) and Western blot analysis (b) for PKCα in murine and human podocytes depicts induction of protein in a time course experiment after stimulation with high glucose (30 mM) and mannitol as osmotic control for up to 24 hrs (results are representative for 3 independent experiments). Scale bars of quantitative PCR summarizes means of high glucose (white bars) and Mannitol (black bars) of n = 3 independent experiments. (*p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

29) Product Images from "Chronic Ethanol Feeding Modulates Inflammatory Mediators, Activation of Nuclear Factor-κB, and Responsiveness to Endotoxin in Murine Kupffer Cells and Circulating Leukocytes"

Article Title: Chronic Ethanol Feeding Modulates Inflammatory Mediators, Activation of Nuclear Factor-κB, and Responsiveness to Endotoxin in Murine Kupffer Cells and Circulating Leukocytes

Journal: Mediators of Inflammation

doi: 10.1155/2014/808695

NF- κ B target gene expression in isolated Kupffer cells in response to LPS after ethanol feeding F4/80 + macrophages were isolated from livers of ethanol-, pair-fed mice, respectively, and stimulated with LPS (10 μ g/mL, Figure 7 ) for 2 h and 24 h. Supernatants were measured for mRNA levels of IL-6 (a), TNF- α (b), NOS2 (c), CXCL-1 (d), and MMP-9 (e) by RT-PCR. Data (mean ± SEM) are expressed as fold change compared with corresponding medium controls (unstimulated) and are representative of five to eight separate experiments. * P
Figure Legend Snippet: NF- κ B target gene expression in isolated Kupffer cells in response to LPS after ethanol feeding F4/80 + macrophages were isolated from livers of ethanol-, pair-fed mice, respectively, and stimulated with LPS (10 μ g/mL, Figure 7 ) for 2 h and 24 h. Supernatants were measured for mRNA levels of IL-6 (a), TNF- α (b), NOS2 (c), CXCL-1 (d), and MMP-9 (e) by RT-PCR. Data (mean ± SEM) are expressed as fold change compared with corresponding medium controls (unstimulated) and are representative of five to eight separate experiments. * P

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

NF- κ B target gene expression in isolated Kupffer cells in response to TNF- α after ethanol feeding F4/80 + macrophages were isolated from livers of ethanol-, pair-fed mice, respectively, and stimulated with TNF- α (500 ng/mL) for 2 h and 24 h. Supernatants were measured for mRNA levels of IL-6 (a), TNF- α (b), NOS2 (c), CXCL-1 (d), and MMP-9 (e) by RT-PCR. Data (mean ± SEM) are expressed as fold change compared with corresponding medium controls (unstimulated) and are representative of five to eight separate experiments. * P
Figure Legend Snippet: NF- κ B target gene expression in isolated Kupffer cells in response to TNF- α after ethanol feeding F4/80 + macrophages were isolated from livers of ethanol-, pair-fed mice, respectively, and stimulated with TNF- α (500 ng/mL) for 2 h and 24 h. Supernatants were measured for mRNA levels of IL-6 (a), TNF- α (b), NOS2 (c), CXCL-1 (d), and MMP-9 (e) by RT-PCR. Data (mean ± SEM) are expressed as fold change compared with corresponding medium controls (unstimulated) and are representative of five to eight separate experiments. * P

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

Proinflammatory cytokine production of F4/80 − hepatic macrophages following ethanol feeding in response to LPS or TNF- α . Kupffer cells were isolated from cis -NF- κ B EGFP mice after 4 weeks of pair feeding regime, as described in Section 2 , and stimulated with 10 μ g/mL LPS or 500 ng/mL TNF- α . Culture supernatants were collected at 2 h, 4 h, and 24 h after stimulation. IL-6 (a) and TNF- α (b) were measured by cytometric bead array (CBA). Data (mean ± SEM) are representative of five to eight independent experiments. * P
Figure Legend Snippet: Proinflammatory cytokine production of F4/80 − hepatic macrophages following ethanol feeding in response to LPS or TNF- α . Kupffer cells were isolated from cis -NF- κ B EGFP mice after 4 weeks of pair feeding regime, as described in Section 2 , and stimulated with 10 μ g/mL LPS or 500 ng/mL TNF- α . Culture supernatants were collected at 2 h, 4 h, and 24 h after stimulation. IL-6 (a) and TNF- α (b) were measured by cytometric bead array (CBA). Data (mean ± SEM) are representative of five to eight independent experiments. * P

Techniques Used: Isolation, Mouse Assay, Crocin Bleaching Assay

30) Product Images from "Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica"

Article Title: Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica

Journal: PLoS ONE

doi: 10.1371/journal.pone.0151083

Cell culture supernatants from BRSV stimulated PBMC induce expression of IL-8, MUC5B and MUC5AC by BT cells. PBMC from control (nonvaccinated) and BRSV vaccinated cows were isolated and stimulated with heat-killed BRSV for 6 days as in Fig 2 . Cell culture supernatants were then diluted 1:1 in cMEM and added to confluent BT cells in 96 well plates for 24 hours. RNA was the isolated from the BT and analyzed by qPCR for expression of IL-8 (A), MUC5B (B) and MUC5AC (C). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results were pooled from two independent experiments. Data represent means ± SEM.
Figure Legend Snippet: Cell culture supernatants from BRSV stimulated PBMC induce expression of IL-8, MUC5B and MUC5AC by BT cells. PBMC from control (nonvaccinated) and BRSV vaccinated cows were isolated and stimulated with heat-killed BRSV for 6 days as in Fig 2 . Cell culture supernatants were then diluted 1:1 in cMEM and added to confluent BT cells in 96 well plates for 24 hours. RNA was the isolated from the BT and analyzed by qPCR for expression of IL-8 (A), MUC5B (B) and MUC5AC (C). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results were pooled from two independent experiments. Data represent means ± SEM.

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

31) Product Images from "Elevation of CpG frequencies in influenza A genome attenuates pathogenicity but enhances host response to infection"

Article Title: Elevation of CpG frequencies in influenza A genome attenuates pathogenicity but enhances host response to infection

Journal: eLife

doi: 10.7554/eLife.12735

Ratio of segment 5 and segment 2 RNA sequences in purified virions Quantitation of segment 5 and segment 2 RNA by qPCR in purified virions of WT and mutant IAV strains. The y-axis records mean values of two biological replicates with values normalised to the ratio observed in WT virus; error bars show SEMs. DOI: http://dx.doi.org/10.7554/eLife.12735.008
Figure Legend Snippet: Ratio of segment 5 and segment 2 RNA sequences in purified virions Quantitation of segment 5 and segment 2 RNA by qPCR in purified virions of WT and mutant IAV strains. The y-axis records mean values of two biological replicates with values normalised to the ratio observed in WT virus; error bars show SEMs. DOI: http://dx.doi.org/10.7554/eLife.12735.008

Techniques Used: Purification, Quantitation Assay, Real-time Polymerase Chain Reaction, Mutagenesis

32) Product Images from "Colony-stimulating factor (CSF) 1 receptor blockade reduces inflammation in human and murine models of rheumatoid arthritis"

Article Title: Colony-stimulating factor (CSF) 1 receptor blockade reduces inflammation in human and murine models of rheumatoid arthritis

