mir 145 specific primer  (Promega)

 
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    GoTaq PCR Core Systems
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    Bundles of GoTaq DNA Polymerase Mg free Buffers Mg Chloride and PCR Nucleotide Mix
    Catalog Number:
    m7660
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    Category:
    Nucleic Acid Extraction Analysis PCR Endpoint PCR
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    Structured Review

    Promega mir 145 specific primer
    <t>miR-145</t> targets the 3′-UTR of OPG and suppresses its expression. (A-C) miR-145 mimics or ASO-miR-145 were co-transfected with pmirGLO or pmirGLO vectors containing wt or mut 3′-UTRs of OPG into MG-63 cells. Following 48 h, cells were harvested and assayed for dual luciferase activity. The results were normalized to Renilla activity. The effects of miR-145 on endogenous OPG expression were determined by (D) reverse transcription-quantitative polymerase chain reaction and (E and F) western blotting. The mRNA and protein expression levels of OPG were normalized to the levels of β-actin mRNA and GAPDH protein, respectively. **P
    Bundles of GoTaq DNA Polymerase Mg free Buffers Mg Chloride and PCR Nucleotide Mix
    https://www.bioz.com/result/mir 145 specific primer/product/Promega
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    Images

    1) Product Images from "Estrogen stimulates osteoprotegerin expression via the suppression of miR-145 expression in MG-63 cells"

    Article Title: Estrogen stimulates osteoprotegerin expression via the suppression of miR-145 expression in MG-63 cells

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.6168

    miR-145 targets the 3′-UTR of OPG and suppresses its expression. (A-C) miR-145 mimics or ASO-miR-145 were co-transfected with pmirGLO or pmirGLO vectors containing wt or mut 3′-UTRs of OPG into MG-63 cells. Following 48 h, cells were harvested and assayed for dual luciferase activity. The results were normalized to Renilla activity. The effects of miR-145 on endogenous OPG expression were determined by (D) reverse transcription-quantitative polymerase chain reaction and (E and F) western blotting. The mRNA and protein expression levels of OPG were normalized to the levels of β-actin mRNA and GAPDH protein, respectively. **P
    Figure Legend Snippet: miR-145 targets the 3′-UTR of OPG and suppresses its expression. (A-C) miR-145 mimics or ASO-miR-145 were co-transfected with pmirGLO or pmirGLO vectors containing wt or mut 3′-UTRs of OPG into MG-63 cells. Following 48 h, cells were harvested and assayed for dual luciferase activity. The results were normalized to Renilla activity. The effects of miR-145 on endogenous OPG expression were determined by (D) reverse transcription-quantitative polymerase chain reaction and (E and F) western blotting. The mRNA and protein expression levels of OPG were normalized to the levels of β-actin mRNA and GAPDH protein, respectively. **P

    Techniques Used: Expressing, Allele-specific Oligonucleotide, Transfection, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot

    E2 decreases mature miR-145 levels. (A) miRNA target sites in the OPG 3′-UTR were analyzed by TargetScan. The potential miR-145 site is presented. (B) MG-63 cells were treated with the indicated concentrations of E2 for 48 h, miR-145 levels were determined by reverse transcription-quantitative polymerase chain reaction. U6 was used as an internal control. **P
    Figure Legend Snippet: E2 decreases mature miR-145 levels. (A) miRNA target sites in the OPG 3′-UTR were analyzed by TargetScan. The potential miR-145 site is presented. (B) MG-63 cells were treated with the indicated concentrations of E2 for 48 h, miR-145 levels were determined by reverse transcription-quantitative polymerase chain reaction. U6 was used as an internal control. **P

    Techniques Used: Real-time Polymerase Chain Reaction

    Overexpression of miR-145 compromises the induction of OPG by estrogen. MG-63 cells were transfected with miR-145 mimics or NC. A total of 24 h post-transfection, the cells were treated with 10 nM E2 for 48 h, and the OPG protein levels secreted into the medium and OPG mRNA were measured by (A) ELISA and (B) reverse transcription-quantitative polymerase chain reaction. The mRNA expression levels of OPG were normalized to the mRNA expression levels of β-actin. **P
    Figure Legend Snippet: Overexpression of miR-145 compromises the induction of OPG by estrogen. MG-63 cells were transfected with miR-145 mimics or NC. A total of 24 h post-transfection, the cells were treated with 10 nM E2 for 48 h, and the OPG protein levels secreted into the medium and OPG mRNA were measured by (A) ELISA and (B) reverse transcription-quantitative polymerase chain reaction. The mRNA expression levels of OPG were normalized to the mRNA expression levels of β-actin. **P

    Techniques Used: Over Expression, Transfection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing

    2) Product Images from "Differential isoform expression and alternative splicing in sex determination in mice"

    Article Title: Differential isoform expression and alternative splicing in sex determination in mice

    Journal: BMC Genomics

    doi: 10.1186/s12864-019-5572-x

    Differentially expressed genes (DEG) in male and female gonads at E11 and E12 and analysis of gene clustering. ( a ) Quantitative PCR analysis of Sry expression in male embryonic day 11 and 12.5 (E11-E12.5) gonads. Biological triplicate results are presented as mean ± SEM. Bars with different superscripts differ significantly (ANOVA, P
    Figure Legend Snippet: Differentially expressed genes (DEG) in male and female gonads at E11 and E12 and analysis of gene clustering. ( a ) Quantitative PCR analysis of Sry expression in male embryonic day 11 and 12.5 (E11-E12.5) gonads. Biological triplicate results are presented as mean ± SEM. Bars with different superscripts differ significantly (ANOVA, P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    3) Product Images from "Cells Lacking the Fumarase Tumor Suppressor Are Protected from Apoptosis through a Hypoxia-Inducible Factor-Independent, AMPK-Dependent Mechanism"

    Article Title: Cells Lacking the Fumarase Tumor Suppressor Are Protected from Apoptosis through a Hypoxia-Inducible Factor-Independent, AMPK-Dependent Mechanism

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.06160-11

    HIF-1α and HIF-2α are stabilized in FH-defective cells but are dispensable for cell protection from apoptosis. (A) Knocking down FH by interfering RNA (FH shRNA) in HK-2 cells resulted in nuclear (nuc.) but not cytoplasmic (cyt.) accumulation of HIF-1α and HIF-2α under normoxic conditions, comparable to that obtained by treating cells with 200 μM cobalt chloride (CoCl 2 ) for 24 h. To study the role of HIF-1α, the latter was down modulated by stable expression of a specific interfering shRNA (HIF-1α shRNA). The same Western blot was reprobed with a monoclonal GAPDH antibody to show equal loading. (B) HIF-1α down modulation by HIF-1α shRNA was also detectable at the mRNA level, as measured by quantitative real-time PCR. The mean fold change of HIF-1α gene expression was calculated using the following formula: ΔΔ C T = [( C T HIF-1 α − C T cyclophilin A for FH shRNA cells, HIF-1α shRNA cells, or HIF-1α shRNA and FH shRNA cells) − ( C T HIF-1 α − C T cyclophilin A for FH shRNA cells)]. (C) FH-defective HK-2 cells remained viable after treatment with 20 μM CDDP for 72 h and were not affected by the simultaneous down modulation of HIF-1α. Also, the response of FH-proficient HK-2 cells was not affected by HIF-1α knockdown. (D) Mouse Fh1 was knocked down by means of stable expression of a specific interfering RNA (Fh shRNA) in the Hif-1β/Arnt-proficient mouse hepatoma Hepa-1C1c7 cell line; its mutant derivative subclone c4, which lacks a functional Hif-1β/Arnt protein; and a revertant clone of the c4 subclone, vT{2} cells, in which the functionality of the Arnt protein is restored by cDNA reexpression. Western blots were probed with a polyclonal anti-human FH antibody, which also labels the mouse protein, and with a monoclonal anti-mouse GAPDH antibody to show equal loading. (E) The aforementioned cells were protected from treatment with 20 μM CDDP for 72 h when FH was knocked down, irrespective of the functionality of Hif-1β/Arnt. (F) FH-defective HK-2 cells accumulated ROS even in the absence of added hydrogen peroxide (H 2 O 2 ). ROS-positive cells were measured as the percentage of cells which accumulated the red fluorescent dye MitoSOX. (G to I) FH-defective HK-2 cells remained similarly viable when treated with 20 μM CDDP for 48 h in the presence of the indicated concentrations of either N -acetylcysteine (NAC) (G), sodium pyruvate (H), or the LDH inhibitor oxamate (I) or in the presence of vehicles used to dissolve the compounds (−).
    Figure Legend Snippet: HIF-1α and HIF-2α are stabilized in FH-defective cells but are dispensable for cell protection from apoptosis. (A) Knocking down FH by interfering RNA (FH shRNA) in HK-2 cells resulted in nuclear (nuc.) but not cytoplasmic (cyt.) accumulation of HIF-1α and HIF-2α under normoxic conditions, comparable to that obtained by treating cells with 200 μM cobalt chloride (CoCl 2 ) for 24 h. To study the role of HIF-1α, the latter was down modulated by stable expression of a specific interfering shRNA (HIF-1α shRNA). The same Western blot was reprobed with a monoclonal GAPDH antibody to show equal loading. (B) HIF-1α down modulation by HIF-1α shRNA was also detectable at the mRNA level, as measured by quantitative real-time PCR. The mean fold change of HIF-1α gene expression was calculated using the following formula: ΔΔ C T = [( C T HIF-1 α − C T cyclophilin A for FH shRNA cells, HIF-1α shRNA cells, or HIF-1α shRNA and FH shRNA cells) − ( C T HIF-1 α − C T cyclophilin A for FH shRNA cells)]. (C) FH-defective HK-2 cells remained viable after treatment with 20 μM CDDP for 72 h and were not affected by the simultaneous down modulation of HIF-1α. Also, the response of FH-proficient HK-2 cells was not affected by HIF-1α knockdown. (D) Mouse Fh1 was knocked down by means of stable expression of a specific interfering RNA (Fh shRNA) in the Hif-1β/Arnt-proficient mouse hepatoma Hepa-1C1c7 cell line; its mutant derivative subclone c4, which lacks a functional Hif-1β/Arnt protein; and a revertant clone of the c4 subclone, vT{2} cells, in which the functionality of the Arnt protein is restored by cDNA reexpression. Western blots were probed with a polyclonal anti-human FH antibody, which also labels the mouse protein, and with a monoclonal anti-mouse GAPDH antibody to show equal loading. (E) The aforementioned cells were protected from treatment with 20 μM CDDP for 72 h when FH was knocked down, irrespective of the functionality of Hif-1β/Arnt. (F) FH-defective HK-2 cells accumulated ROS even in the absence of added hydrogen peroxide (H 2 O 2 ). ROS-positive cells were measured as the percentage of cells which accumulated the red fluorescent dye MitoSOX. (G to I) FH-defective HK-2 cells remained similarly viable when treated with 20 μM CDDP for 48 h in the presence of the indicated concentrations of either N -acetylcysteine (NAC) (G), sodium pyruvate (H), or the LDH inhibitor oxamate (I) or in the presence of vehicles used to dissolve the compounds (−).

