quantitative real time pcr analysis total rna  (Thermo Fisher)


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    Thermo Fisher quantitative real time pcr analysis total rna
    Kinetics of cytokine production (A) and mRNA expression (B) in mouse macrophages. Macrophages from WT mice and TLR2-deficient mice were untreated or treated with heat-killed S. aureus (MOI of 5), and then culture supernatants were harvested at different time intervals and assayed for TNF-α, IL-6 and IL-10 concentrations using ELISAs. Total <t>RNA</t> in these cells was also isolated and analyzed using real-time <t>PCR</t> for the mRNA expression of the cytokines at different time points. The results were normalized to GAPDH gene expression and are shown as fold changes relative to gene expression in untreated control cells. Data are the mean ± SD from 3 independent experiments. * p
    Quantitative Real Time Pcr Analysis Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 591 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Contribution of Toll-Like Receptor 2 to the Innate Response against Staphylococcus aureus Infection in Mice"

    Article Title: Contribution of Toll-Like Receptor 2 to the Innate Response against Staphylococcus aureus Infection in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074287

    Kinetics of cytokine production (A) and mRNA expression (B) in mouse macrophages. Macrophages from WT mice and TLR2-deficient mice were untreated or treated with heat-killed S. aureus (MOI of 5), and then culture supernatants were harvested at different time intervals and assayed for TNF-α, IL-6 and IL-10 concentrations using ELISAs. Total RNA in these cells was also isolated and analyzed using real-time PCR for the mRNA expression of the cytokines at different time points. The results were normalized to GAPDH gene expression and are shown as fold changes relative to gene expression in untreated control cells. Data are the mean ± SD from 3 independent experiments. * p
    Figure Legend Snippet: Kinetics of cytokine production (A) and mRNA expression (B) in mouse macrophages. Macrophages from WT mice and TLR2-deficient mice were untreated or treated with heat-killed S. aureus (MOI of 5), and then culture supernatants were harvested at different time intervals and assayed for TNF-α, IL-6 and IL-10 concentrations using ELISAs. Total RNA in these cells was also isolated and analyzed using real-time PCR for the mRNA expression of the cytokines at different time points. The results were normalized to GAPDH gene expression and are shown as fold changes relative to gene expression in untreated control cells. Data are the mean ± SD from 3 independent experiments. * p

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

    2) Product Images from "Functional Redundancy of Two Pax-Like Proteins in Transcriptional Activation of Cyst Wall Protein Genes in Giardia lamblia"

    Article Title: Functional Redundancy of Two Pax-Like Proteins in Transcriptional Activation of Cyst Wall Protein Genes in Giardia lamblia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030614

    Recruitment of Pax2 to the cwp1-3 and myb2 promoters. (A) Microarray analysis. Microarray data were obtained from the 5′▵5N-Pac and pPPax1 (or pPPax2) cell lines during vegetative growth. Fold changes are shown as the ratio of transcript levels in the pPPax1 (or pPPax2) cell line relative to the 5′▵5N-Pac cell line. Results are expressed as the means ± standard error of at least three separate experiments. (B) Pax2 and Pax1 overexpression generated similar gene expression patterns. The Venn diagrams illustrate the overlap of altered gene expression between the Pax2 and Pax1 overexpressing cells. Thirty eight and 185 genes were up-regulated (i.e. increased levels of gene expression relative to the control) in the Pax1 and Pax2 overexpressing cells, respectively. Among them, nineteen genes overlap. Fifty four and 172 genes were down-regulated in the Pax1 and Pax2 overexpressing cells, respectively. Among them, thirty genes overlap. (C) ChIP assays. The non-transfected WB cells were cultured in growth medium for 24 h and then subjected to ChIP assays. Anti-Pax2 antibody was used to assess binding of Pax2 to endogenous gene promoters. Preimmune serum was used as a negative control. Immunoprecipitated chromatin was analyzed by PCR using primers that amplify the 5′-flanking region of specific genes. At least three independent experiments were performed. Representative results are shown. Immunoprecipitated products of Pax2 yielded more PCR products of pax2 , cwp1 , cwp2 , cwp3 , myb2 , and ran promoters, indicating that Pax2 was bound to these promoters. The 18S ribosomal RNA gene promoter was used as a negative control for our ChIP analysis.
    Figure Legend Snippet: Recruitment of Pax2 to the cwp1-3 and myb2 promoters. (A) Microarray analysis. Microarray data were obtained from the 5′▵5N-Pac and pPPax1 (or pPPax2) cell lines during vegetative growth. Fold changes are shown as the ratio of transcript levels in the pPPax1 (or pPPax2) cell line relative to the 5′▵5N-Pac cell line. Results are expressed as the means ± standard error of at least three separate experiments. (B) Pax2 and Pax1 overexpression generated similar gene expression patterns. The Venn diagrams illustrate the overlap of altered gene expression between the Pax2 and Pax1 overexpressing cells. Thirty eight and 185 genes were up-regulated (i.e. increased levels of gene expression relative to the control) in the Pax1 and Pax2 overexpressing cells, respectively. Among them, nineteen genes overlap. Fifty four and 172 genes were down-regulated in the Pax1 and Pax2 overexpressing cells, respectively. Among them, thirty genes overlap. (C) ChIP assays. The non-transfected WB cells were cultured in growth medium for 24 h and then subjected to ChIP assays. Anti-Pax2 antibody was used to assess binding of Pax2 to endogenous gene promoters. Preimmune serum was used as a negative control. Immunoprecipitated chromatin was analyzed by PCR using primers that amplify the 5′-flanking region of specific genes. At least three independent experiments were performed. Representative results are shown. Immunoprecipitated products of Pax2 yielded more PCR products of pax2 , cwp1 , cwp2 , cwp3 , myb2 , and ran promoters, indicating that Pax2 was bound to these promoters. The 18S ribosomal RNA gene promoter was used as a negative control for our ChIP analysis.