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-016-0973-6

Colony-stimulating factor-1 (CSF-1), IL-34 and CSF1 receptor ( CSF1R ) are expressed in synovial tissue from patients with rheumatoid arthritis ( RA ) and psoriatic arthritis ( PsA ). a Quantification of relative IL-34, CSF-1 and CSF1R mRNA expression in synovial tissue from 6 patients with RA and 6 with PsA. Quantitative PCR data are shown as relative quantity ( RQ ), as described in “ Methods ”. Data are presented as scatter plots, where each plot represents an individual value, bars represent the mean, and error bars indicate the standard error of the mean (SEM). b , c Immunohistochemical analyses of RA, PsA and OA synovial tissue stained with control rabbit, anti-IL34 ( b ) and anti-CSF1 antibodies ( c ). d Quantitative analysis of IL-34 and CSF-1 staining in synovial tissue. Synovial sections from 15 patients with RA, 15 with PsA and 7 patients with osteoarthritis ( OA ) were stained with antibodies against IL-34 and CSF-1 antibodies as above, and the integrated optical density ( IOD/mm 2 ) corrected for cellularity was calculated by digital image analysis. Each plot represents an individual value, bars represent the mean, and error bars indicate the SEM. * P
Figure Legend Snippet: Colony-stimulating factor-1 (CSF-1), IL-34 and CSF1 receptor ( CSF1R ) are expressed in synovial tissue from patients with rheumatoid arthritis ( RA ) and psoriatic arthritis ( PsA ). a Quantification of relative IL-34, CSF-1 and CSF1R mRNA expression in synovial tissue from 6 patients with RA and 6 with PsA. Quantitative PCR data are shown as relative quantity ( RQ ), as described in “ Methods ”. Data are presented as scatter plots, where each plot represents an individual value, bars represent the mean, and error bars indicate the standard error of the mean (SEM). b , c Immunohistochemical analyses of RA, PsA and OA synovial tissue stained with control rabbit, anti-IL34 ( b ) and anti-CSF1 antibodies ( c ). d Quantitative analysis of IL-34 and CSF-1 staining in synovial tissue. Synovial sections from 15 patients with RA, 15 with PsA and 7 patients with osteoarthritis ( OA ) were stained with antibodies against IL-34 and CSF-1 antibodies as above, and the integrated optical density ( IOD/mm 2 ) corrected for cellularity was calculated by digital image analysis. Each plot represents an individual value, bars represent the mean, and error bars indicate the SEM. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining

33) Product Images from "Colony-stimulating factor (CSF) 1 receptor blockade reduces inflammation in human and murine models of rheumatoid arthritis"

Article Title: Colony-stimulating factor (CSF) 1 receptor blockade reduces inflammation in human and murine models of rheumatoid arthritis

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-016-0973-6

Anti-colony stimulating factor-1 receptor (anti-CSFR1) antibody reduces the production of inflammatory mediators in synovial tissue in rheumatoid arthritis (RA). a IL-6 ELISA production in supernatants of RA synovial tissue after 24-h incubation in medium alone or increasing concentrations of colony stimulating factor-1 ( CSF-1 ) or IL-34 (n = 7). b - d IL-6 ELISA production in supernatants of RA synovial tissue after 4 days of incubation in medium alone in the presence of IgG1 or anti-IL-34 antibody (Ab) (5 μg/ml for both, n = 5) ( b ), anti-CSF-1 Ab (5 μg/ml, n = 3) ( c ), or increasing concentrations of IgG4 or huAB1 (n = 5) ( d ). e , f Multiplex analysis of protein production in supernatants of RA synovial tissue after 4 days of incubation in medium alone in the presence of 1 μg/ml IgG4 or 1 μg/ml huAB1 (n = 4). Boxes represent the 25th–75th percentiles, lines within the boxes mark the median value, and lines outside the boxes denote the 10th and 90th percentiles. * P
Figure Legend Snippet: Anti-colony stimulating factor-1 receptor (anti-CSFR1) antibody reduces the production of inflammatory mediators in synovial tissue in rheumatoid arthritis (RA). a IL-6 ELISA production in supernatants of RA synovial tissue after 24-h incubation in medium alone or increasing concentrations of colony stimulating factor-1 ( CSF-1 ) or IL-34 (n = 7). b - d IL-6 ELISA production in supernatants of RA synovial tissue after 4 days of incubation in medium alone in the presence of IgG1 or anti-IL-34 antibody (Ab) (5 μg/ml for both, n = 5) ( b ), anti-CSF-1 Ab (5 μg/ml, n = 3) ( c ), or increasing concentrations of IgG4 or huAB1 (n = 5) ( d ). e , f Multiplex analysis of protein production in supernatants of RA synovial tissue after 4 days of incubation in medium alone in the presence of 1 μg/ml IgG4 or 1 μg/ml huAB1 (n = 4). Boxes represent the 25th–75th percentiles, lines within the boxes mark the median value, and lines outside the boxes denote the 10th and 90th percentiles. * P

Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation, Multiplex Assay

Prophylactic treatment with anti-colony stimulating factor-1 receptor ( anti-CSF1R ) prevents pathological progression in collagen-induced arthritis ( CIA ).  a  Daily global arthritic scores for mice treated intraperitoneally every 3 days with vehicle, Enbrel, or muAb5. Treatment continued three times weekly from day 0 with control vehicle ( triangles ), Enbrel (10 mg/kg) ( circles ) or muAb5 (30 mg/kg) ( squares ).  b  Representative images of pathological appearances of the joints in the indicated treatment group, visualized by H  E staining. Note the representative areas of synovial cellular infiltration and pannus formation ( arrows ).  c  Inflammation ( I ), pannus formation ( PF ), cartilage damage ( CD ), and bone damage ( BD ) scores for mice in each treatment group. Data are mean ± standard error of the mean (SEM) for each group (n = 12 animals per group).  # P
Figure Legend Snippet: Prophylactic treatment with anti-colony stimulating factor-1 receptor ( anti-CSF1R ) prevents pathological progression in collagen-induced arthritis ( CIA ). a Daily global arthritic scores for mice treated intraperitoneally every 3 days with vehicle, Enbrel, or muAb5. Treatment continued three times weekly from day 0 with control vehicle ( triangles ), Enbrel (10 mg/kg) ( circles ) or muAb5 (30 mg/kg) ( squares ). b Representative images of pathological appearances of the joints in the indicated treatment group, visualized by H E staining. Note the representative areas of synovial cellular infiltration and pannus formation ( arrows ). c Inflammation ( I ), pannus formation ( PF ), cartilage damage ( CD ), and bone damage ( BD ) scores for mice in each treatment group. Data are mean ± standard error of the mean (SEM) for each group (n = 12 animals per group). # P

Techniques Used: Mouse Assay, Staining

Therapeutic treatment with anti-colony stimulating factor-1 receptor ( anti-CSF1R ) suppresses bone destruction and pannus formation in collagen-induced arthritis ( CIA ).  a  Histopathological assessment of CIA in mice treated with vehicle, Enbrel, or muAB5 after the initiation of arthritis. * P
Figure Legend Snippet: Therapeutic treatment with anti-colony stimulating factor-1 receptor ( anti-CSF1R ) suppresses bone destruction and pannus formation in collagen-induced arthritis ( CIA ). a Histopathological assessment of CIA in mice treated with vehicle, Enbrel, or muAB5 after the initiation of arthritis. * P

Techniques Used: Mouse Assay

Colony-stimulating factor-1 (CSF-1), IL-34 and CSF1 receptor ( CSF1R ) are expressed in synovial tissue from patients with rheumatoid arthritis ( RA ) and psoriatic arthritis ( PsA ). a Quantification of relative IL-34, CSF-1 and CSF1R mRNA expression in synovial tissue from 6 patients with RA and 6 with PsA. Quantitative PCR data are shown as relative quantity ( RQ ), as described in “ Methods ”. Data are presented as scatter plots, where each plot represents an individual value, bars represent the mean, and error bars indicate the standard error of the mean (SEM). b , c Immunohistochemical analyses of RA, PsA and OA synovial tissue stained with control rabbit, anti-IL34 ( b ) and anti-CSF1 antibodies ( c ). d Quantitative analysis of IL-34 and CSF-1 staining in synovial tissue. Synovial sections from 15 patients with RA, 15 with PsA and 7 patients with osteoarthritis ( OA ) were stained with antibodies against IL-34 and CSF-1 antibodies as above, and the integrated optical density ( IOD/mm 2 ) corrected for cellularity was calculated by digital image analysis. Each plot represents an individual value, bars represent the mean, and error bars indicate the SEM. * P
Figure Legend Snippet: Colony-stimulating factor-1 (CSF-1), IL-34 and CSF1 receptor ( CSF1R ) are expressed in synovial tissue from patients with rheumatoid arthritis ( RA ) and psoriatic arthritis ( PsA ). a Quantification of relative IL-34, CSF-1 and CSF1R mRNA expression in synovial tissue from 6 patients with RA and 6 with PsA. Quantitative PCR data are shown as relative quantity ( RQ ), as described in “ Methods ”. Data are presented as scatter plots, where each plot represents an individual value, bars represent the mean, and error bars indicate the standard error of the mean (SEM). b , c Immunohistochemical analyses of RA, PsA and OA synovial tissue stained with control rabbit, anti-IL34 ( b ) and anti-CSF1 antibodies ( c ). d Quantitative analysis of IL-34 and CSF-1 staining in synovial tissue. Synovial sections from 15 patients with RA, 15 with PsA and 7 patients with osteoarthritis ( OA ) were stained with antibodies against IL-34 and CSF-1 antibodies as above, and the integrated optical density ( IOD/mm 2 ) corrected for cellularity was calculated by digital image analysis. Each plot represents an individual value, bars represent the mean, and error bars indicate the SEM. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining

IL-34 and colony stimulating factor-1 ( CSF-1 ) macrophages have similar phenotypic characteristics. a Fluorescence-activated cell sorting (FACS) analysis of Annexin V-propidium iodide ( PI ) staining in macrophages differentiated for 7 days in granulocyte-macrophage colony-stimulating factor ( GM-CSF ) (5 ng/ml), CSF-1 (25 ng/ml) or IL-34 (25 ng/ml) from peripheral blood and synovial fluid of healthy donors ( HD ) and patients with rheumatoid arthritis (RA). Data are presented as percentage of double-positive cells and represent the mean ± standard error of the mean (SEM) of 3–4 independent experiments. b , c FACS analysis of expression of macrophage surface markers CD14, CD163, CD206, and CD64 in macrophages differentiated for 7 days in GM-CSF, CSF-1 or IL-34 derived from monocytes of buffy coat ( b ) or monocytes from peripheral blood of HD or patients with RA ( PB ), or from synovial fluid from patients with RA ( SF ) ( c ). Data are presented as the geometric mean ( geo mean ) and represent the mean ± SEM of 4–5 independent experiments per marker. * P
Figure Legend Snippet: IL-34 and colony stimulating factor-1 ( CSF-1 ) macrophages have similar phenotypic characteristics. a Fluorescence-activated cell sorting (FACS) analysis of Annexin V-propidium iodide ( PI ) staining in macrophages differentiated for 7 days in granulocyte-macrophage colony-stimulating factor ( GM-CSF ) (5 ng/ml), CSF-1 (25 ng/ml) or IL-34 (25 ng/ml) from peripheral blood and synovial fluid of healthy donors ( HD ) and patients with rheumatoid arthritis (RA). Data are presented as percentage of double-positive cells and represent the mean ± standard error of the mean (SEM) of 3–4 independent experiments. b , c FACS analysis of expression of macrophage surface markers CD14, CD163, CD206, and CD64 in macrophages differentiated for 7 days in GM-CSF, CSF-1 or IL-34 derived from monocytes of buffy coat ( b ) or monocytes from peripheral blood of HD or patients with RA ( PB ), or from synovial fluid from patients with RA ( SF ) ( c ). Data are presented as the geometric mean ( geo mean ) and represent the mean ± SEM of 4–5 independent experiments per marker. * P

Techniques Used: Fluorescence, FACS, Staining, Expressing, Derivative Assay, Marker

Gene expression in macrophages differentiated from synovial fluid monocytes in rheumatoid arthritis (RA). Analyses of mRNA expression levels of genes involved in extracellular matrix remodeling formation in colony-stimulating factor-1 ( CSF-1 )- or IL-34-differentiated macrophages (Mφ) derived from monocytes of synovial fluid in RA (n = 3). Data are shown as relative quantity ( RQ ) in CSF-1 Mφ, as described in “ Methods ”. * P
Figure Legend Snippet: Gene expression in macrophages differentiated from synovial fluid monocytes in rheumatoid arthritis (RA). Analyses of mRNA expression levels of genes involved in extracellular matrix remodeling formation in colony-stimulating factor-1 ( CSF-1 )- or IL-34-differentiated macrophages (Mφ) derived from monocytes of synovial fluid in RA (n = 3). Data are shown as relative quantity ( RQ ) in CSF-1 Mφ, as described in “ Methods ”. * P

Techniques Used: Expressing, Derivative Assay

Gene expression in differentiated macrophages. a mRNA expression profiles of 336 genes involved in angiogenesis, extracellular matrix remodeling, and osteoclast formation in granulocyte-macrophage colony-stimulating factor ( GM-CSF )-, colony-stimulating factor-1 ( CSF-1 )- or IL-34-differentiated macrophages (Mφ) derived from monocytes of buffy coats (n = 3). Data are presented in an unsupervised clustergram. b Analyses of mRNA expression levels of selected genes analyzed in a . Data are shown as relative quantity ( RQ ) in relation to GM-CSF Mφ, as described in “ Methods ”
Figure Legend Snippet: Gene expression in differentiated macrophages. a mRNA expression profiles of 336 genes involved in angiogenesis, extracellular matrix remodeling, and osteoclast formation in granulocyte-macrophage colony-stimulating factor ( GM-CSF )-, colony-stimulating factor-1 ( CSF-1 )- or IL-34-differentiated macrophages (Mφ) derived from monocytes of buffy coats (n = 3). Data are presented in an unsupervised clustergram. b Analyses of mRNA expression levels of selected genes analyzed in a . Data are shown as relative quantity ( RQ ) in relation to GM-CSF Mφ, as described in “ Methods ”

Techniques Used: Expressing, Derivative Assay

34) Product Images from "The oncogene EVI1 enhances transcriptional and biological responses of human myeloid cells to all-trans retinoic acid"

Article Title: The oncogene EVI1 enhances transcriptional and biological responses of human myeloid cells to all-trans retinoic acid

Journal: Cell Cycle

doi: 10.4161/15384101.2014.946869

Evi1 and ATRA cooperatively regulate gene expression in HL-60 cells. ( A ) Immunoblot analysis demonstrating expression of ectopic Evi1 in HL-60_Evi1 cells. ( B ) Intranuclear staining and flow cytometry showing expression of ectopic Evi1 in HL-60_Evi1 cells on the single cell level. Red, EVI1 antibody; gray, isotype control. ( C ) Heatmap representing the expression (z-score) of genes whose induction by ATRA was enhanced by, or observed only in the presence of, Evi1 (category 1, CAT1) in HL-60 cells. In this model system, no genes fulfilled the criteria for inclusion into categories 2-4.
Figure Legend Snippet: Evi1 and ATRA cooperatively regulate gene expression in HL-60 cells. ( A ) Immunoblot analysis demonstrating expression of ectopic Evi1 in HL-60_Evi1 cells. ( B ) Intranuclear staining and flow cytometry showing expression of ectopic Evi1 in HL-60_Evi1 cells on the single cell level. Red, EVI1 antibody; gray, isotype control. ( C ) Heatmap representing the expression (z-score) of genes whose induction by ATRA was enhanced by, or observed only in the presence of, Evi1 (category 1, CAT1) in HL-60 cells. In this model system, no genes fulfilled the criteria for inclusion into categories 2-4.

Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry

Evi1 enhances ATRA induced apoptosis and differentiation in HL-60 cells. ( A ) HL-60_vec and HL-60_Evi1 cells were treated with solvent or ATRA for 5 days, stained with Annexin V and 7AAD, and analyzed by flow cytometry. Double negative cells were classified as viable (white portions of bars), Annexin V positive 7AAD negative cells as early apoptotic (gray portions of bars), and double positive cells as late apoptotic/secondary necrotic (black portions of bars). 89 Results represent means +/- SEs of 8 independent experiments. ( B ) HL-60_vec cells (white bars) and HL-60_Evi1 cells (black bars) were treated with solvent or ATRA for 5 days, stained with PE-conjugated CD11b or isotype control antibodies, and subjected to flow cytometry. Results are expressed as the means of the PE fluorescence of CD11b stainings relative to that of the respective isotype controls and to untreated HL-60_vec cells, and represent the means + SEs of 7 independent experiments. Significance was calculated using Student's 2-tailed t-test (*, P
Figure Legend Snippet: Evi1 enhances ATRA induced apoptosis and differentiation in HL-60 cells. ( A ) HL-60_vec and HL-60_Evi1 cells were treated with solvent or ATRA for 5 days, stained with Annexin V and 7AAD, and analyzed by flow cytometry. Double negative cells were classified as viable (white portions of bars), Annexin V positive 7AAD negative cells as early apoptotic (gray portions of bars), and double positive cells as late apoptotic/secondary necrotic (black portions of bars). 89 Results represent means +/- SEs of 8 independent experiments. ( B ) HL-60_vec cells (white bars) and HL-60_Evi1 cells (black bars) were treated with solvent or ATRA for 5 days, stained with PE-conjugated CD11b or isotype control antibodies, and subjected to flow cytometry. Results are expressed as the means of the PE fluorescence of CD11b stainings relative to that of the respective isotype controls and to untreated HL-60_vec cells, and represent the means + SEs of 7 independent experiments. Significance was calculated using Student's 2-tailed t-test (*, P

Techniques Used: Staining, Flow Cytometry, Cytometry, Fluorescence

EVI1 and ATRA synergistically induce GDF15 protein in myeloid and in epithelial cells. ( A ) U937_vec and U937_EVI1 cells were treated with solvent or ATRA for 3 or 5 days, and the intracellular precursor and secreted mature forms of GDF15 were detected in whole cell extracts (day 3) and culture supernatants (day 5), respectively, by immunoblot analysis. Both forms are disulfide linked homodimers, and the corresponding 35 kDa and 15 kDa monomers are detected after reducing SDS-PAGE. ( B ) U937_vec and U937_EVI1 cells were treated with solvent or ATRA for 3 or 5 days, and mature GDF15 was detected in culture supernatants by ELISA. Results represent means + SEs from 3 independent experiments. Significance was calculated using Student's 2-tailed t-test (*, P
Figure Legend Snippet: EVI1 and ATRA synergistically induce GDF15 protein in myeloid and in epithelial cells. ( A ) U937_vec and U937_EVI1 cells were treated with solvent or ATRA for 3 or 5 days, and the intracellular precursor and secreted mature forms of GDF15 were detected in whole cell extracts (day 3) and culture supernatants (day 5), respectively, by immunoblot analysis. Both forms are disulfide linked homodimers, and the corresponding 35 kDa and 15 kDa monomers are detected after reducing SDS-PAGE. ( B ) U937_vec and U937_EVI1 cells were treated with solvent or ATRA for 3 or 5 days, and mature GDF15 was detected in culture supernatants by ELISA. Results represent means + SEs from 3 independent experiments. Significance was calculated using Student's 2-tailed t-test (*, P

Techniques Used: SDS Page, Enzyme-linked Immunosorbent Assay

Experimental down-regulation of GDF15 counteracts EVI1 enhancement of the ATRA induced cell cycle arrest. ( A ) U937_vec and U937_EVI1 cells infected with a control shRNA targeting Renilla luciferase (shRen) or shRNAs against GDF15 (shGDF15-1, shGDF15-2) were treated with solvent or ATRA for 24 h, and GDF15 mRNA levels were measured by qRT-PCR. GDF15 expression was normalized to that of B2M using the ΔΔ Ct method 88 and ATRA treated U937_EVI1_shRen cells as a calibrator. Results represent means + SEs from 3 independent experiments. ( B ) U937_vec and U937_EVI1 cells containing shRen, shGDF15-1, or shGDF15-2 were treated with solvent or ATRA for 5 days, and the GDF15 precursor was detected by immunoblot analysis. α-tubulin was used as a loading control. ( C ) U937_vec and U937_EVI1 cells containing shRen, shGDF15-1, or shGDF15-2 were treated with solvent or ATRA for 5 days, and mature GDF15 was detected in culture supernatants by ELISA. Results represent means + SEs from 5 independent experiments. Due to variation in factors results were not significant, but the enhancement of the ATRA induction by EVI1 in shRen infected cells, as well as the knockdown of GDF15 by both shRNAs were consistently seen in all experiments. ( D ) U937_vec and U937_EVI1 cells containing shRen, shGDF15-1, or shGDF15-2 were treated with solvent or ATRA for 5 d and subjected to cell cycle analysis by propidium iodide staining and flow cytometry. Results represent means + SEs from 7 independent experiments. Significance was calculated using Student's 2-tailed t-test (*, P
Figure Legend Snippet: Experimental down-regulation of GDF15 counteracts EVI1 enhancement of the ATRA induced cell cycle arrest. ( A ) U937_vec and U937_EVI1 cells infected with a control shRNA targeting Renilla luciferase (shRen) or shRNAs against GDF15 (shGDF15-1, shGDF15-2) were treated with solvent or ATRA for 24 h, and GDF15 mRNA levels were measured by qRT-PCR. GDF15 expression was normalized to that of B2M using the ΔΔ Ct method 88 and ATRA treated U937_EVI1_shRen cells as a calibrator. Results represent means + SEs from 3 independent experiments. ( B ) U937_vec and U937_EVI1 cells containing shRen, shGDF15-1, or shGDF15-2 were treated with solvent or ATRA for 5 days, and the GDF15 precursor was detected by immunoblot analysis. α-tubulin was used as a loading control. ( C ) U937_vec and U937_EVI1 cells containing shRen, shGDF15-1, or shGDF15-2 were treated with solvent or ATRA for 5 days, and mature GDF15 was detected in culture supernatants by ELISA. Results represent means + SEs from 5 independent experiments. Due to variation in factors results were not significant, but the enhancement of the ATRA induction by EVI1 in shRen infected cells, as well as the knockdown of GDF15 by both shRNAs were consistently seen in all experiments. ( D ) U937_vec and U937_EVI1 cells containing shRen, shGDF15-1, or shGDF15-2 were treated with solvent or ATRA for 5 d and subjected to cell cycle analysis by propidium iodide staining and flow cytometry. Results represent means + SEs from 7 independent experiments. Significance was calculated using Student's 2-tailed t-test (*, P

Techniques Used: Infection, shRNA, Luciferase, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Cycle Assay, Staining, Flow Cytometry, Cytometry

EVI1 and ATRA regulate gene expression both in an independent and a cooperative manner in human myeloid cells. ( A ) Numbers of genes significantly up- or down-regulated in response to ATRA in U937_vec cells, HL-60_vec cells, or both. ( B ) Numbers of genes significantly up- or downregulated in U937_EVI1 vs. U937_vec cells and in HL-60_Evi1 versus HL-60_vec cells. ( C ) Heatmap representing the expression (z-score) of genes whose induction by ATRA was enhanced by, or observed only in the presence of, EVI1 (category 1, CAT 1), whose repression by ATRA was counteracted by EVI1 (category 2, CAT 2), whose induction by ATRA was counteracted by EVI1 (category 3, CAT 3), and whose repression by ATRA was enhanced by, or observed only in the presence of, EVI1 (category 4, CAT 4) in U937 cells.
Figure Legend Snippet: EVI1 and ATRA regulate gene expression both in an independent and a cooperative manner in human myeloid cells. ( A ) Numbers of genes significantly up- or down-regulated in response to ATRA in U937_vec cells, HL-60_vec cells, or both. ( B ) Numbers of genes significantly up- or downregulated in U937_EVI1 vs. U937_vec cells and in HL-60_Evi1 versus HL-60_vec cells. ( C ) Heatmap representing the expression (z-score) of genes whose induction by ATRA was enhanced by, or observed only in the presence of, EVI1 (category 1, CAT 1), whose repression by ATRA was counteracted by EVI1 (category 2, CAT 2), whose induction by ATRA was counteracted by EVI1 (category 3, CAT 3), and whose repression by ATRA was enhanced by, or observed only in the presence of, EVI1 (category 4, CAT 4) in U937 cells.