    Techniques Used: shRNA, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Mutagenesis, Functional Assay

    4) Product Images from "A Magnaporthe grisea Cyclophilin Acts as a Virulence Determinant during Plant Infection"

    Article Title: A Magnaporthe grisea Cyclophilin Acts as a Virulence Determinant during Plant Infection

    Journal: The Plant Cell

    doi: 10.1105/tpc.010389

    Reintroduction of CYP1 into a Δcyp1 Mutant Restores Virulence and CsA Sensitivity. (A) Rice blast assays were performed by spraying conidial suspensions of uniform concentration (10 5 conidia/mL) onto seedlings of rice cv CO-39. Leaf 1 is from a plant inoculated with Δcyp1 mutant TR33. Leaf 2 is from a plant inoculated with the isogenic wild-type strain Guy11. Leaf 3 is from a plant inoculated with MV42, a transformant of Δcyp1 mutant TR33 that carries a single copy of a 1.1-kb PstI fragment of CYP1 . Leaf 4 is from a plant inoculated with MV72, a transformant of Δcyp1 mutant TR33 that carries a single copy of a 4.3-kb XhoI fragment of CYP1 . CsA sensitivity assays were performed by incubating mycelial plugs of Magnaporthe on CM agar medium containing 100 μg/mL CsA and incubating plates at 24°C for 5 days. (B) Plate 1 was inoculated with Δcyp1 mutant TR33, plate 2 was inoculated with Guy11, and plates 3 to 6 were inoculated with transformants MV37, MV39, MV40, and MV42, respectively, each of which carries a single copy of a 1.1-kb PstI fragment of CYP1 . (C) Plate 1 was inoculated with Δcyp1 mutant TR33, plate 2 was inoculated with Guy11, and plates 3 to 10 were inoculated with transformants MV44, MV67, MV70, MV72, MV73, and MV74, respectively, each of which carries a single copy of a 4.3-kb XhoI fragment of CYP1 .
    Figure Legend Snippet: Reintroduction of CYP1 into a Δcyp1 Mutant Restores Virulence and CsA Sensitivity. (A) Rice blast assays were performed by spraying conidial suspensions of uniform concentration (10 5 conidia/mL) onto seedlings of rice cv CO-39. Leaf 1 is from a plant inoculated with Δcyp1 mutant TR33. Leaf 2 is from a plant inoculated with the isogenic wild-type strain Guy11. Leaf 3 is from a plant inoculated with MV42, a transformant of Δcyp1 mutant TR33 that carries a single copy of a 1.1-kb PstI fragment of CYP1 . Leaf 4 is from a plant inoculated with MV72, a transformant of Δcyp1 mutant TR33 that carries a single copy of a 4.3-kb XhoI fragment of CYP1 . CsA sensitivity assays were performed by incubating mycelial plugs of Magnaporthe on CM agar medium containing 100 μg/mL CsA and incubating plates at 24°C for 5 days. (B) Plate 1 was inoculated with Δcyp1 mutant TR33, plate 2 was inoculated with Guy11, and plates 3 to 6 were inoculated with transformants MV37, MV39, MV40, and MV42, respectively, each of which carries a single copy of a 1.1-kb PstI fragment of CYP1 . (C) Plate 1 was inoculated with Δcyp1 mutant TR33, plate 2 was inoculated with Guy11, and plates 3 to 10 were inoculated with transformants MV44, MV67, MV70, MV72, MV73, and MV74, respectively, each of which carries a single copy of a 4.3-kb XhoI fragment of CYP1 .

    Techniques Used: Mutagenesis, Concentration Assay

    Model Showing the Predicted Cellular Roles of CYP1 Cyclophilin in Magnaporthe. In this model, CYP1 cyclophilin regulates virulence-associated functions, including penetration peg formation and cellular turgor generation, and also is required for efficient sporulation. Some of these functions may involve an interaction between CYP1 and calcineurin, perhaps to regulate calcineurin assembly and activity, but there are likely to be a number of other targets with which CYP1 interacts. The addition of exogenous CsA to Magnaporthe leads to the formation of a CYP1-CsA complex, which inactivates calcineurin and prevents calcium/calmodulin-dependent protein phosphatase signaling. Acute sensitivity to CsA indicates that calcineurin is required for appressorium morphogenesis in addition to regulating hyphal growth and development. CAM, calmodulin; CNA, calcineurin A catalytic subunit; CNB, calcineurin B regulatory subunit; CsA, cyclosporin A; CYP1, CYP1 -encoded cyclophilin.
    Figure Legend Snippet: Model Showing the Predicted Cellular Roles of CYP1 Cyclophilin in Magnaporthe. In this model, CYP1 cyclophilin regulates virulence-associated functions, including penetration peg formation and cellular turgor generation, and also is required for efficient sporulation. Some of these functions may involve an interaction between CYP1 and calcineurin, perhaps to regulate calcineurin assembly and activity, but there are likely to be a number of other targets with which CYP1 interacts. The addition of exogenous CsA to Magnaporthe leads to the formation of a CYP1-CsA complex, which inactivates calcineurin and prevents calcium/calmodulin-dependent protein phosphatase signaling. Acute sensitivity to CsA indicates that calcineurin is required for appressorium morphogenesis in addition to regulating hyphal growth and development. CAM, calmodulin; CNA, calcineurin A catalytic subunit; CNB, calcineurin B regulatory subunit; CsA, cyclosporin A; CYP1, CYP1 -encoded cyclophilin.