    Techniques Used: Microarray, Over Expression, Generated, Expressing, Chromatin Immunoprecipitation, Transfection, Western Blot, Cell Culture, Binding Assay, Negative Control, Immunoprecipitation, Polymerase Chain Reaction

    Interaction between ERK1 and Pax2 (or Pax1). (A) Co-immunoprecipitation assay. The 5′Δ5N-Pac, pPERK1, and pPERK1m stable transfectants were cultured in encystation medium for 24 h. Proteins from cell lysates were immunoprecipitated using anti-HA antibody conjugated to beads. The precipitates were analyzed by Western blot with anti-HA, anti-Pax2, or anti-Pax1 antibody as indicated. (B) Expression of HA tagged ERK1, Pax2, and Pax1 proteins in whole cell extracts. The 5′Δ5N-Pac, pPERK1, and pPERK1m stable transfectants were cultured in encystation medium for 24 h (Enc, encystation) and then subjected to Western blot analysis. The blot was probed by anti-HA, anti-Pax2, anti-Pax1, and anti-RAN antibody. Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. (C) RT-PCR analysis of gene expression in the ERK1- and ERK1m-overexpressing cell line. The 5′Δ5N-Pac, pPERK1, and pPERK1m stable transfectants were cultured in encystation medium for 24 h (Enc, encystation) and then subjected to RT-PCR analysis. PCR was performed using primers specific for pax2 , pax1 , ran , and 18S ribosomal RNA genes.
    Figure Legend Snippet: Interaction between ERK1 and Pax2 (or Pax1). (A) Co-immunoprecipitation assay. The 5′Δ5N-Pac, pPERK1, and pPERK1m stable transfectants were cultured in encystation medium for 24 h. Proteins from cell lysates were immunoprecipitated using anti-HA antibody conjugated to beads. The precipitates were analyzed by Western blot with anti-HA, anti-Pax2, or anti-Pax1 antibody as indicated. (B) Expression of HA tagged ERK1, Pax2, and Pax1 proteins in whole cell extracts. The 5′Δ5N-Pac, pPERK1, and pPERK1m stable transfectants were cultured in encystation medium for 24 h (Enc, encystation) and then subjected to Western blot analysis. The blot was probed by anti-HA, anti-Pax2, anti-Pax1, and anti-RAN antibody. Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. (C) RT-PCR analysis of gene expression in the ERK1- and ERK1m-overexpressing cell line. The 5′Δ5N-Pac, pPERK1, and pPERK1m stable transfectants were cultured in encystation medium for 24 h (Enc, encystation) and then subjected to RT-PCR analysis. PCR was performed using primers specific for pax2 , pax1 , ran , and 18S ribosomal RNA genes.