Techniques Used: Expressing

qRT-PCR confirms cooperative gene regulation by EVI1 and ATRA in U937 cells. RNA from U937_vec and U937_EVI1 cells that had been treated with solvent or ATRA for 24 h was reverse transcribed and subjected to qRT-PCR for the indicated genes. Gene expression relative to the housekeeping gene B2M and to solvent treated U937_vec cells was calculated using the ΔΔ Ct method. 88 Results represent means + SEs from at least 3 independent experiments; all of these were also independent of the microarray experiments. In the absence of ATRA, TGM2 mRNA levels were below the detection limit. Significance was calculated using Student's 2-tailed t-test (*, P
Figure Legend Snippet: qRT-PCR confirms cooperative gene regulation by EVI1 and ATRA in U937 cells. RNA from U937_vec and U937_EVI1 cells that had been treated with solvent or ATRA for 24 h was reverse transcribed and subjected to qRT-PCR for the indicated genes. Gene expression relative to the housekeeping gene B2M and to solvent treated U937_vec cells was calculated using the ΔΔ Ct method. 88 Results represent means + SEs from at least 3 independent experiments; all of these were also independent of the microarray experiments. In the absence of ATRA, TGM2 mRNA levels were below the detection limit. Significance was calculated using Student's 2-tailed t-test (*, P

Techniques Used: Quantitative RT-PCR, Expressing, Microarray

EVI1 enhances ATRA induced cell cycle arrest and apoptosis in U937 cells. ( A , B ) U937_vec cells (white bars) and U937_EVI1 cells (black bars) were treated with solvent or ATRA and counted at the indicated time points ( A ), or subjected to cell cycle analysis by propidium iodide staining and flow cytometry on day 5 ( B ). ( C ) U937_vec and U937_EVI1 cells were treated with solvent or ATRA for 7 days, stained with Annexin V and 7AAD, and analyzed by flow cytometry. Double negative cells were classified as viable (white portions of bars), Annexin V positive 7AAD negative cells as early apoptotic (gray portions of bars), and double positive cells as late apoptotic/secondary necrotic (black portions of bars). 89 Results represent means + SEs from at least 3 independent experiments. Significance was calculated using Student's 2-tailed t-test (*, P
Figure Legend Snippet: EVI1 enhances ATRA induced cell cycle arrest and apoptosis in U937 cells. ( A , B ) U937_vec cells (white bars) and U937_EVI1 cells (black bars) were treated with solvent or ATRA and counted at the indicated time points ( A ), or subjected to cell cycle analysis by propidium iodide staining and flow cytometry on day 5 ( B ). ( C ) U937_vec and U937_EVI1 cells were treated with solvent or ATRA for 7 days, stained with Annexin V and 7AAD, and analyzed by flow cytometry. Double negative cells were classified as viable (white portions of bars), Annexin V positive 7AAD negative cells as early apoptotic (gray portions of bars), and double positive cells as late apoptotic/secondary necrotic (black portions of bars). 89 Results represent means + SEs from at least 3 independent experiments. Significance was calculated using Student's 2-tailed t-test (*, P

Techniques Used: Cell Cycle Assay, Staining, Flow Cytometry, Cytometry

35) Product Images from "Transcription Profile of Aging and Cognition-Related Genes in the Medial Prefrontal Cortex"

Article Title: Transcription Profile of Aging and Cognition-Related Genes in the Medial Prefrontal Cortex

Journal: Frontiers in Aging Neuroscience

doi: 10.3389/fnagi.2016.00113

Comparison between RT-qPCR and RNA-seq . Six genes were selected for validation experiments using a subset of animals. Each panel provides the mPFC expression determined by RT-qPCR (left, ΔΔCT values) and RNA-seq (right, counts). Two-tailed t -tests confirmed increased expression of Arc, Fos, Egr1, Egr2, and Egr4 in AI, relative to AU rats. Gene expression for young animals is provided for comparison to aged animals. For two genes, Lin7b and Egr4 , age differences were confirmed ( *** p
Figure Legend Snippet: Comparison between RT-qPCR and RNA-seq . Six genes were selected for validation experiments using a subset of animals. Each panel provides the mPFC expression determined by RT-qPCR (left, ΔΔCT values) and RNA-seq (right, counts). Two-tailed t -tests confirmed increased expression of Arc, Fos, Egr1, Egr2, and Egr4 in AI, relative to AU rats. Gene expression for young animals is provided for comparison to aged animals. For two genes, Lin7b and Egr4 , age differences were confirmed ( *** p

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

Region of the mPFC and white matter (WM) collected for RNA-seq . The right panel provides a schematic of a coronal slice +2.7 anterior to bregma diagram as adapted from Paxinos and Watson ( 1986 ) and illustrates the region of the mPFC and white matter collected for RNA-seq. The left panel shows a coronal slice from this same region.
Figure Legend Snippet: Region of the mPFC and white matter (WM) collected for RNA-seq . The right panel provides a schematic of a coronal slice +2.7 anterior to bregma diagram as adapted from Paxinos and Watson ( 1986 ) and illustrates the region of the mPFC and white matter collected for RNA-seq. The left panel shows a coronal slice from this same region.

Techniques Used: RNA Sequencing Assay

36) Product Images from "Loss of Ezh2 promotes a midbrain-to-forebrain identity switch by direct gene derepression and Wnt-dependent regulation"

Article Title: Loss of Ezh2 promotes a midbrain-to-forebrain identity switch by direct gene derepression and Wnt-dependent regulation

Journal: BMC Biology

doi: 10.1186/s12915-015-0210-9

Neural progenitor cell proliferation is controlled by Ezh2-mediated repression of cell cycle and Wnt/β-catenin signaling inhibitors. ( a ) Microarray analysis of three dissected E10.5 control and mutant midbrains identified 126 differentially expressed genes (≥1.75×, P ≤0.01), the majority of which (114) are upregulated upon Ezh2 ablation. Genes further analyzed are indicated. ( b ) qRT-PCR for Ezh2 , cell cycle regulators Cdkn2a and Cdkn2c , and Wnt signaling inhibitors Wif1 and Dkk2 on control and mutant E11.5 midbrains confirms microarray data. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, * P ≤0.05, Student’s t -test. ( c ) Chromatin immunoprecipitation confirms the presence of H3K27me3 at the transcription start site (±500 bp) of Cdkn2a , Cdkn2c , Wif1 , and Dkk2 . Intergenic region Int1 serves as unmethylated negative control. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( d – e ) In situ hybridization for Cdkn2a ( d ) and Wif1 ( e ) mRNA illustrates increased gene expression in Ezh2 mutants. ( f ) Immunostaining for β-galactosidase + cells on the BAT- gal Wnt/β-catenin signaling reporter line demonstrates diminished signaling in Ezh2-deficient midbrains. n ≥3 in each group, ** P ≤0.01, Student’s t -test. Cartoon insert indicates area of analysis for f and g . ( g ) Immunostaining against CyclinD1 and qRT-PCR. ( h ) Ccnd1 and Lef1 Wnt signaling downstream targets show decreased expression upon Ezh2 ablation. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( i ) H E staining of E12.5 sagittal midbrain sections of controls and Wnt/β-catenin signaling-ablated embryos. Mutant embryos exhibit reduced neuroepithelium thickness indicated with grey brackets in the magnifications. DAPI staining serves as nuclear marker: f , g ; Scale bars: d , e , 100 μm; f , g , 40 μm; i , 400 μm; Error bars indicate SD; ctrl, Control; dMB, Dorsal midbrain; vMB, Ventral midbrain
Figure Legend Snippet: Neural progenitor cell proliferation is controlled by Ezh2-mediated repression of cell cycle and Wnt/β-catenin signaling inhibitors. ( a ) Microarray analysis of three dissected E10.5 control and mutant midbrains identified 126 differentially expressed genes (≥1.75×, P ≤0.01), the majority of which (114) are upregulated upon Ezh2 ablation. Genes further analyzed are indicated. ( b ) qRT-PCR for Ezh2 , cell cycle regulators Cdkn2a and Cdkn2c , and Wnt signaling inhibitors Wif1 and Dkk2 on control and mutant E11.5 midbrains confirms microarray data. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, * P ≤0.05, Student’s t -test. ( c ) Chromatin immunoprecipitation confirms the presence of H3K27me3 at the transcription start site (±500 bp) of Cdkn2a , Cdkn2c , Wif1 , and Dkk2 . Intergenic region Int1 serves as unmethylated negative control. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( d – e ) In situ hybridization for Cdkn2a ( d ) and Wif1 ( e ) mRNA illustrates increased gene expression in Ezh2 mutants. ( f ) Immunostaining for β-galactosidase + cells on the BAT- gal Wnt/β-catenin signaling reporter line demonstrates diminished signaling in Ezh2-deficient midbrains. n ≥3 in each group, ** P ≤0.01, Student’s t -test. Cartoon insert indicates area of analysis for f and g . ( g ) Immunostaining against CyclinD1 and qRT-PCR. ( h ) Ccnd1 and Lef1 Wnt signaling downstream targets show decreased expression upon Ezh2 ablation. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( i ) H E staining of E12.5 sagittal midbrain sections of controls and Wnt/β-catenin signaling-ablated embryos. Mutant embryos exhibit reduced neuroepithelium thickness indicated with grey brackets in the magnifications. DAPI staining serves as nuclear marker: f , g ; Scale bars: d , e , 100 μm; f , g , 40 μm; i , 400 μm; Error bars indicate SD; ctrl, Control; dMB, Dorsal midbrain; vMB, Ventral midbrain