    Techniques Used: Activity Assay, Chick Chorioallantoic Membrane Assay

    CYP1 Encodes Two mRNA Transcripts and Is Highly Expressed in Vegetative Growth in Culture and during Rice Blast Disease. (A) Mycelial cultures of Magnaporthe Guy11 and the Δpmk1 ) were grown in rich growth medium (CM) for 48 hr, and mycelium was removed and transferred to growth medium lacking nitrate (−N) or glucose (−G) or supplemented with 0.4 M NaCl (hyperosmotic stress [HS]) or transferred to CM in the control experiment. Cultures were grown for another 24 hr, RNA was removed, and RNA gel blots were prepared. Blots were probed with a 1.5-kb EcoRI-XhoI insert from the CYP1 cDNA clone pMV1. Transcripts of ∼990 and ∼840 bp were detected. (B) RT-PCR amplification of CYP1 using gene-specific primer pairs. Total RNA was extracted from Magnaporthe and reverse transcribed to cDNA using an oligo(dT) primer. PCR was performed on the resulting cDNA templates (lanes 2, 4, 6, 8, 10, and 12) and genomic DNA templates (lanes 1, 3, 5 7, 9, and 11). Primer pairs were designed to amplify intron-containing fragments from genomic DNA and shorter intron-free amplicons from cDNA templates. Primer pair CC5-1 and CC3-1 specific to the shorter CYP1 transcript were predicted to amplify a 599-bp fragment from genomic DNA and a 400-bp fragment from cDNA, respectively. Primer pair MC5-1 and CC3-1 were predicted to amplify an 874-bp fragment from DNA and a 580-bp fragment from cDNA, respectively. Lanes 1 to 4, amplification from Magnaporthe wild-type strain Guy11; lanes 5 to 8, amplification from Δcyp1 strain TR33; lanes 9 to 12, amplification from CYP1-complemented Δcyp1 transformant MV42. The 200-bp amplicon is a fragment of the Magnaporthe actin gene that was amplified as a positive control from every template. (C) Twenty-day-old rice seedlings of cv CO-39 were inoculated with conidia of Magnaporthe strain Guy11, and rice blast disease was allowed to progress until symptoms became apparent 96 hr later. RNA was extracted from rice seedlings at 0, 24, 48, 72, and 96 hr. RNA gel blots were probed with a 1.5-kb EcoRI-XhoI insert from the CYP1 cDNA clone pMV1. An equivalent RNA gel blot of fractionated RNA from a Magnaporthe mycelial culture (CM) was hybridized to the same probe under the same conditions, and the autoradiograph was exposed for an equal period.
    Figure Legend Snippet: CYP1 Encodes Two mRNA Transcripts and Is Highly Expressed in Vegetative Growth in Culture and during Rice Blast Disease. (A) Mycelial cultures of Magnaporthe Guy11 and the Δpmk1 ) were grown in rich growth medium (CM) for 48 hr, and mycelium was removed and transferred to growth medium lacking nitrate (−N) or glucose (−G) or supplemented with 0.4 M NaCl (hyperosmotic stress [HS]) or transferred to CM in the control experiment. Cultures were grown for another 24 hr, RNA was removed, and RNA gel blots were prepared. Blots were probed with a 1.5-kb EcoRI-XhoI insert from the CYP1 cDNA clone pMV1. Transcripts of ∼990 and ∼840 bp were detected. (B) RT-PCR amplification of CYP1 using gene-specific primer pairs. Total RNA was extracted from Magnaporthe and reverse transcribed to cDNA using an oligo(dT) primer. PCR was performed on the resulting cDNA templates (lanes 2, 4, 6, 8, 10, and 12) and genomic DNA templates (lanes 1, 3, 5 7, 9, and 11). Primer pairs were designed to amplify intron-containing fragments from genomic DNA and shorter intron-free amplicons from cDNA templates. Primer pair CC5-1 and CC3-1 specific to the shorter CYP1 transcript were predicted to amplify a 599-bp fragment from genomic DNA and a 400-bp fragment from cDNA, respectively. Primer pair MC5-1 and CC3-1 were predicted to amplify an 874-bp fragment from DNA and a 580-bp fragment from cDNA, respectively. Lanes 1 to 4, amplification from Magnaporthe wild-type strain Guy11; lanes 5 to 8, amplification from Δcyp1 strain TR33; lanes 9 to 12, amplification from CYP1-complemented Δcyp1 transformant MV42. The 200-bp amplicon is a fragment of the Magnaporthe actin gene that was amplified as a positive control from every template. (C) Twenty-day-old rice seedlings of cv CO-39 were inoculated with conidia of Magnaporthe strain Guy11, and rice blast disease was allowed to progress until symptoms became apparent 96 hr later. RNA was extracted from rice seedlings at 0, 24, 48, 72, and 96 hr. RNA gel blots were probed with a 1.5-kb EcoRI-XhoI insert from the CYP1 cDNA clone pMV1. An equivalent RNA gel blot of fractionated RNA from a Magnaporthe mycelial culture (CM) was hybridized to the same probe under the same conditions, and the autoradiograph was exposed for an equal period.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Positive Control, Western Blot, Autoradiography

    CYP1 Mediates Sensitivity to CsA in Magnaporthe. (A) Vegetative growth inhibition of Magnaporthe by CsA was assayed by inoculating 3 × 10 3 conidia onto the surface of a CM agar plate. Filter paper discs containing CsA in 1% DMSO were placed on the culture, and the zones of growth inhibition were observed. The bar chart shows the diameter of the zone of inhibition after exposure to four concentrations of CsA. Each bar represents the mean of three independent replications of the experiment with three plates per experiment, and the error bars represent standard deviations. (B) Effect of CsA on appressorium development by Magnaporthe. Each bar represents the mean of three independent experiments, and the error bars represent standard deviations. (C) Magnaporthe appressorium formation on plastic cover slips after exposure to 10 μg/mL CsA by Guy11 and Δcyp1 mutant TR33. A dome-shaped appressorium is marked with an arrowhead. Bar = 10 μm.
    Figure Legend Snippet: CYP1 Mediates Sensitivity to CsA in Magnaporthe. (A) Vegetative growth inhibition of Magnaporthe by CsA was assayed by inoculating 3 × 10 3 conidia onto the surface of a CM agar plate. Filter paper discs containing CsA in 1% DMSO were placed on the culture, and the zones of growth inhibition were observed. The bar chart shows the diameter of the zone of inhibition after exposure to four concentrations of CsA. Each bar represents the mean of three independent replications of the experiment with three plates per experiment, and the error bars represent standard deviations. (B) Effect of CsA on appressorium development by Magnaporthe. Each bar represents the mean of three independent experiments, and the error bars represent standard deviations. (C) Magnaporthe appressorium formation on plastic cover slips after exposure to 10 μg/mL CsA by Guy11 and Δcyp1 mutant TR33. A dome-shaped appressorium is marked with an arrowhead. Bar = 10 μm.

    Techniques Used: Inhibition, Mutagenesis

    Cellular Distribution of Lipid Droplets during Appressorium Morphogenesis by Magnaporthe Strain Guy11 and the Δcyp1 Mutant TR33. ). Photographs were taken 0 and 12 hr after inoculation. Hoffman modulation contrast images (left) and epifluorescence images of Nile red–stained material (right) are presented. (A) and (B) Guy11 conidium at 0 hr. (C) and (D) Δcyp1 mutant TR33 conidium at 0 hr. (E) and (F) Guy11 developing appressoria after 12 hr. (G) and (H) Δcyp1 mutant TR33 developing appressoria after 12 hr. Bars = 10 μm for all panels.
    Figure Legend Snippet: Cellular Distribution of Lipid Droplets during Appressorium Morphogenesis by Magnaporthe Strain Guy11 and the Δcyp1 Mutant TR33. ). Photographs were taken 0 and 12 hr after inoculation. Hoffman modulation contrast images (left) and epifluorescence images of Nile red–stained material (right) are presented. (A) and (B) Guy11 conidium at 0 hr. (C) and (D) Δcyp1 mutant TR33 conidium at 0 hr. (E) and (F) Guy11 developing appressoria after 12 hr. (G) and (H) Δcyp1 mutant TR33 developing appressoria after 12 hr. Bars = 10 μm for all panels.