    Techniques Used: Co-Immunoprecipitation Assay, Cell Culture, Immunoprecipitation, Western Blot, Expressing, SDS Page, Staining, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Activation of cwp1-3 and myb2 gene expression in the Pax2 overexpressing cell line. (A) Overexpression of Pax2 increased the levels of CWP1 protein. The 5′▵5N-Pac, pPPax2, and pPPax2m1-3 stable transfectants were cultured in growth medium and then subjected to SDS-PAGE and Western blot. The blot was probed by anti-Pax2, anti-HA and anti-CWP1 antibody. Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. Representative results are shown. (B) RT-PCR analysis of gene expression in the Pax2 and Pax2m1-3 overexpressing cell lines. The 5′▵5N-Pac, pPPax2, and pPPax2m1-3 stable transfectants were cultured in growth medium and then subjected to RT-PCR analysis using primers specific for pax2-ha , pax2 , cwp1 , cwp2 , cwp3 , myb2 , ran , and 18S ribosomal RNA genes. (C) Quantitative real-time PCR analysis of gene expression in the Pax2 and Pax2m1-3 overexpressing cell lines. Real-time PCR was performed using primers specific for pax2-ha , pax2 , cwp1 , cwp2 , cwp3 , myb2 , ran , and 18S ribosomal RNA genes. Similar mRNA levels of the ran and 18S ribosomal RNA genes for these samples were detected. Transcript levels were normalized to 18S ribosomal RNA levels. Fold changes in mRNA expression are shown as the ratio of transcript levels in the pPPax2 or pPPax2m1-3 cell line relative to the 5′▵5N-Pac cell line. Results are expressed as the means ± standard error of at least three separate experiments. (D) Cyst count. The 5′▵5N-Pac, 5′▵5N-Pac, pPPax2, and pPPax2m1-3 stable transfectants were cultured in growth medium and then subjected to cyst count as described under “ Methods ”. The sum of total cysts is expressed as relative expression level over control. Values are shown as means ± standard error.
    Figure Legend Snippet: Activation of cwp1-3 and myb2 gene expression in the Pax2 overexpressing cell line. (A) Overexpression of Pax2 increased the levels of CWP1 protein. The 5′▵5N-Pac, pPPax2, and pPPax2m1-3 stable transfectants were cultured in growth medium and then subjected to SDS-PAGE and Western blot. The blot was probed by anti-Pax2, anti-HA and anti-CWP1 antibody. Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. Representative results are shown. (B) RT-PCR analysis of gene expression in the Pax2 and Pax2m1-3 overexpressing cell lines. The 5′▵5N-Pac, pPPax2, and pPPax2m1-3 stable transfectants were cultured in growth medium and then subjected to RT-PCR analysis using primers specific for pax2-ha , pax2 , cwp1 , cwp2 , cwp3 , myb2 , ran , and 18S ribosomal RNA genes. (C) Quantitative real-time PCR analysis of gene expression in the Pax2 and Pax2m1-3 overexpressing cell lines. Real-time PCR was performed using primers specific for pax2-ha , pax2 , cwp1 , cwp2 , cwp3 , myb2 , ran , and 18S ribosomal RNA genes. Similar mRNA levels of the ran and 18S ribosomal RNA genes for these samples were detected. Transcript levels were normalized to 18S ribosomal RNA levels. Fold changes in mRNA expression are shown as the ratio of transcript levels in the pPPax2 or pPPax2m1-3 cell line relative to the 5′▵5N-Pac cell line. Results are expressed as the means ± standard error of at least three separate experiments. (D) Cyst count. The 5′▵5N-Pac, 5′▵5N-Pac, pPPax2, and pPPax2m1-3 stable transfectants were cultured in growth medium and then subjected to cyst count as described under “ Methods ”. The sum of total cysts is expressed as relative expression level over control. Values are shown as means ± standard error.

    Techniques Used: Activation Assay, Expressing, Over Expression, Cell Culture, SDS Page, Western Blot, Staining, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Analysis of pax2 gene expression. (A) RT-PCR and quantitative real-time PCR analysis of pax2 gene expression. RNA samples were prepared from G. lamblia wild-type non-transfected WB cells cultured in growth (Veg, vegetative growth) or encystation medium and harvested at 24 h (Enc, encystation). RT-PCR was performed using primers specific for pax2 , cwp1 , ran , and 18S ribosomal RNA genes. Ribosomal RNA quality and loading controls are shown in the bottom panel. Representative results are shown on the left. Real-time PCR was performed using primers specific for pax2 , cwp1 , ran and 18S ribosomal RNA genes. Transcript levels were normalized to 18S ribosomal RNA levels. Fold changes in mRNA expression are shown as the ratio of transcript levels in encysting cells relative to vegetative cells. Results are expressed as the means ± standard error of at least three separate experiments (right panel). (B) Pax2 protein levels in different stages. The wild-type non-transfected WB cells were cultured in growth (Veg, vegetative growth) or encystation medium for 24 h (Enc, encystation) and then subjected to SDS-PAGE and Western blot. The blot was probed by anti-Pax2, anti-RAN, or anti-α tubulin antibody. Representative results are shown. Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. (C) Diagrams of the 5′▵5N-Pac and pPPax2 plasmid. The pac gene (open box) is under the control of the 5′- and 3′-flanking regions of the gdh gene (striated box). In construct pPPax2, the pax2 gene is under the control of its own 5′-flanking region (open box) and the 3′-flanking region of the ran gene (dotted box). The filled black box indicates the coding sequence of the HA epitope tag. (D) Pax2 protein levels in pPPax2 stable transfectants. The pPPax2 stable transfectants were cultured in growth (Veg, vegetative growth) or encystation medium for 24 h (Enc, encystation) and then subjected to SDS-PAGE and Western blot. HA-tagged Pax2 protein was detected using an anti-HA antibody by Western blot analysis. The blot was also probed by anti-RAN or anti-α tubulin antibody. Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. (E) Nuclear localization of Pax2. The pPPax2 stable transfectants were cultured in growth (Veg, left panels) or encystation medium for 24 h (Enc, right panels), and then subjected to immunofluorescence analysis using anti-HA antibody for detection. The product of pPPax2 localizes to the nuclei in both vegetative and encysting trophozoites (upper panels). The middle panels show the DAPI staining of cell nuclei. The bottom panels are the merged images of the DAPI staining and images of Pax2-HA.
    Figure Legend Snippet: Analysis of pax2 gene expression. (A) RT-PCR and quantitative real-time PCR analysis of pax2 gene expression. RNA samples were prepared from G. lamblia wild-type non-transfected WB cells cultured in growth (Veg, vegetative growth) or encystation medium and harvested at 24 h (Enc, encystation). RT-PCR was performed using primers specific for pax2 , cwp1 , ran , and 18S ribosomal RNA genes. Ribosomal RNA quality and loading controls are shown in the bottom panel. Representative results are shown on the left. Real-time PCR was performed using primers specific for pax2 , cwp1 , ran and 18S ribosomal RNA genes. Transcript levels were normalized to 18S ribosomal RNA levels. Fold changes in mRNA expression are shown as the ratio of transcript levels in encysting cells relative to vegetative cells. Results are expressed as the means ± standard error of at least three separate experiments (right panel). (B) Pax2 protein levels in different stages. The wild-type non-transfected WB cells were cultured in growth (Veg, vegetative growth) or encystation medium for 24 h (Enc, encystation) and then subjected to SDS-PAGE and Western blot. The blot was probed by anti-Pax2, anti-RAN, or anti-α tubulin antibody. Representative results are shown. Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. (C) Diagrams of the 5′▵5N-Pac and pPPax2 plasmid. The pac gene (open box) is under the control of the 5′- and 3′-flanking regions of the gdh gene (striated box). In construct pPPax2, the pax2 gene is under the control of its own 5′-flanking region (open box) and the 3′-flanking region of the ran gene (dotted box). The filled black box indicates the coding sequence of the HA epitope tag. (D) Pax2 protein levels in pPPax2 stable transfectants. The pPPax2 stable transfectants were cultured in growth (Veg, vegetative growth) or encystation medium for 24 h (Enc, encystation) and then subjected to SDS-PAGE and Western blot. HA-tagged Pax2 protein was detected using an anti-HA antibody by Western blot analysis. The blot was also probed by anti-RAN or anti-α tubulin antibody. Equal amounts of protein loading were confirmed by SDS-PAGE and Coomassie blue staining. (E) Nuclear localization of Pax2. The pPPax2 stable transfectants were cultured in growth (Veg, left panels) or encystation medium for 24 h (Enc, right panels), and then subjected to immunofluorescence analysis using anti-HA antibody for detection. The product of pPPax2 localizes to the nuclei in both vegetative and encysting trophozoites (upper panels). The middle panels show the DAPI staining of cell nuclei. The bottom panels are the merged images of the DAPI staining and images of Pax2-HA.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Cell Culture, SDS Page, Staining, Plasmid Preparation, Construct, Sequencing, Immunofluorescence