Techniques Used: Microarray, Mutagenesis, Quantitative RT-PCR, Chromatin Immunoprecipitation, Negative Control, In Situ Hybridization, Expressing, Immunostaining, Staining, Marker

37) Product Images from "Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4"

Article Title: Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4

Journal: Scientific Reports

doi: 10.1038/srep30529

Viperin expression inhibits RABV replication. ( A ) Viperin inhibits RABV replication in viperin-eGFP-transfected BHK-21 cells. The viperin stably expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.1. Virus titres were determined at 24, 36, and 48 hpi. ( B ) RABV proteins in the infected viperin stably expressing BHK-21 cells were detected by Western blotting. ( C ) The N protein/actin, P protein/actin and M protein/actin ratios in Figure 2F were measured using Li-Cor Odyssey 3.0 analytical software version 29. ( D ) RNA expression levels of viperin. rRC-HL vRNA and N mRNA expression levels were detected by qRT-PCR at 24, 36, and 48 hpi. Viperin-expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.01. Data were normalized to β-actin expression and are presented as relative fold expression values to each control cell population infected with rRC-HL.
Figure Legend Snippet: Viperin expression inhibits RABV replication. ( A ) Viperin inhibits RABV replication in viperin-eGFP-transfected BHK-21 cells. The viperin stably expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.1. Virus titres were determined at 24, 36, and 48 hpi. ( B ) RABV proteins in the infected viperin stably expressing BHK-21 cells were detected by Western blotting. ( C ) The N protein/actin, P protein/actin and M protein/actin ratios in Figure 2F were measured using Li-Cor Odyssey 3.0 analytical software version 29. ( D ) RNA expression levels of viperin. rRC-HL vRNA and N mRNA expression levels were detected by qRT-PCR at 24, 36, and 48 hpi. Viperin-expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.01. Data were normalized to β-actin expression and are presented as relative fold expression values to each control cell population infected with rRC-HL.

Techniques Used: Expressing, Transfection, Stable Transfection, Infection, Western Blot, Software, RNA Expression, Quantitative RT-PCR

38) Product Images from "Ionic Liquids Impact the Bioenergy Feedstock-Degrading Microbiome and Transcription of Enzymes Relevant to Polysaccharide Hydrolysis"

Article Title: Ionic Liquids Impact the Bioenergy Feedstock-Degrading Microbiome and Transcription of Enzymes Relevant to Polysaccharide Hydrolysis

Journal: mSystems

doi: 10.1128/mSystems.00120-16

Relative abundances of phyla in the microbial communities under conditions of various IL concentrations. (A and B) DNA sequence data were binned and assigned to the nearest taxonomic classification based on full metagenome analysis (A) and iTag 16S ribosomal RNA analysis (B). (C) Distance tree showing separation of metagenomes.
Figure Legend Snippet: Relative abundances of phyla in the microbial communities under conditions of various IL concentrations. (A and B) DNA sequence data were binned and assigned to the nearest taxonomic classification based on full metagenome analysis (A) and iTag 16S ribosomal RNA analysis (B). (C) Distance tree showing separation of metagenomes.

Techniques Used: Sequencing

39) Product Images from "Mesenchymal stromal cells reset the scatter factor system and cytokine network in experimental kidney transplantation"

Article Title: Mesenchymal stromal cells reset the scatter factor system and cytokine network in experimental kidney transplantation

Journal: BMC Immunology

doi: 10.1186/s12865-014-0044-1

Semiquantitative MSPmRNA expression in PBMC, MSC in basal condition evaluated by reverse transcription (RT)–PCR that used 1 mg of RNA as the template. PBMC known for their capacity to produce MSP were used as controls. Columns indicate MSP mRNA expression normalized to the beta-actin expression and converted into fold change. *p
Figure Legend Snippet: Semiquantitative MSPmRNA expression in PBMC, MSC in basal condition evaluated by reverse transcription (RT)–PCR that used 1 mg of RNA as the template. PBMC known for their capacity to produce MSP were used as controls. Columns indicate MSP mRNA expression normalized to the beta-actin expression and converted into fold change. *p

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

MSP mRNA expression of control and allografted rats. Groups are defined as in Figure 8 . MSP mRNA expression in renal tissue analyzed by RT PCR in all groups of rats. Columns indicate MSP mRNA expression normalized to the beta-actin expression and converted into fold change. *p
Figure Legend Snippet: MSP mRNA expression of control and allografted rats. Groups are defined as in Figure 8 . MSP mRNA expression in renal tissue analyzed by RT PCR in all groups of rats. Columns indicate MSP mRNA expression normalized to the beta-actin expression and converted into fold change. *p

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

40) Product Images from "FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice"

Article Title: FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0105349

Detailed analysis of adipose tissue. All data are collected from 30 weeks old mice. *indicate significant p-values between Lep ob/ob ;Fto +/+ and Lep ob/ob ; Fto −/− . a) Weights of different fat pads from female mice (n = 13, 16, 18, 16). b) Area size of epigonadal fat cells from female mice (n = 4, 4, 8, 7). c+d) Expression analysis for different marker genes of epigonadal adipose tissue (n = 6, 4, 5, 5, 5). Following p-values were calculated: between Lep ob/ob ;Fto +/− and Lep ob/ob ; Fto −/− : PPARγ2: p = 0,08, Adiponectin: p = 0,21, TNFα:p = 0,06, IL-6:p = 0,03. Data are presented as mean. Error bars indicate the SEM.
Figure Legend Snippet: Detailed analysis of adipose tissue. All data are collected from 30 weeks old mice. *indicate significant p-values between Lep ob/ob ;Fto +/+ and Lep ob/ob ; Fto −/− . a) Weights of different fat pads from female mice (n = 13, 16, 18, 16). b) Area size of epigonadal fat cells from female mice (n = 4, 4, 8, 7). c+d) Expression analysis for different marker genes of epigonadal adipose tissue (n = 6, 4, 5, 5, 5). Following p-values were calculated: between Lep ob/ob ;Fto +/− and Lep ob/ob ; Fto −/− : PPARγ2: p = 0,08, Adiponectin: p = 0,21, TNFα:p = 0,06, IL-6:p = 0,03. Data are presented as mean. Error bars indicate the SEM.

Techniques Used: Mouse Assay, Expressing, Marker

Related Articles

Real-time Polymerase Chain Reaction:

Article Title: Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy
Article Snippet: .. RNA isolation and qPCR analysis RNA from FAPs was extracted using miRNeasy Micro kit (Qiagen) following the manufacturer’s protocol, including DNase treatment of the samples. cDNA was synthetized using QuantiTect Reverse Transcription kit (Qiagen) and analysed by real-time quantitative PCR (RT-PCR) using SYBR Green PCR Master Mix (Applied Biosystems), using primers provided in Supplementary Table . .. Relative expression was calculated using 2−∆ct method , Gapdh housekeeping gene expression was used to normalize gene expression.