    Techniques Used: Mutagenesis, Staining

    Targeted Replacement of the CYP1 Gene Results in a Reduction in Virulence. (A) Restriction map of the CYP1 locus showing the orientation of the CYP1 open reading frame. A 4.3-kb XhoI fragment spanning the locus was subcloned to create pCYP1X, and a 1.1-kb PstI fragment, which contained the entire CYP1 open reading frame, was removed. This was replaced with a 1.0-kb PstI fragment containing a hygromycin B resistance gene cassette ( HPH ). The construct pΔcyp1 was linearized and transformed into Magnaporthe Guy11. Transformants were selected that had undergone homologous recombination. B, BamHI; H, HindIII; P, PstI; X, XhoI. (B) DNA gel blot analysis of pΔcyp1 Magnaporthe transformants. Genomic DNA was digested with BamHI and HindIII and probed with the 1.1-kb PstI CYP1 fragment (left) and the 1-kb HPH cassette (right). Lane 1, Guy11; lanes 2, 3, and 5, transformants showing ectopic insertion of pΔcyp1; lane 4, Δcyp1 transformant TR33. (C) Rice blast symptoms produced by Δcyp1 mutant TR33 and the isogenic wild-type strain Guy11 on rice cv CO-39. Leaves 1 and 2 were from plants that had been inoculated with a suspension of Guy11 conidia at a concentration of 10 4 conidia/mL. Leaf 3 was from a plant inoculated with a suspension of Guy11 conidia at a concentration of 10 5 conidia/mL. Leaves 4 and 5 were from plants that had been inoculated with a suspension of Δcyp1 mutant TR33 conidia at a concentration of 10 4 conidia/mL. Leaf 6 was from a plant inoculated with a suspension of Δcyp1 TR33 conidia at 10 5 conidia/mL.
    Figure Legend Snippet: Targeted Replacement of the CYP1 Gene Results in a Reduction in Virulence. (A) Restriction map of the CYP1 locus showing the orientation of the CYP1 open reading frame. A 4.3-kb XhoI fragment spanning the locus was subcloned to create pCYP1X, and a 1.1-kb PstI fragment, which contained the entire CYP1 open reading frame, was removed. This was replaced with a 1.0-kb PstI fragment containing a hygromycin B resistance gene cassette ( HPH ). The construct pΔcyp1 was linearized and transformed into Magnaporthe Guy11. Transformants were selected that had undergone homologous recombination. B, BamHI; H, HindIII; P, PstI; X, XhoI. (B) DNA gel blot analysis of pΔcyp1 Magnaporthe transformants. Genomic DNA was digested with BamHI and HindIII and probed with the 1.1-kb PstI CYP1 fragment (left) and the 1-kb HPH cassette (right). Lane 1, Guy11; lanes 2, 3, and 5, transformants showing ectopic insertion of pΔcyp1; lane 4, Δcyp1 transformant TR33. (C) Rice blast symptoms produced by Δcyp1 mutant TR33 and the isogenic wild-type strain Guy11 on rice cv CO-39. Leaves 1 and 2 were from plants that had been inoculated with a suspension of Guy11 conidia at a concentration of 10 4 conidia/mL. Leaf 3 was from a plant inoculated with a suspension of Guy11 conidia at a concentration of 10 5 conidia/mL. Leaves 4 and 5 were from plants that had been inoculated with a suspension of Δcyp1 mutant TR33 conidia at a concentration of 10 4 conidia/mL. Leaf 6 was from a plant inoculated with a suspension of Δcyp1 TR33 conidia at 10 5 conidia/mL.

    Techniques Used: Construct, Transformation Assay, Homologous Recombination, Western Blot, Produced, Mutagenesis, Concentration Assay

    Predicted Amino Acid Sequence and Phylogenetic Analysis of the Magnaporthe CYP1 Gene. (A) Alignment of the predicted CYP1 cyclophilin-encoding gene product with cyclophilins from Uromyces fabae , Tolypocladium niveum , Neurospora crassa , Fusarium sporotrichoides , Schizosaccharomyces pombe , Saccharomyces cerevisiae ( CPH1 and CYPC ), and Candida albicans ( CYPH ). Identical amino acids are highlighted against a black background, conserved residues are shown on a dark gray background, and similar amino acids are shown on a light gray background. CYP1 , T. niveum CYP , and the N. crassa CYPH genes contain N-terminal extensions encoding a mitochondrial form of the cyclophilin. (B) ). Branch strengths were tested by 100 repetitions of the bootstrap algorithm with branch swapping (numbers in parenthesis show the percentage bootstrap value). Lengths of branches are shown. The tree was rooted using the midpoint method. Abbreviations and numbers correspond to gene names and GenBank accession numbers, respectively. Organisms are as follows: Tn, T. niveum ; Ti, Tolypocladium inflatum ; Cs, Cordyceps subsessilis ; Fs, F. sporotrichoides ; Nc, N. crassa ; Bg, Botrytis cinerea ; Tm, Trichophyton mentagrophytes ; An, Aspergillus nidulans ; Af, Aspergillus fumigatus ; Mg, M. grisea ; Hs, Homo sapiens ; Cn, Cryptococcus neoformans ; Sc, S. cerevisiae ; Ca, Candida albicans ; Sp, S. pombe ; Mf, Malassezia furfur ; Myg, Mycosphaerella graminicola ; At, Arabidopsis thaliana ; Dm, Drosophila melanogaster ; Uf, U. fabae .
    Figure Legend Snippet: Predicted Amino Acid Sequence and Phylogenetic Analysis of the Magnaporthe CYP1 Gene. (A) Alignment of the predicted CYP1 cyclophilin-encoding gene product with cyclophilins from Uromyces fabae , Tolypocladium niveum , Neurospora crassa , Fusarium sporotrichoides , Schizosaccharomyces pombe , Saccharomyces cerevisiae ( CPH1 and CYPC ), and Candida albicans ( CYPH ). Identical amino acids are highlighted against a black background, conserved residues are shown on a dark gray background, and similar amino acids are shown on a light gray background. CYP1 , T. niveum CYP , and the N. crassa CYPH genes contain N-terminal extensions encoding a mitochondrial form of the cyclophilin. (B) ). Branch strengths were tested by 100 repetitions of the bootstrap algorithm with branch swapping (numbers in parenthesis show the percentage bootstrap value). Lengths of branches are shown. The tree was rooted using the midpoint method. Abbreviations and numbers correspond to gene names and GenBank accession numbers, respectively. Organisms are as follows: Tn, T. niveum ; Ti, Tolypocladium inflatum ; Cs, Cordyceps subsessilis ; Fs, F. sporotrichoides ; Nc, N. crassa ; Bg, Botrytis cinerea ; Tm, Trichophyton mentagrophytes ; An, Aspergillus nidulans ; Af, Aspergillus fumigatus ; Mg, M. grisea ; Hs, Homo sapiens ; Cn, Cryptococcus neoformans ; Sc, S. cerevisiae ; Ca, Candida albicans ; Sp, S. pombe ; Mf, Malassezia furfur ; Myg, Mycosphaerella graminicola ; At, Arabidopsis thaliana ; Dm, Drosophila melanogaster ; Uf, U. fabae .

    Techniques Used: Sequencing

    Ultrastructure of Conidia Produced by Magnaporthe Strain Guy11 and the Δcyp1 Mutant TR33. . (A) Median section of a three-celled conidium of Guy11 showing the distribution of numerous lipid bodies (arrowheads). A vacuole with heterogenous contents fills much of the central cell. (B) Median section of a three-celled conidium of the Δcyp1 mutant TR33 showing a large central vacuole and fewer lipid bodies, a small number of which are visible around the periphery of the central cell. Bar in (B) = 5 μm for (A) and (B) .
    Figure Legend Snippet: Ultrastructure of Conidia Produced by Magnaporthe Strain Guy11 and the Δcyp1 Mutant TR33. . (A) Median section of a three-celled conidium of Guy11 showing the distribution of numerous lipid bodies (arrowheads). A vacuole with heterogenous contents fills much of the central cell. (B) Median section of a three-celled conidium of the Δcyp1 mutant TR33 showing a large central vacuole and fewer lipid bodies, a small number of which are visible around the periphery of the central cell. Bar in (B) = 5 μm for (A) and (B) .