    3) Product Images from "Prdx6 retards senescence and restores trabecular meshwork cell health by regulating reactive oxygen species"

    Article Title: Prdx6 retards senescence and restores trabecular meshwork cell health by regulating reactive oxygen species

    Journal: Cell Death Discovery

    doi: 10.1038/cddiscovery.2017.60

    Aging TM cells exposed to H 2 O 2 displayed reduced Prdx6 expression, increased TGF β s and accumulation of ECM proteins with elevated ROS levels and reduced cell viability. ( a ) Total RNA were isolated from TM cells of different ages exposed to H 2 O 2 and processed for real-time PCR with specific primers as indicated. Histogram values are mean±S.D. of three independent experiments, showing significant reduction of Prdx6 expression in aging TM cells, in age-dependent manner (* P
    Figure Legend Snippet: Aging TM cells exposed to H 2 O 2 displayed reduced Prdx6 expression, increased TGF β s and accumulation of ECM proteins with elevated ROS levels and reduced cell viability. ( a ) Total RNA were isolated from TM cells of different ages exposed to H 2 O 2 and processed for real-time PCR with specific primers as indicated. Histogram values are mean±S.D. of three independent experiments, showing significant reduction of Prdx6 expression in aging TM cells, in age-dependent manner (* P

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

    4) Product Images from "Effect of salt stress on ion concentration, proline content, antioxidant enzyme activities and gene expression in tomato cultivars"

    Article Title: Effect of salt stress on ion concentration, proline content, antioxidant enzyme activities and gene expression in tomato cultivars

    Journal: AoB Plants

    doi: 10.1093/aobpla/plw055

    Relative gene expression of tomato HKT1;1 and HKT1;2 in response to salt stress in leaves and roots San Miguel, Perfect peel HF1 and Mouna HF1 genotypes. Total RNA was purified from tissues of tomato plants treated with 150 mM NaCl for 0 h, 6 h, 12 h and 7 days. Transcript level was analyzed by qRT-PCR using primers indicated in Table 1 . Tomato Actin gene was used as reference gene. Error bars show the standard error between three replicates performed. Bars with different letters within each panel are significantly different at P > 0.05 according to Tukey's test.
    Figure Legend Snippet: Relative gene expression of tomato HKT1;1 and HKT1;2 in response to salt stress in leaves and roots San Miguel, Perfect peel HF1 and Mouna HF1 genotypes. Total RNA was purified from tissues of tomato plants treated with 150 mM NaCl for 0 h, 6 h, 12 h and 7 days. Transcript level was analyzed by qRT-PCR using primers indicated in Table 1 . Tomato Actin gene was used as reference gene. Error bars show the standard error between three replicates performed. Bars with different letters within each panel are significantly different at P > 0.05 according to Tukey's test.