Article Title: Exercise intervention alters HDL subclass distribution and function in obese women
Article Snippet: .. Cells were then stimulated with 20 ng/ml murine tumour necrosis factor alpha (TNF-α) (PeproTech, 315-01A) for 5 h. Following RNA isolation and cDNA synthesis, cDNA was amplified for 25 cycles using the RT2 SYBR Green qPCR kit (Qiagen, 330,500) in the RotorGene6000 (Corbit Lifesciences) to quantify expression levels of VCAM and GAPDH. .. Data is presented as relative reduction in VCAM expression compared to an untreated control.

Article Title: East Indian Sandalwood Oil Is a Phosphodiesterase Inhibitor: A New Therapeutic Option in the Treatment of Inflammatory Skin Disease
Article Snippet: .. Quantitative Real-Time PCR (qRT-PCR) for mRNA Expression of PDE Isoforms Total RNA was isolated from DF, BEAS-2B, A549, and THP-1 cells using RNeasy kit (QIAGEN, MD, United States) according to the vendor’s protocol. cDNA was synthesized using Super Script First-Strand synthesis system for RT-PCR (Invitrogen). .. PDE4A, PDE4B, PDE4C, PDE4D, PDE7A, and PDE7B were amplified using real-time PCR system ABI-PRISM 7900 (Applied Biosystems, Foster City, CA, United States).

Amplification:

Article Title: Exercise intervention alters HDL subclass distribution and function in obese women
Article Snippet: .. Cells were then stimulated with 20 ng/ml murine tumour necrosis factor alpha (TNF-α) (PeproTech, 315-01A) for 5 h. Following RNA isolation and cDNA synthesis, cDNA was amplified for 25 cycles using the RT2 SYBR Green qPCR kit (Qiagen, 330,500) in the RotorGene6000 (Corbit Lifesciences) to quantify expression levels of VCAM and GAPDH. .. Data is presented as relative reduction in VCAM expression compared to an untreated control.

Synthesized:

Article Title: East Indian Sandalwood Oil Is a Phosphodiesterase Inhibitor: A New Therapeutic Option in the Treatment of Inflammatory Skin Disease
Article Snippet: .. Quantitative Real-Time PCR (qRT-PCR) for mRNA Expression of PDE Isoforms Total RNA was isolated from DF, BEAS-2B, A549, and THP-1 cells using RNeasy kit (QIAGEN, MD, United States) according to the vendor’s protocol. cDNA was synthesized using Super Script First-Strand synthesis system for RT-PCR (Invitrogen). .. PDE4A, PDE4B, PDE4C, PDE4D, PDE7A, and PDE7B were amplified using real-time PCR system ABI-PRISM 7900 (Applied Biosystems, Foster City, CA, United States).

Isolation:

Article Title: Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632
Article Snippet: .. After removal of the feeder cells with Versene, RNA from SBK at passage 5 and 6 for the +Y group and 4, 5, and 6 for the -Y group was isolated using the RNeasy kit (Qiagen). ..

Article Title: PUMILIO hyperactivity drives premature aging of Norad-deficient mice
Article Snippet: .. RNA isolation and quantitative reverse transcription PCR (qRT-PCR) Total RNA was isolated from cells or tissues using the miRNeasy Mini Kit (Qiagen) following the manufacturer’s instructions including an on-column DNAse I digest to remove genomic DNA contamination. .. Complementary DNA (cDNA) was generated from 1 μg of total RNA using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen) according to the manufacturer’s protocol.

Article Title: Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy
Article Snippet: .. RNA isolation and qPCR analysis RNA from FAPs was extracted using miRNeasy Micro kit (Qiagen) following the manufacturer’s protocol, including DNase treatment of the samples. cDNA was synthetized using QuantiTect Reverse Transcription kit (Qiagen) and analysed by real-time quantitative PCR (RT-PCR) using SYBR Green PCR Master Mix (Applied Biosystems), using primers provided in Supplementary Table . .. Relative expression was calculated using 2−∆ct method , Gapdh housekeeping gene expression was used to normalize gene expression.

Article Title: Exercise intervention alters HDL subclass distribution and function in obese women
Article Snippet: .. Cells were then stimulated with 20 ng/ml murine tumour necrosis factor alpha (TNF-α) (PeproTech, 315-01A) for 5 h. Following RNA isolation and cDNA synthesis, cDNA was amplified for 25 cycles using the RT2 SYBR Green qPCR kit (Qiagen, 330,500) in the RotorGene6000 (Corbit Lifesciences) to quantify expression levels of VCAM and GAPDH. .. Data is presented as relative reduction in VCAM expression compared to an untreated control.

Article Title: East Indian Sandalwood Oil Is a Phosphodiesterase Inhibitor: A New Therapeutic Option in the Treatment of Inflammatory Skin Disease
Article Snippet: .. Quantitative Real-Time PCR (qRT-PCR) for mRNA Expression of PDE Isoforms Total RNA was isolated from DF, BEAS-2B, A549, and THP-1 cells using RNeasy kit (QIAGEN, MD, United States) according to the vendor’s protocol. cDNA was synthesized using Super Script First-Strand synthesis system for RT-PCR (Invitrogen). .. PDE4A, PDE4B, PDE4C, PDE4D, PDE7A, and PDE7B were amplified using real-time PCR system ABI-PRISM 7900 (Applied Biosystems, Foster City, CA, United States).

Quantitative RT-PCR:

Article Title: PUMILIO hyperactivity drives premature aging of Norad-deficient mice
Article Snippet: .. RNA isolation and quantitative reverse transcription PCR (qRT-PCR) Total RNA was isolated from cells or tissues using the miRNeasy Mini Kit (Qiagen) following the manufacturer’s instructions including an on-column DNAse I digest to remove genomic DNA contamination. .. Complementary DNA (cDNA) was generated from 1 μg of total RNA using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen) according to the manufacturer’s protocol.

Article Title: East Indian Sandalwood Oil Is a Phosphodiesterase Inhibitor: A New Therapeutic Option in the Treatment of Inflammatory Skin Disease
Article Snippet: .. Quantitative Real-Time PCR (qRT-PCR) for mRNA Expression of PDE Isoforms Total RNA was isolated from DF, BEAS-2B, A549, and THP-1 cells using RNeasy kit (QIAGEN, MD, United States) according to the vendor’s protocol. cDNA was synthesized using Super Script First-Strand synthesis system for RT-PCR (Invitrogen). .. PDE4A, PDE4B, PDE4C, PDE4D, PDE7A, and PDE7B were amplified using real-time PCR system ABI-PRISM 7900 (Applied Biosystems, Foster City, CA, United States).

SYBR Green Assay:

Article Title: Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy
Article Snippet: .. RNA isolation and qPCR analysis RNA from FAPs was extracted using miRNeasy Micro kit (Qiagen) following the manufacturer’s protocol, including DNase treatment of the samples. cDNA was synthetized using QuantiTect Reverse Transcription kit (Qiagen) and analysed by real-time quantitative PCR (RT-PCR) using SYBR Green PCR Master Mix (Applied Biosystems), using primers provided in Supplementary Table . .. Relative expression was calculated using 2−∆ct method , Gapdh housekeeping gene expression was used to normalize gene expression.

Article Title: Exercise intervention alters HDL subclass distribution and function in obese women
Article Snippet: .. Cells were then stimulated with 20 ng/ml murine tumour necrosis factor alpha (TNF-α) (PeproTech, 315-01A) for 5 h. Following RNA isolation and cDNA synthesis, cDNA was amplified for 25 cycles using the RT2 SYBR Green qPCR kit (Qiagen, 330,500) in the RotorGene6000 (Corbit Lifesciences) to quantify expression levels of VCAM and GAPDH. .. Data is presented as relative reduction in VCAM expression compared to an untreated control.