    Techniques Used: Produced, Mutagenesis

    5) Product Images from "Anti-inflammatory effects of sargachromenol-rich ethanolic extract of Myagropsis myagroides on lipopolysaccharide-stimulated BV-2 cells"

    Article Title: Anti-inflammatory effects of sargachromenol-rich ethanolic extract of Myagropsis myagroides on lipopolysaccharide-stimulated BV-2 cells

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-14-231

    Effect of MME on the translocation and activation of NF-κB in LPS-stimulated BV-2 cells. (A) Cells were treated with and without MME for 2 h followed by LPS stimulation for 30 min. NF-κB/p65 subunits were probed by anti-NF-κB antibody and Alexa Fluor® 488-conjugated secondary antibody. The nuclei were stained by DAPI and the images were captured by confocal microscopy (×40). (B) Cells pretreated with different concentrations of MME for 2 h were stimulated with LPS for 30 min. Cytosolic and nuclear extracts were prepared and analyzed using Western blot by using corresponding antibodies. (C) Cells were co-transfected with 2 μg of NF-κB promoter-containing luciferase DNA along with 40 ng of control pRL-TK DNA for 40 h. Transfected cells were pretreated with various concentrations of MME for 2 h and then stimulated with LPS for 6 h. Cell lysates were prepared and used for reporter gene assay. Data are means ± SDs of three independent experiments. a p
    Figure Legend Snippet: Effect of MME on the translocation and activation of NF-κB in LPS-stimulated BV-2 cells. (A) Cells were treated with and without MME for 2 h followed by LPS stimulation for 30 min. NF-κB/p65 subunits were probed by anti-NF-κB antibody and Alexa Fluor® 488-conjugated secondary antibody. The nuclei were stained by DAPI and the images were captured by confocal microscopy (×40). (B) Cells pretreated with different concentrations of MME for 2 h were stimulated with LPS for 30 min. Cytosolic and nuclear extracts were prepared and analyzed using Western blot by using corresponding antibodies. (C) Cells were co-transfected with 2 μg of NF-κB promoter-containing luciferase DNA along with 40 ng of control pRL-TK DNA for 40 h. Transfected cells were pretreated with various concentrations of MME for 2 h and then stimulated with LPS for 6 h. Cell lysates were prepared and used for reporter gene assay. Data are means ± SDs of three independent experiments. a p

    Techniques Used: Translocation Assay, Activation Assay, Staining, Confocal Microscopy, Western Blot, Transfection, Luciferase, Reporter Gene Assay

    6) Product Images from "Docosahexaenoic Acid Inhibits Cerulein-Induced Acute Pancreatitis in Rats"

    Article Title: Docosahexaenoic Acid Inhibits Cerulein-Induced Acute Pancreatitis in Rats

    Journal: Nutrients

    doi: 10.3390/nu9070744

    The effect of DHA on the serum level of IL-6, and the levels of IL-6 mRNA and protein in the pancreas. ( A ) The level of IL-6 in the serum was determined using ELISA ( B ) RT-PCR was performed on reverse-transcribed RNA isolated from the pancreatic tissue. The mRNA level of IL-6 was normalized to that of GAPDH; ( C ) The protein level of IL-6 in the pancreas was determined using ELISA and expressed as pg/mg protein. Values are mean ± S.E. for the 10 rats in each group. * p
    Figure Legend Snippet: The effect of DHA on the serum level of IL-6, and the levels of IL-6 mRNA and protein in the pancreas. ( A ) The level of IL-6 in the serum was determined using ELISA ( B ) RT-PCR was performed on reverse-transcribed RNA isolated from the pancreatic tissue. The mRNA level of IL-6 was normalized to that of GAPDH; ( C ) The protein level of IL-6 in the pancreas was determined using ELISA and expressed as pg/mg protein. Values are mean ± S.E. for the 10 rats in each group. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Isolation

    7) Product Images from "Low doses of cholera toxin and its mediator cAMP induce CTLA-2 secretion by dendritic cells to enhance regulatory T cell conversion"

    Article Title: Low doses of cholera toxin and its mediator cAMP induce CTLA-2 secretion by dendritic cells to enhance regulatory T cell conversion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0178114

    Cathepsin L expression in CT/cAMP-DCs does not influence iTreg conversion. (A) BM-DCs were differentially matured with the indicated stimuli for 10 h (CT lo 0.1 μg/ml; cAMP 100 μM; LPS 0.1 μg/ml). Total cell lysates were analyzed for mature Ctsl protein content by Western Blot. One representative experiment is shown. (B) Densitometric analysis of Western blot data of 3 mice per group from 3 independent experiments performed like the one shown in A, normalized to β-actin and relative to untreated control. (C) BM-DCs were stimulated with CT lo or cAMP for 4 h. CTLA-2α and -2β mRNA expression was determined by RT-sqPCR, representative experiment, (D) Densitometric analysis of mRNA data of 3 mice per group from 3 independent experiments performed like the one shown in C, normalized to β-actin and relative to WT untreated control. (E) OT-II T cells were co-cultured with WT or Ctsl -/- BM-DCs-treated with CT lo or cAMP for 4 h in the presence of 100 ng/ml OVA 327-339 peptide and 2 ng/ml TGF-β. After 5 days cells were analyzed by flow cytometry for Foxp3 and CD25 expression in a CD4 + Vβ5 + gate. Data represent the change in the frequency of iTreg conversion using Ctsl -/- DCs normalized to WT DCs of n = 5 experiments. Error bars represent mean ± SD. (B) One Way ANOVA, Dunnett post-test. (E) Two-tailed Student’s t test. ns: not significant.
    Figure Legend Snippet: Cathepsin L expression in CT/cAMP-DCs does not influence iTreg conversion. (A) BM-DCs were differentially matured with the indicated stimuli for 10 h (CT lo 0.1 μg/ml; cAMP 100 μM; LPS 0.1 μg/ml). Total cell lysates were analyzed for mature Ctsl protein content by Western Blot. One representative experiment is shown. (B) Densitometric analysis of Western blot data of 3 mice per group from 3 independent experiments performed like the one shown in A, normalized to β-actin and relative to untreated control. (C) BM-DCs were stimulated with CT lo or cAMP for 4 h. CTLA-2α and -2β mRNA expression was determined by RT-sqPCR, representative experiment, (D) Densitometric analysis of mRNA data of 3 mice per group from 3 independent experiments performed like the one shown in C, normalized to β-actin and relative to WT untreated control. (E) OT-II T cells were co-cultured with WT or Ctsl -/- BM-DCs-treated with CT lo or cAMP for 4 h in the presence of 100 ng/ml OVA 327-339 peptide and 2 ng/ml TGF-β. After 5 days cells were analyzed by flow cytometry for Foxp3 and CD25 expression in a CD4 + Vβ5 + gate. Data represent the change in the frequency of iTreg conversion using Ctsl -/- DCs normalized to WT DCs of n = 5 experiments. Error bars represent mean ± SD. (B) One Way ANOVA, Dunnett post-test. (E) Two-tailed Student’s t test. ns: not significant.

    Techniques Used: Expressing, Western Blot, Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, Two Tailed Test

    CT and cAMP induces both CTLA-2α and -2β during DC maturation. BM-DCs were differentially matured with the indicated stimuli for 16h unless otherwise specified. (A) Principal component analysis comparing untreated BM-DCs, DCs matured for 6h with the Th2-inducing stimuli TNF, the Trypanosoma brucei antigens Mitat or mVSG, and the Th1-inducing LPS from our previous study [ 17 ] with CT hi (1 μg/ml) matured Th17-inducing DCs investigated in this study. (B) Heat map displaying the top 25 genes regulated in DCs after CT hi or TNF stimulation compared with untreated DCs. Data show single time point values of a single microarray. (C) Densitometric analysis of CTLA-2α and -2β expression levels determined by RT-sqPCR normalized to β-actin and relative to untreated control for n = 4 experiments; CT lo (0.1 μg/ml). (D) CT hi stimulation of BM-DCs ± the cAMP inhibitor KH7 before RT-sqPCR normalized to β-actin of n = 3 independent and pooled experiments. (E) DC maturation analysis by flow cytometry of CD86 and MHC-II on CD11c positive cells. (F) Statistical evaluation of D with respect to untreated control for n = 5 experiments. (G) IL-1β, IL-6 and IL-23 secretion by differentially stimulated DCs measured by ELISA n = 3 experiments. Statistical analysis was performed with respect to untreated control. Error bars represent mean ± SD. One Way ANOVA, Dunnett post-test. *p
    Figure Legend Snippet: CT and cAMP induces both CTLA-2α and -2β during DC maturation. BM-DCs were differentially matured with the indicated stimuli for 16h unless otherwise specified. (A) Principal component analysis comparing untreated BM-DCs, DCs matured for 6h with the Th2-inducing stimuli TNF, the Trypanosoma brucei antigens Mitat or mVSG, and the Th1-inducing LPS from our previous study [ 17 ] with CT hi (1 μg/ml) matured Th17-inducing DCs investigated in this study. (B) Heat map displaying the top 25 genes regulated in DCs after CT hi or TNF stimulation compared with untreated DCs. Data show single time point values of a single microarray. (C) Densitometric analysis of CTLA-2α and -2β expression levels determined by RT-sqPCR normalized to β-actin and relative to untreated control for n = 4 experiments; CT lo (0.1 μg/ml). (D) CT hi stimulation of BM-DCs ± the cAMP inhibitor KH7 before RT-sqPCR normalized to β-actin of n = 3 independent and pooled experiments. (E) DC maturation analysis by flow cytometry of CD86 and MHC-II on CD11c positive cells. (F) Statistical evaluation of D with respect to untreated control for n = 5 experiments. (G) IL-1β, IL-6 and IL-23 secretion by differentially stimulated DCs measured by ELISA n = 3 experiments. Statistical analysis was performed with respect to untreated control. Error bars represent mean ± SD. One Way ANOVA, Dunnett post-test. *p