    Techniques Used: Expressing, Purification, Quantitative RT-PCR

    Relative gene expression of tomato LeNHX1 , LeNHX3 and LeNHX4 in response to salt stress in in leaves and roots of San Miguel, Perfect peel HF1 and Mouna HF1 genotypes. Total RNA was purified from tissues of tomato plants treated with 150 mM NaCl for 0 h, 6 h, 12 h and 7 days. Transcript level was analyzed by qRT-PCR using primers indicated in Table 1 . Tomato Actin gene was used as reference gene. Error bars show the standard error between three replicates performed. Bars with different letters within each panel are significantly different at P > 0.05 according to Tukey's test.
    Figure Legend Snippet: Relative gene expression of tomato LeNHX1 , LeNHX3 and LeNHX4 in response to salt stress in in leaves and roots of San Miguel, Perfect peel HF1 and Mouna HF1 genotypes. Total RNA was purified from tissues of tomato plants treated with 150 mM NaCl for 0 h, 6 h, 12 h and 7 days. Transcript level was analyzed by qRT-PCR using primers indicated in Table 1 . Tomato Actin gene was used as reference gene. Error bars show the standard error between three replicates performed. Bars with different letters within each panel are significantly different at P > 0.05 according to Tukey's test.

    Techniques Used: Expressing, Purification, Quantitative RT-PCR

    Relative gene expression of tomato ERF9 , ERF16 and ERF80 in response to salt stress in leaves and roots San Miguel, Perfect peel HF1 and Mouna HF1genotypes. Total RNA was purified from tissues of tomato plants treated with 150 mM NaCl for 0 h, 6 h, 12 h and 7 days. Transcript level was analyzed by qRT-PCR using primers indicated in Table 1 . Tomato Actin gene was used as reference gene. Error bars show the standard error between three replicates performed. Bars with different letters within each panel are significantly different at P > 0.05 according to Tukey's test.
    Figure Legend Snippet: Relative gene expression of tomato ERF9 , ERF16 and ERF80 in response to salt stress in leaves and roots San Miguel, Perfect peel HF1 and Mouna HF1genotypes. Total RNA was purified from tissues of tomato plants treated with 150 mM NaCl for 0 h, 6 h, 12 h and 7 days. Transcript level was analyzed by qRT-PCR using primers indicated in Table 1 . Tomato Actin gene was used as reference gene. Error bars show the standard error between three replicates performed. Bars with different letters within each panel are significantly different at P > 0.05 according to Tukey's test.

    Techniques Used: Expressing, Purification, Quantitative RT-PCR

    Relative gene expression of tomato WRKY8 , WRKY31 and WRKY39 in response to salt stress in leaves and roots of San Miguel, Perfect peel HF1 and Mouna HF1 genotypes. Total RNA was purified from tissues of tomato plants treated with 150 mM NaCl for 0 h, 6 h, 12 h and 7 days. Transcript level was analyzed by qRT-PCR using primers indicated in Table 1 . Tomato Actin gene was used as reference gene. Error bars show the standard error between three replicates performed. Bars with different letters within each panel are significantly different at P > 0.05 according to Tukey's test.
    Figure Legend Snippet: Relative gene expression of tomato WRKY8 , WRKY31 and WRKY39 in response to salt stress in leaves and roots of San Miguel, Perfect peel HF1 and Mouna HF1 genotypes. Total RNA was purified from tissues of tomato plants treated with 150 mM NaCl for 0 h, 6 h, 12 h and 7 days. Transcript level was analyzed by qRT-PCR using primers indicated in Table 1 . Tomato Actin gene was used as reference gene. Error bars show the standard error between three replicates performed. Bars with different letters within each panel are significantly different at P > 0.05 according to Tukey's test.

    Techniques Used: Expressing, Purification, Quantitative RT-PCR

    5) Product Images from "LncRNA SNHG7 sponges miR-216b to promote proliferation and liver metastasis of colorectal cancer through upregulating GALNT1"

    Article Title: LncRNA SNHG7 sponges miR-216b to promote proliferation and liver metastasis of colorectal cancer through upregulating GALNT1