Polymerase Chain Reaction:

Article Title: PUMILIO hyperactivity drives premature aging of Norad-deficient mice
Article Snippet: .. RNA isolation and quantitative reverse transcription PCR (qRT-PCR) Total RNA was isolated from cells or tissues using the miRNeasy Mini Kit (Qiagen) following the manufacturer’s instructions including an on-column DNAse I digest to remove genomic DNA contamination. .. Complementary DNA (cDNA) was generated from 1 μg of total RNA using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen) according to the manufacturer’s protocol.

Article Title: Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy
Article Snippet: .. RNA isolation and qPCR analysis RNA from FAPs was extracted using miRNeasy Micro kit (Qiagen) following the manufacturer’s protocol, including DNase treatment of the samples. cDNA was synthetized using QuantiTect Reverse Transcription kit (Qiagen) and analysed by real-time quantitative PCR (RT-PCR) using SYBR Green PCR Master Mix (Applied Biosystems), using primers provided in Supplementary Table . .. Relative expression was calculated using 2−∆ct method , Gapdh housekeeping gene expression was used to normalize gene expression.

Expressing:

Article Title: Occurrence of cagA+vacA s1a m1 i1 Helicobacter pylori in farm animals in Egypt and ability to survive in experimentally contaminated UHT milk
Article Snippet: .. Detection of mRNA expression of vacA and cagA by reverse transcription polymerase chain reaction (RT-PCR) RNA was extracted from the UHT milk samples and mouse gastric mucosa using the RNeasy mini kit (Qiagen) with DNase treatment step using RNase-free DNase set (Qiagen). .. The concentration of the extracted RNA was measured by Qubit® 2.0 Fluorometer (Thermo Fisher Scientific).

Article Title: Exercise intervention alters HDL subclass distribution and function in obese women
Article Snippet: .. Cells were then stimulated with 20 ng/ml murine tumour necrosis factor alpha (TNF-α) (PeproTech, 315-01A) for 5 h. Following RNA isolation and cDNA synthesis, cDNA was amplified for 25 cycles using the RT2 SYBR Green qPCR kit (Qiagen, 330,500) in the RotorGene6000 (Corbit Lifesciences) to quantify expression levels of VCAM and GAPDH. .. Data is presented as relative reduction in VCAM expression compared to an untreated control.

Article Title: East Indian Sandalwood Oil Is a Phosphodiesterase Inhibitor: A New Therapeutic Option in the Treatment of Inflammatory Skin Disease
Article Snippet: .. Quantitative Real-Time PCR (qRT-PCR) for mRNA Expression of PDE Isoforms Total RNA was isolated from DF, BEAS-2B, A549, and THP-1 cells using RNeasy kit (QIAGEN, MD, United States) according to the vendor’s protocol. cDNA was synthesized using Super Script First-Strand synthesis system for RT-PCR (Invitrogen). .. PDE4A, PDE4B, PDE4C, PDE4D, PDE7A, and PDE7B were amplified using real-time PCR system ABI-PRISM 7900 (Applied Biosystems, Foster City, CA, United States).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Occurrence of cagA+vacA s1a m1 i1 Helicobacter pylori in farm animals in Egypt and ability to survive in experimentally contaminated UHT milk
Article Snippet: .. Detection of mRNA expression of vacA and cagA by reverse transcription polymerase chain reaction (RT-PCR) RNA was extracted from the UHT milk samples and mouse gastric mucosa using the RNeasy mini kit (Qiagen) with DNase treatment step using RNase-free DNase set (Qiagen). .. The concentration of the extracted RNA was measured by Qubit® 2.0 Fluorometer (Thermo Fisher Scientific).

Article Title: Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy
Article Snippet: .. RNA isolation and qPCR analysis RNA from FAPs was extracted using miRNeasy Micro kit (Qiagen) following the manufacturer’s protocol, including DNase treatment of the samples. cDNA was synthetized using QuantiTect Reverse Transcription kit (Qiagen) and analysed by real-time quantitative PCR (RT-PCR) using SYBR Green PCR Master Mix (Applied Biosystems), using primers provided in Supplementary Table . .. Relative expression was calculated using 2−∆ct method , Gapdh housekeeping gene expression was used to normalize gene expression.

Article Title: East Indian Sandalwood Oil Is a Phosphodiesterase Inhibitor: A New Therapeutic Option in the Treatment of Inflammatory Skin Disease
Article Snippet: .. Quantitative Real-Time PCR (qRT-PCR) for mRNA Expression of PDE Isoforms Total RNA was isolated from DF, BEAS-2B, A549, and THP-1 cells using RNeasy kit (QIAGEN, MD, United States) according to the vendor’s protocol. cDNA was synthesized using Super Script First-Strand synthesis system for RT-PCR (Invitrogen). .. PDE4A, PDE4B, PDE4C, PDE4D, PDE7A, and PDE7B were amplified using real-time PCR system ABI-PRISM 7900 (Applied Biosystems, Foster City, CA, United States).

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    Qiagen rnase free dnase set
    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including <t>DNase</t> digestion). Optional steps are indicated by dotted arrows. Note that <t>RNase</t> digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
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    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Journal: Frontiers in Microbiology

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    doi: 10.3389/fmicb.2016.01588

    Figure Lengend Snippet: Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Article Snippet: The following DNases were tested for their ability to remove amplifiable DNA from TNA samples: DNase I (Sigma), RNase-Free DNase Set (QIAGEN), RNase-Free DNase I (Epicentre Biotechnologies) and TURBO DNA-free DNase Kit (Ambion, Life Technologies).

    Techniques: Environmental Sampling, Purification, Real-time Polymerase Chain Reaction, Lysis

    BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.

    Journal: Immunity

    Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells

    doi: 10.1016/j.immuni.2018.01.013

    Figure Lengend Snippet: BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.

    Article Snippet: Total RNA was extracted and on-column digestion with DNase (QIAGEN, Cat#79254) was performed, followed by elution with 10μl of RNase-free water.

    Techniques: Binding Assay, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Modification, Marker

    Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P

    Journal: Nature Communications

    Article Title: Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza

    doi: 10.1038/s41467-018-04792-7

    Figure Lengend Snippet: Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P

    Article Snippet: Extracted RNA were treated with DNase I (QIAGEN, Cat# 79254, USA) according to the manufacturer’s protocol and purified by RNeasy Mini Kit (QIAGEN, Cat# 74106, USA) to exclude plasmid DNA contamination.

    Techniques: Activity Assay, Plasmid Preparation, RNA Expression, Transfection, Quantitative RT-PCR, Negative Control, Infection, Plaque Assay

    Enzymatic digestion of decellularized ECM scaffolds releases small RNA molecules. ( A ) Nucleic acid extracted from untreated UBM (no digest) and pepsin-, proteinase K–, or collagenase-treated UBM was exposed to RNase A, DNase I, or no-nuclease treatment (control). ( B ) Electropherogram depicting the small RNA pattern of nucleic acid in fluorescence units (FU) before (top panel) and after (bottom panel) DNase I treatment. ( C ) Electropherogram depicting small RNA pattern from the indicated samples in FU. ( D ) A subset of nucleic molecules in biologic scaffolds is protected from nuclease degradation.

    Journal: Science Advances

    Article Title: Matrix-bound nanovesicles within ECM bioscaffolds

    doi: 10.1126/sciadv.1600502

    Figure Lengend Snippet: Enzymatic digestion of decellularized ECM scaffolds releases small RNA molecules. ( A ) Nucleic acid extracted from untreated UBM (no digest) and pepsin-, proteinase K–, or collagenase-treated UBM was exposed to RNase A, DNase I, or no-nuclease treatment (control). ( B ) Electropherogram depicting the small RNA pattern of nucleic acid in fluorescence units (FU) before (top panel) and after (bottom panel) DNase I treatment. ( C ) Electropherogram depicting small RNA pattern from the indicated samples in FU. ( D ) A subset of nucleic molecules in biologic scaffolds is protected from nuclease degradation.

    Article Snippet: RNase-free DNase was obtained from Qiagen.

    Techniques: Fluorescence