    Techniques Used: Microarray, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    8) Product Images from "Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages: Implications for calcification-related chronic inflammatory diseases"

    Article Title: Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages: Implications for calcification-related chronic inflammatory diseases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177829

    GRP is present in THP-1 MoM extracellular vesicles (EVs) at protein and mRNA levels. EVs were isolated from THP-1 MoM cell conditioned media by differential ultracentrifugation at 100.000 xg and characterized by TEM (A), Western blot for the exosomal marker CD9, GAPDH and total GRP (tGRP) (B), and qualitative analysis of GRP, MGP and GAPDH mRNA (C). Scale bar in panel (a) represents 200 nm.
    Figure Legend Snippet: GRP is present in THP-1 MoM extracellular vesicles (EVs) at protein and mRNA levels. EVs were isolated from THP-1 MoM cell conditioned media by differential ultracentrifugation at 100.000 xg and characterized by TEM (A), Western blot for the exosomal marker CD9, GAPDH and total GRP (tGRP) (B), and qualitative analysis of GRP, MGP and GAPDH mRNA (C). Scale bar in panel (a) represents 200 nm.

    Techniques Used: Isolation, Transmission Electron Microscopy, Western Blot, Marker

    Overexpression of GRP in THP-1 cells rescues hydroxyapatite induced inflammation. THP-1 cells were transiently transfected with GRP mRNA for 24 h and either maintained in control conditions (C, T) or stimulated with HA (C-HA, T-HA) for additional 48 h. Gene expression of GRP (A) and the inflammatory marker genes NFkB (B) and TNFα (C) were determined by qPCR (A), and levels of TNFα accumulation were measured by ELISA in conditioned culture media (D). (A-C) Data are presented as means (n = 6) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Tukey's test. Statistical significance was defined as P
    Figure Legend Snippet: Overexpression of GRP in THP-1 cells rescues hydroxyapatite induced inflammation. THP-1 cells were transiently transfected with GRP mRNA for 24 h and either maintained in control conditions (C, T) or stimulated with HA (C-HA, T-HA) for additional 48 h. Gene expression of GRP (A) and the inflammatory marker genes NFkB (B) and TNFα (C) were determined by qPCR (A), and levels of TNFα accumulation were measured by ELISA in conditioned culture media (D). (A-C) Data are presented as means (n = 6) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Tukey's test. Statistical significance was defined as P

    Techniques Used: Over Expression, Transfection, Expressing, Marker, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    GRP and MGP are up-regulated in hydroxyapatite stimulated THP-1 MoM cells. Differentiated THP-1 macrophage cells were stimulated with 250 μg/ml of synthetic hydroxyapatite nano-crystals and harvested for RNA extraction at determined time points during 12h. Relative gene expression analysis of the inflammatory marker IL-1β, GRP and MGP was determined by qPCR at indicated time points. Gene expression levels are relative to the expression of control untreated cells during each time point. GAPDH was used as housekeeping gene and data is presented as means (n = 3) ± standard error of duplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Tukey's test. Statistical significance was defined as P
    Figure Legend Snippet: GRP and MGP are up-regulated in hydroxyapatite stimulated THP-1 MoM cells. Differentiated THP-1 macrophage cells were stimulated with 250 μg/ml of synthetic hydroxyapatite nano-crystals and harvested for RNA extraction at determined time points during 12h. Relative gene expression analysis of the inflammatory marker IL-1β, GRP and MGP was determined by qPCR at indicated time points. Gene expression levels are relative to the expression of control untreated cells during each time point. GAPDH was used as housekeeping gene and data is presented as means (n = 3) ± standard error of duplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Tukey's test. Statistical significance was defined as P

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

    Coating of BCPs with GRP reduces TNFα production in THP-1 MoM when compared with naked crystals. Differentiated THP-1 MoM cells were treated with 100 μg/ml of BCP crystals or BCPs-coated with cGRP/ucGRP (PMC/cGRP; PMC/ucGRP) proteins for 24 h, and non-stimulated cells were used as control (C). Control non-stimulated cells were also treated with 1.5 μg/ml of cGRP/ucGRP proteins for comparison. Conditioned media were collected and used to determine TNFα accumulation through ELISA assays. Data are presented as means (n = 3) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Dunnett's test. Statistical significance was defined as P
    Figure Legend Snippet: Coating of BCPs with GRP reduces TNFα production in THP-1 MoM when compared with naked crystals. Differentiated THP-1 MoM cells were treated with 100 μg/ml of BCP crystals or BCPs-coated with cGRP/ucGRP (PMC/cGRP; PMC/ucGRP) proteins for 24 h, and non-stimulated cells were used as control (C). Control non-stimulated cells were also treated with 1.5 μg/ml of cGRP/ucGRP proteins for comparison. Conditioned media were collected and used to determine TNFα accumulation through ELISA assays. Data are presented as means (n = 3) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Dunnett's test. Statistical significance was defined as P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    γ-carboxylated GRP and MGP are produced in THP-1 cell line. THP-1 and THP-1 MoM differentiated with 25 ng/ml of PMA during 48h were cultured in control conditions and harvested for RNA and protein extraction. (A) Qualitative gene expression analysis of GRP, MGP, VKOR and GGCX by RT-PCR in undifferentiated THP-1 representing monocyte cells (THP-1), and in differentiated THP-1 representing macrophages (THP-1 MoM). (B) Western blot analysis of thirty μg of total RIPA protein extracts of THP-1 and THP-1 MoM cells using the conformation-specific antibodies recognizing γ-carboxylated GRP (cGRP) and MGP (cMGP). Position of relevant molecular mass markers (kDa) is indicated on the right side.
    Figure Legend Snippet: γ-carboxylated GRP and MGP are produced in THP-1 cell line. THP-1 and THP-1 MoM differentiated with 25 ng/ml of PMA during 48h were cultured in control conditions and harvested for RNA and protein extraction. (A) Qualitative gene expression analysis of GRP, MGP, VKOR and GGCX by RT-PCR in undifferentiated THP-1 representing monocyte cells (THP-1), and in differentiated THP-1 representing macrophages (THP-1 MoM). (B) Western blot analysis of thirty μg of total RIPA protein extracts of THP-1 and THP-1 MoM cells using the conformation-specific antibodies recognizing γ-carboxylated GRP (cGRP) and MGP (cMGP). Position of relevant molecular mass markers (kDa) is indicated on the right side.

    Techniques Used: Produced, Cell Culture, Protein Extraction, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Overexpression of GRP in THP-1 cells rescues LPS induced inflammation. THP-1 cells were transiently transfected with GRP mRNA during 24, 48 and 72 h, and GRP overexpression was evaluated by measuring levels of GRP mRNA (A) and protein production (B). (A) GRP Normalized expression was determined by qPCR in non-transfected (C) and transfected (T) cells at the indicated time points. Data are presented as means (n = 3) ± standard error of duplicates of two independent experiments. Student’s t-test was used for comparison between C and T groups at each time point. Statistical significance was defined as P
    Figure Legend Snippet: Overexpression of GRP in THP-1 cells rescues LPS induced inflammation. THP-1 cells were transiently transfected with GRP mRNA during 24, 48 and 72 h, and GRP overexpression was evaluated by measuring levels of GRP mRNA (A) and protein production (B). (A) GRP Normalized expression was determined by qPCR in non-transfected (C) and transfected (T) cells at the indicated time points. Data are presented as means (n = 3) ± standard error of duplicates of two independent experiments. Student’s t-test was used for comparison between C and T groups at each time point. Statistical significance was defined as P

    Techniques Used: Over Expression, Transfection, Expressing, Real-time Polymerase Chain Reaction

    GRP reduces TNFα and PGE2 production in THP-1 MoM cells stimulated with LPS. (A) Differentiated THP-1 MoM cells were treated with 0.5 μg/ml, 0.75 μg/ml and 1.5 μg/ml of purified cGRP and ucGRP proteins for 24 h, followed by exposure to 50 ng/ml LPS for additional 24 h. Cells treated with 2 μM dexamethasone (DXM) were used as a positive anti-inflammatory control, and non-stimulated cells (C) as controls to LPS stimulation. Conditioned cell culture media were collected and used to determine TNFα accumulation by ELISA assays. Data are presented as means (n = 3) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Dunnett's test. Statistical significance was defined as P
    Figure Legend Snippet: GRP reduces TNFα and PGE2 production in THP-1 MoM cells stimulated with LPS. (A) Differentiated THP-1 MoM cells were treated with 0.5 μg/ml, 0.75 μg/ml and 1.5 μg/ml of purified cGRP and ucGRP proteins for 24 h, followed by exposure to 50 ng/ml LPS for additional 24 h. Cells treated with 2 μM dexamethasone (DXM) were used as a positive anti-inflammatory control, and non-stimulated cells (C) as controls to LPS stimulation. Conditioned cell culture media were collected and used to determine TNFα accumulation by ELISA assays. Data are presented as means (n = 3) ± standard error of triplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Dunnett's test. Statistical significance was defined as P