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0759-7

    SNHG7 acted as a ceRNA by sponging miR-216b and regulated GALNT1 expression indirectly. a The differential expression of GALNT1 in CRC tissues and adjacent normal colon tissues was examined. Expressional levels of GALNT1 in CRC cell lines and FHC cell. b Pearson’s correlation curve identified positive correlation between SNHG7 and GALNT1 in CRC tissues. c The expressional levels of GALNT1 mRNA and protein, E-cadherin and Vimentin proteins in CRC cells transfected with siSNHG7 were evaluated by qRT-PCR and western blot. d The levels of GALNT1 mRNA and protein, E-cadherin and Vimentin proteins in CRC cells transfected with pcDNA/SNHG7 were evaluated by qRT-PCR and western blot. e The predicted binding sites of miR-216b to the SNHG7 sequence were shown. f The differential expression of miR-216b in CRC tissues and adjacent normal colon tissues was analyzed. g The levels of miR-216b was investigated in CRC cell lines and human normal colon cell line by qRT-PCR. h Pearson’s correlation curve revealed the negative relevance between SNHG7 and miR-216b expression. i RNA-IP was performed in SW620 and SW480 cells transfected with NC mimic and miR-216b mimic. SNHG7 expression was detected using qRT-PCR. j Luciferase activity of 293T cells cotransfected with miR-216b mimic and luciferase reporters containing SNHG7-Wt or SNHG7-Mut transcript were analyzed. k The predicted binding sites of miR-216b to the GALNT1 sequence were shown. l Pearson’s correlation curve revealed the negative relevance between GALNT1 and miR-216b levels. m Luciferase activity of 293T cells cotransfected with miR-216b mimic and luciferase reporters containing GALLNT1-Wt or GALNT1-Mut transcript were performed. n The levels of GALNT1 transfected with miR-216b inhibitor or siSNHG7 in SW620 cell were analyzed by qRT-PCR and western blot. o The levels of GALNT1 transfected with miR-216b mimic or pcDNA/SNHG7 in SW480 cells were analyzed by qRT-PCR and western blot. The error bars in all graphs represented SD, and each experiment was repeated three times. * p
    Figure Legend Snippet: SNHG7 acted as a ceRNA by sponging miR-216b and regulated GALNT1 expression indirectly. a The differential expression of GALNT1 in CRC tissues and adjacent normal colon tissues was examined. Expressional levels of GALNT1 in CRC cell lines and FHC cell. b Pearson’s correlation curve identified positive correlation between SNHG7 and GALNT1 in CRC tissues. c The expressional levels of GALNT1 mRNA and protein, E-cadherin and Vimentin proteins in CRC cells transfected with siSNHG7 were evaluated by qRT-PCR and western blot. d The levels of GALNT1 mRNA and protein, E-cadherin and Vimentin proteins in CRC cells transfected with pcDNA/SNHG7 were evaluated by qRT-PCR and western blot. e The predicted binding sites of miR-216b to the SNHG7 sequence were shown. f The differential expression of miR-216b in CRC tissues and adjacent normal colon tissues was analyzed. g The levels of miR-216b was investigated in CRC cell lines and human normal colon cell line by qRT-PCR. h Pearson’s correlation curve revealed the negative relevance between SNHG7 and miR-216b expression. i RNA-IP was performed in SW620 and SW480 cells transfected with NC mimic and miR-216b mimic. SNHG7 expression was detected using qRT-PCR. j Luciferase activity of 293T cells cotransfected with miR-216b mimic and luciferase reporters containing SNHG7-Wt or SNHG7-Mut transcript were analyzed. k The predicted binding sites of miR-216b to the GALNT1 sequence were shown. l Pearson’s correlation curve revealed the negative relevance between GALNT1 and miR-216b levels. m Luciferase activity of 293T cells cotransfected with miR-216b mimic and luciferase reporters containing GALLNT1-Wt or GALNT1-Mut transcript were performed. n The levels of GALNT1 transfected with miR-216b inhibitor or siSNHG7 in SW620 cell were analyzed by qRT-PCR and western blot. o The levels of GALNT1 transfected with miR-216b mimic or pcDNA/SNHG7 in SW480 cells were analyzed by qRT-PCR and western blot. The error bars in all graphs represented SD, and each experiment was repeated three times. * p

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Binding Assay, Sequencing, Luciferase, Activity Assay

    6) Product Images from "Cyclooxygenase-2 Is a Target of MicroRNA-16 in Human Hepatoma Cells"

    Article Title: Cyclooxygenase-2 Is a Target of MicroRNA-16 in Human Hepatoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0050935

    miR-16 regulates COX-2 expression in HCC cell lines. WRL68 and Hep3B cells were transfected with: 30 nM siRNA anti-COX2 (siCOX-2) or 50 nM of miR-16, miR-16 inhibitor (In-miR-16), miR negative control (miR-NC) or miR negative control inhibitor (In-miR-NC). ( A–B ) COX-2 protein was analyzed by Western blot 48 h after transfection and normalized against α-tubulin protein. COX-2 mRNA and miR-16 expression were analyzed by real-time PCR. COX-2 mRNA and miR-16 expression were normalized against 36b4 mRNA and U6 RNA levels, respectively. Relative expression of each sample refers to control as 1 (cells transfected only with lipofectamine). ( C–D ) PGE 2 concentration was determined by enzyme immunoassay in the supernatant of the cells. Data are reported as means±SD of four independent experiments. *p
    Figure Legend Snippet: miR-16 regulates COX-2 expression in HCC cell lines. WRL68 and Hep3B cells were transfected with: 30 nM siRNA anti-COX2 (siCOX-2) or 50 nM of miR-16, miR-16 inhibitor (In-miR-16), miR negative control (miR-NC) or miR negative control inhibitor (In-miR-NC). ( A–B ) COX-2 protein was analyzed by Western blot 48 h after transfection and normalized against α-tubulin protein. COX-2 mRNA and miR-16 expression were analyzed by real-time PCR. COX-2 mRNA and miR-16 expression were normalized against 36b4 mRNA and U6 RNA levels, respectively. Relative expression of each sample refers to control as 1 (cells transfected only with lipofectamine). ( C–D ) PGE 2 concentration was determined by enzyme immunoassay in the supernatant of the cells. Data are reported as means±SD of four independent experiments. *p

    Techniques Used: Expressing, Transfection, Negative Control, Western Blot, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay

    HuR antagonizes the downregulation of COX-2 expression caused by miR-16 in hepatoma cell lines. WRL68 cell extracts (500 µg per lane) were immunoprecipitated with HuR or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 ( A ) or miR-16 primers ( B ). PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The abundance of the transcripts present in WRL68 cells after HuR immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; unbound, unbound mRNA after immunoprecipitation with HuR antibody; bound, bound mRNA after immunoprecipitation with HuR antibody; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( C–D ) WRL68 and Hep3B cell lines were transfected with miR-16 or In-miR-16 (50 nM). COX-2 and HuR protein levels were analyzed by Western Blot. ( E–F ) WRL68 and Hep3B cell lines were cotransfected with miR-16 (50 nM) and pcDNA3-HuR-GFP expression vector (4 µg). COX-2 and HuR protein levels were analyzed by Western Blot. Data are reported as means±SD of three independent experiments. *p
    Figure Legend Snippet: HuR antagonizes the downregulation of COX-2 expression caused by miR-16 in hepatoma cell lines. WRL68 cell extracts (500 µg per lane) were immunoprecipitated with HuR or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 ( A ) or miR-16 primers ( B ). PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The abundance of the transcripts present in WRL68 cells after HuR immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; unbound, unbound mRNA after immunoprecipitation with HuR antibody; bound, bound mRNA after immunoprecipitation with HuR antibody; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( C–D ) WRL68 and Hep3B cell lines were transfected with miR-16 or In-miR-16 (50 nM). COX-2 and HuR protein levels were analyzed by Western Blot. ( E–F ) WRL68 and Hep3B cell lines were cotransfected with miR-16 (50 nM) and pcDNA3-HuR-GFP expression vector (4 µg). COX-2 and HuR protein levels were analyzed by Western Blot. Data are reported as means±SD of three independent experiments. *p

    Techniques Used: Expressing, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Electrophoresis, Staining, Transfection, Western Blot, Plasmid Preparation

    miR-16 and COX-2 correlate inversely in HCC cell lines. Cells were plated in 100-mm dishes and grown to 60–70% confluence in culture medium supplemented with 10% FBS. ( A ) Total cellular extracts were prepared from HCC cells and protein (30–50 µg/lane) was analyzed by Western blot. A representative Western blot showing COX-2 protein. The expression of target protein was normalized to that of α-tubulin. Densitometric analysis of COX-2 expression (black bars) is referring to HH as 1 and expressed as relative expression (RE). Total RNA was prepared from HCC cell lines and COX-2 mRNA was analyzed by real-time PCR. COX-2 mRNA amounts (white bars), normalized to the expression of 36b4 mRNA, and miR-16 expression (grey bars), normalized against U6 RNA levels, were calculated. Values represent fold change relative to human hepatocytes (HH) as 1. Data are reported as means±SD of three independent experiments. ** p
    Figure Legend Snippet: miR-16 and COX-2 correlate inversely in HCC cell lines. Cells were plated in 100-mm dishes and grown to 60–70% confluence in culture medium supplemented with 10% FBS. ( A ) Total cellular extracts were prepared from HCC cells and protein (30–50 µg/lane) was analyzed by Western blot. A representative Western blot showing COX-2 protein. The expression of target protein was normalized to that of α-tubulin. Densitometric analysis of COX-2 expression (black bars) is referring to HH as 1 and expressed as relative expression (RE). Total RNA was prepared from HCC cell lines and COX-2 mRNA was analyzed by real-time PCR. COX-2 mRNA amounts (white bars), normalized to the expression of 36b4 mRNA, and miR-16 expression (grey bars), normalized against U6 RNA levels, were calculated. Values represent fold change relative to human hepatocytes (HH) as 1. Data are reported as means±SD of three independent experiments. ** p

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

    miR-16 binds COX-2 mRNA and inhibits its translation. ( A ) WRL68 cell extracts (500 µg per lane) were immunoprecipitated with Ago-2 or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 primers. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The presence of COX-2 mRNA in WRL68 cell transfected with miR-16 or Lipofectamine after Ago2 immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( B ) Scheme of pGL3-empty, pGL3-seed and pGL3-mut reporter vectors. In pGL3-seed, the putative binding site of miR-16 on COX-2 mRNA 3′-UTR region (as detected by RNAhybrid software) was introduced downstream luciferase gene. In pGL3-mut this region was mutated in order to avoid the binding between miR-16 and Luc mRNA. ( C–D ) A luciferase assay was carried out on HuH-7 and HepG2 cell lines using pGL3-seed and pGL3-mut reporter vectors. Firefly luciferase activity was evaluated 48 h after co-transfection with pGL3-empty/seed/mut (750 ng), miR-16 (50 nM), In-miR-16 (50 nM) and miR-NC (50 nM) as indicated. Data were normalized against renilla luciferase activity (all samples were co-transfected with 50 ng pRL vector and refer to the positive control, pGL3 empty vector). Data are reported as means±SD of three independent experiments. *p
    Figure Legend Snippet: miR-16 binds COX-2 mRNA and inhibits its translation. ( A ) WRL68 cell extracts (500 µg per lane) were immunoprecipitated with Ago-2 or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 primers. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The presence of COX-2 mRNA in WRL68 cell transfected with miR-16 or Lipofectamine after Ago2 immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( B ) Scheme of pGL3-empty, pGL3-seed and pGL3-mut reporter vectors. In pGL3-seed, the putative binding site of miR-16 on COX-2 mRNA 3′-UTR region (as detected by RNAhybrid software) was introduced downstream luciferase gene. In pGL3-mut this region was mutated in order to avoid the binding between miR-16 and Luc mRNA. ( C–D ) A luciferase assay was carried out on HuH-7 and HepG2 cell lines using pGL3-seed and pGL3-mut reporter vectors. Firefly luciferase activity was evaluated 48 h after co-transfection with pGL3-empty/seed/mut (750 ng), miR-16 (50 nM), In-miR-16 (50 nM) and miR-NC (50 nM) as indicated. Data were normalized against renilla luciferase activity (all samples were co-transfected with 50 ng pRL vector and refer to the positive control, pGL3 empty vector). Data are reported as means±SD of three independent experiments. *p