    Techniques Used: Purification, Cell Culture, Enzyme-linked Immunosorbent Assay

    GRP and MGP are up-regulated in LPS stimulated THP-1 cells. THP-1 and differentiated THP-1 macrophage cells were stimulated with 100 ng/ml of LPS and harvested for RNA extraction at determined time points during 12h. Relative gene expression analysis of the inflammatory marker IL-1β, GRP, MGP and GGCX was determined by qPCR in monocytes (THP-1 cells) (A) and in differentiated THP-1 macrophages (B) at indicated time points. Gene expression levels are relative to the expression of control untreated cells during each time point. GAPDH was used as housekeeping gene and data is presented as means (n = 4) ± standard error of duplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Tukey's test. Statistical significance was defined as P
    Figure Legend Snippet: GRP and MGP are up-regulated in LPS stimulated THP-1 cells. THP-1 and differentiated THP-1 macrophage cells were stimulated with 100 ng/ml of LPS and harvested for RNA extraction at determined time points during 12h. Relative gene expression analysis of the inflammatory marker IL-1β, GRP, MGP and GGCX was determined by qPCR in monocytes (THP-1 cells) (A) and in differentiated THP-1 macrophages (B) at indicated time points. Gene expression levels are relative to the expression of control untreated cells during each time point. GAPDH was used as housekeeping gene and data is presented as means (n = 4) ± standard error of duplicates of two independent experiments. Ordinary one-way ANOVA was used and multiple comparisons were achieved with Tukey's test. Statistical significance was defined as P

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

    9) Product Images from "PcaO Positively Regulates pcaHG of the ?-Ketoadipate Pathway in Corynebacterium glutamicum ▿"

    Article Title: PcaO Positively Regulates pcaHG of the ?-Ketoadipate Pathway in Corynebacterium glutamicum ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01338-09

    Determination of the transcription start site (TSS) of  pcaHG  (A) and diagram of intergenic sequences between  pcaHG  and  ncgl2316  (B). In panel A, the transcription start base C is indicated by a triangle. Primer extension was performed using DNase I-treated RNA (10 μg) isolated from cells cultivated in mineral salts medium containing protocatechuate. The labeled primer (2315PE) was complementary to a sequence 96 bp downstream of the translation start codon of  pcaH . The sequencing products of  PpcaHG  obtained with the labeled primer (lanes G, A, C, and T) were simultaneously electrophoresed with the reverse transcribed products. In panel B, the palindromic sequences (Rep1, Rep2, Rep3, and Rep4), putative ribosome binding site (RBS), and putative −10 and −35 regions are indicated.
    Figure Legend Snippet: Determination of the transcription start site (TSS) of pcaHG (A) and diagram of intergenic sequences between pcaHG and ncgl2316 (B). In panel A, the transcription start base C is indicated by a triangle. Primer extension was performed using DNase I-treated RNA (10 μg) isolated from cells cultivated in mineral salts medium containing protocatechuate. The labeled primer (2315PE) was complementary to a sequence 96 bp downstream of the translation start codon of pcaH . The sequencing products of PpcaHG obtained with the labeled primer (lanes G, A, C, and T) were simultaneously electrophoresed with the reverse transcribed products. In panel B, the palindromic sequences (Rep1, Rep2, Rep3, and Rep4), putative ribosome binding site (RBS), and putative −10 and −35 regions are indicated.

    Techniques Used: Isolation, Labeling, Sequencing, Binding Assay

    10) Product Images from "A Magnaporthe grisea Cyclophilin Acts as a Virulence Determinant during Plant Infection"

    Article Title: A Magnaporthe grisea Cyclophilin Acts as a Virulence Determinant during Plant Infection

    Journal: The Plant Cell

    doi: 10.1105/tpc.010389

    CYP1 Encodes Two mRNA Transcripts and Is Highly Expressed in Vegetative Growth in Culture and during Rice Blast Disease. (A) Mycelial cultures of Magnaporthe Guy11 and the Δpmk1 ) were grown in rich growth medium (CM) for 48 hr, and mycelium was removed and transferred to growth medium lacking nitrate (−N) or glucose (−G) or supplemented with 0.4 M NaCl (hyperosmotic stress [HS]) or transferred to CM in the control experiment. Cultures were grown for another 24 hr, RNA was removed, and RNA gel blots were prepared. Blots were probed with a 1.5-kb EcoRI-XhoI insert from the CYP1 cDNA clone pMV1. Transcripts of ∼990 and ∼840 bp were detected. (B) RT-PCR amplification of CYP1 using gene-specific primer pairs. Total RNA was extracted from Magnaporthe and reverse transcribed to cDNA using an oligo(dT) primer. PCR was performed on the resulting cDNA templates (lanes 2, 4, 6, 8, 10, and 12) and genomic DNA templates (lanes 1, 3, 5 7, 9, and 11). Primer pairs were designed to amplify intron-containing fragments from genomic DNA and shorter intron-free amplicons from cDNA templates. Primer pair CC5-1 and CC3-1 specific to the shorter CYP1 transcript were predicted to amplify a 599-bp fragment from genomic DNA and a 400-bp fragment from cDNA, respectively. Primer pair MC5-1 and CC3-1 were predicted to amplify an 874-bp fragment from DNA and a 580-bp fragment from cDNA, respectively. Lanes 1 to 4, amplification from Magnaporthe wild-type strain Guy11; lanes 5 to 8, amplification from Δcyp1 strain TR33; lanes 9 to 12, amplification from CYP1-complemented Δcyp1 transformant MV42. The 200-bp amplicon is a fragment of the Magnaporthe actin gene that was amplified as a positive control from every template. (C) Twenty-day-old rice seedlings of cv CO-39 were inoculated with conidia of Magnaporthe strain Guy11, and rice blast disease was allowed to progress until symptoms became apparent 96 hr later. RNA was extracted from rice seedlings at 0, 24, 48, 72, and 96 hr. RNA gel blots were probed with a 1.5-kb EcoRI-XhoI insert from the CYP1 cDNA clone pMV1. An equivalent RNA gel blot of fractionated RNA from a Magnaporthe mycelial culture (CM) was hybridized to the same probe under the same conditions, and the autoradiograph was exposed for an equal period.
    Figure Legend Snippet: CYP1 Encodes Two mRNA Transcripts and Is Highly Expressed in Vegetative Growth in Culture and during Rice Blast Disease. (A) Mycelial cultures of Magnaporthe Guy11 and the Δpmk1 ) were grown in rich growth medium (CM) for 48 hr, and mycelium was removed and transferred to growth medium lacking nitrate (−N) or glucose (−G) or supplemented with 0.4 M NaCl (hyperosmotic stress [HS]) or transferred to CM in the control experiment. Cultures were grown for another 24 hr, RNA was removed, and RNA gel blots were prepared. Blots were probed with a 1.5-kb EcoRI-XhoI insert from the CYP1 cDNA clone pMV1. Transcripts of ∼990 and ∼840 bp were detected. (B) RT-PCR amplification of CYP1 using gene-specific primer pairs. Total RNA was extracted from Magnaporthe and reverse transcribed to cDNA using an oligo(dT) primer. PCR was performed on the resulting cDNA templates (lanes 2, 4, 6, 8, 10, and 12) and genomic DNA templates (lanes 1, 3, 5 7, 9, and 11). Primer pairs were designed to amplify intron-containing fragments from genomic DNA and shorter intron-free amplicons from cDNA templates. Primer pair CC5-1 and CC3-1 specific to the shorter CYP1 transcript were predicted to amplify a 599-bp fragment from genomic DNA and a 400-bp fragment from cDNA, respectively. Primer pair MC5-1 and CC3-1 were predicted to amplify an 874-bp fragment from DNA and a 580-bp fragment from cDNA, respectively. Lanes 1 to 4, amplification from Magnaporthe wild-type strain Guy11; lanes 5 to 8, amplification from Δcyp1 strain TR33; lanes 9 to 12, amplification from CYP1-complemented Δcyp1 transformant MV42. The 200-bp amplicon is a fragment of the Magnaporthe actin gene that was amplified as a positive control from every template. (C) Twenty-day-old rice seedlings of cv CO-39 were inoculated with conidia of Magnaporthe strain Guy11, and rice blast disease was allowed to progress until symptoms became apparent 96 hr later. RNA was extracted from rice seedlings at 0, 24, 48, 72, and 96 hr. RNA gel blots were probed with a 1.5-kb EcoRI-XhoI insert from the CYP1 cDNA clone pMV1. An equivalent RNA gel blot of fractionated RNA from a Magnaporthe mycelial culture (CM) was hybridized to the same probe under the same conditions, and the autoradiograph was exposed for an equal period.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Positive Control, Western Blot, Autoradiography

    11) Product Images from "Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases *"

    Article Title: Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.700070

    Synthesized CDR3δ peptides SL1 and SL2 did not specifically bind to the plasmas and PBMCs of SLE patients. A , ELISA assay of SL1 and SL2 peptides binding to the plasmas of SLE patients and controls. SL1-Vm and SL2-Vm are control CDR3δ
    Figure Legend Snippet: Synthesized CDR3δ peptides SL1 and SL2 did not specifically bind to the plasmas and PBMCs of SLE patients. A , ELISA assay of SL1 and SL2 peptides binding to the plasmas of SLE patients and controls. SL1-Vm and SL2-Vm are control CDR3δ

    Techniques Used: Synthesized, Enzyme-linked Immunosorbent Assay, Binding Assay

    12) Product Images from "Anti-inflammatory effects of sargachromenol-rich ethanolic extract of Myagropsis myagroides on lipopolysaccharide-stimulated BV-2 cells"

    Article Title: Anti-inflammatory effects of sargachromenol-rich ethanolic extract of Myagropsis myagroides on lipopolysaccharide-stimulated BV-2 cells

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-14-231

    Effect of MME on the translocation and activation of NF-κB in LPS-stimulated BV-2 cells. (A) Cells were treated with and without MME for 2 h followed by LPS stimulation for 30 min. NF-κB/p65 subunits were probed by anti-NF-κB antibody and Alexa Fluor® 488-conjugated secondary antibody. The nuclei were stained by DAPI and the images were captured by confocal microscopy (×40). (B) Cells pretreated with different concentrations of MME for 2 h were stimulated with LPS for 30 min. Cytosolic and nuclear extracts were prepared and analyzed using Western blot by using corresponding antibodies. (C) Cells were co-transfected with 2 μg of NF-κB promoter-containing luciferase DNA along with 40 ng of control pRL-TK DNA for 40 h. Transfected cells were pretreated with various concentrations of MME for 2 h and then stimulated with LPS for 6 h. Cell lysates were prepared and used for reporter gene assay. Data are means ± SDs of three independent experiments. a p
    Figure Legend Snippet: Effect of MME on the translocation and activation of NF-κB in LPS-stimulated BV-2 cells. (A) Cells were treated with and without MME for 2 h followed by LPS stimulation for 30 min. NF-κB/p65 subunits were probed by anti-NF-κB antibody and Alexa Fluor® 488-conjugated secondary antibody. The nuclei were stained by DAPI and the images were captured by confocal microscopy (×40). (B) Cells pretreated with different concentrations of MME for 2 h were stimulated with LPS for 30 min. Cytosolic and nuclear extracts were prepared and analyzed using Western blot by using corresponding antibodies. (C) Cells were co-transfected with 2 μg of NF-κB promoter-containing luciferase DNA along with 40 ng of control pRL-TK DNA for 40 h. Transfected cells were pretreated with various concentrations of MME for 2 h and then stimulated with LPS for 6 h. Cell lysates were prepared and used for reporter gene assay. Data are means ± SDs of three independent experiments. a p

    Techniques Used: Translocation Assay, Activation Assay, Staining, Confocal Microscopy, Western Blot, Transfection, Luciferase, Reporter Gene Assay

    13) Product Images from "Varicella-Zoster Virus (VZV) Small Noncoding RNAs Antisense to the VZV Latency-Encoded Transcript VLT Enhance Viral Replication"

    Article Title: Varicella-Zoster Virus (VZV) Small Noncoding RNAs Antisense to the VZV Latency-Encoded Transcript VLT Enhance Viral Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.00123-20

    Method used for measuring growth of VZV infectious foci (FOI) in ARPE-19 cells. Ten to thirty ARPE-19 cells infected with VZV66RFP were added to 90% confluent wells of 96-well plates. At 24 h postinfection, cells were transfected either with an LNA antagonist to a scrambled RNA (A and B) or with VZVsncRNA9 (C and D). The RFP fluorescence of entire wells was photographed with an automated microscope, and the micrographs were assembled into a single image. (E) A single fluorescent VZV FOI after image processing. (F) FOI in panel E after application of a threshold for fluorescence (and inversion of the pixel luminance values): the black pixels represent the fluorescence indicating the presence of the virus. The yellow line demarcates the area within which black pixels were counted by the computer in order to generate a value for infection in an FOI. See Materials and Methods for more details. (G to L) Growth of a single focus of infection and its quantification. (G to J) Single FOI at 1 (blue), 2 (magenta), 3 (yellow), and 5 (green) days after transfection. (K) Images were pseudocolored and overlaid in order to visualize the FOI growth. (L) Graph of the quantification of this FOI on these days. Bars, 1 mm (A to D), 250 μm (E and F), and 100 μm (G to K).
    Figure Legend Snippet: Method used for measuring growth of VZV infectious foci (FOI) in ARPE-19 cells. Ten to thirty ARPE-19 cells infected with VZV66RFP were added to 90% confluent wells of 96-well plates. At 24 h postinfection, cells were transfected either with an LNA antagonist to a scrambled RNA (A and B) or with VZVsncRNA9 (C and D). The RFP fluorescence of entire wells was photographed with an automated microscope, and the micrographs were assembled into a single image. (E) A single fluorescent VZV FOI after image processing. (F) FOI in panel E after application of a threshold for fluorescence (and inversion of the pixel luminance values): the black pixels represent the fluorescence indicating the presence of the virus. The yellow line demarcates the area within which black pixels were counted by the computer in order to generate a value for infection in an FOI. See Materials and Methods for more details. (G to L) Growth of a single focus of infection and its quantification. (G to J) Single FOI at 1 (blue), 2 (magenta), 3 (yellow), and 5 (green) days after transfection. (K) Images were pseudocolored and overlaid in order to visualize the FOI growth. (L) Graph of the quantification of this FOI on these days. Bars, 1 mm (A to D), 250 μm (E and F), and 100 μm (G to K).

    Techniques Used: Infection, Transfection, Fluorescence, Microscopy

    Administration of an LNAA to VZVsncRNA9 increases VZV FOI growth and number of infectious plaques. (A) The growth of FOI 1 to 5 days after transfection of an LNAA to VZVsncRNA9 (black bars) compared to control scrambled LNA RNA (gray bars) in a typical experiment is shown in arbitrary (pixel) units. A difference was observed at 2 dpt and became significant by 3 dpt. (B) Average increase in size of individual FOI between 1 and 5 dpt for each of the three experiments performed. (C) The transfection of an antagonist to VZVsncRNA9 increased the growth of FOI by an average of 55% for the three experiments. (D) Progeny virus yields quantified by plaque assays of cells harvested from the LNA antagonist and scrambled LNA RNA-transfected wells 7 dpt. The antagonist increased the plaque count compared to scrambled RNA by an average of 79% ( n = 3). Asterisks indicate statistical significance between control and LNAA-transfected wells (*, P
    Figure Legend Snippet: Administration of an LNAA to VZVsncRNA9 increases VZV FOI growth and number of infectious plaques. (A) The growth of FOI 1 to 5 days after transfection of an LNAA to VZVsncRNA9 (black bars) compared to control scrambled LNA RNA (gray bars) in a typical experiment is shown in arbitrary (pixel) units. A difference was observed at 2 dpt and became significant by 3 dpt. (B) Average increase in size of individual FOI between 1 and 5 dpt for each of the three experiments performed. (C) The transfection of an antagonist to VZVsncRNA9 increased the growth of FOI by an average of 55% for the three experiments. (D) Progeny virus yields quantified by plaque assays of cells harvested from the LNA antagonist and scrambled LNA RNA-transfected wells 7 dpt. The antagonist increased the plaque count compared to scrambled RNA by an average of 79% ( n = 3). Asterisks indicate statistical significance between control and LNAA-transfected wells (*, P

    Techniques Used: Transfection

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