    Techniques Used: Immunoprecipitation, Polymerase Chain Reaction, Amplification, Electrophoresis, Staining, Transfection, Binding Assay, Software, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Positive Control

    Effect of miR-16 on COX-2 mRNA and protein stability. Hep3B cells were transfected with 50 nM miR-16 or miR-NC, or 30 nM siCOX-2. 5 µg/ml actinomycin-D (Act D) or 10 µg/ml cycloheximide (CHX) were added after transfection. ( A–B ) COX-2 protein was analyzed by Western blot at different time after actinomycin-D treatment. Corresponding densitometry analysis is shown and the relative expression of each sample is related to sample at 0 h as 1. ( C ) mRNA COX-2 levels were analyzed by real time PCR. COX-2 mRNA amounts were calculated as relative expression and normalized to the expression of 36b4 mRNA. Values represent fold change relative to sample at 0 h. ( D–E ) COX-2 protein levels were analyzed by Western blot in the presence or absence of cycloheximide. Corresponding densitometric analysis is shown and the relative expression of each sample is related to the value at 0 h as 1. F) Hep3B cells were transfected with 50 nM miR-16, miR-16 inhibitor (In-miR-16) or lipofectamine and permeabilized with digitonine to obtain soluble and pellet fractions enriched in PB as described in Methods. RNA was isolated from each fraction with Trizol reagent, reverse transcriptased, and PCR amplified with COX-2, Xrn1 and actin primers. Input, RNA extracted from cells prior to fractionation. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. G) The presence of COX-2 mRNA in soluble and PB fractions was assessed and fold differences were plotted. Data are reported as means±SD of three independent experiments. **p
    Figure Legend Snippet: Effect of miR-16 on COX-2 mRNA and protein stability. Hep3B cells were transfected with 50 nM miR-16 or miR-NC, or 30 nM siCOX-2. 5 µg/ml actinomycin-D (Act D) or 10 µg/ml cycloheximide (CHX) were added after transfection. ( A–B ) COX-2 protein was analyzed by Western blot at different time after actinomycin-D treatment. Corresponding densitometry analysis is shown and the relative expression of each sample is related to sample at 0 h as 1. ( C ) mRNA COX-2 levels were analyzed by real time PCR. COX-2 mRNA amounts were calculated as relative expression and normalized to the expression of 36b4 mRNA. Values represent fold change relative to sample at 0 h. ( D–E ) COX-2 protein levels were analyzed by Western blot in the presence or absence of cycloheximide. Corresponding densitometric analysis is shown and the relative expression of each sample is related to the value at 0 h as 1. F) Hep3B cells were transfected with 50 nM miR-16, miR-16 inhibitor (In-miR-16) or lipofectamine and permeabilized with digitonine to obtain soluble and pellet fractions enriched in PB as described in Methods. RNA was isolated from each fraction with Trizol reagent, reverse transcriptased, and PCR amplified with COX-2, Xrn1 and actin primers. Input, RNA extracted from cells prior to fractionation. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. G) The presence of COX-2 mRNA in soluble and PB fractions was assessed and fold differences were plotted. Data are reported as means±SD of three independent experiments. **p

    Techniques Used: Transfection, Activated Clotting Time Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Amplification, Fractionation, Electrophoresis, Staining

    7) Product Images from "Isoeugenin, a Novel Nitric Oxide Synthase Inhibitor Isolated from the Rhizomes of Imperata cylindrica"

    Article Title: Isoeugenin, a Novel Nitric Oxide Synthase Inhibitor Isolated from the Rhizomes of Imperata cylindrica

    Journal: Molecules

    doi: 10.3390/molecules201219767

    Inhibitory effect of isoeugenin on LPS-induced TNF-α ( A ); IL-6 ( B ); IL-1β ( C ) mRNA expression in RAW264.7 cells. Total RNA was prepared for the Real Time-PCR analysis of TNF-α, IL-6, IL-1β gene expression from RAW264.7 macrophage cells pretreated with different concentrations (12.5, 25, 50 μg/mL) of isoeugenin for 1 h followed by LPS (1 μg/mL) for 24 h. NOR values were obtained in the absence of LPS and samples. The experiment was repeated three times and similar results were obtained. Values represent means ± S.D. of three independent experiments. ## p
    Figure Legend Snippet: Inhibitory effect of isoeugenin on LPS-induced TNF-α ( A ); IL-6 ( B ); IL-1β ( C ) mRNA expression in RAW264.7 cells. Total RNA was prepared for the Real Time-PCR analysis of TNF-α, IL-6, IL-1β gene expression from RAW264.7 macrophage cells pretreated with different concentrations (12.5, 25, 50 μg/mL) of isoeugenin for 1 h followed by LPS (1 μg/mL) for 24 h. NOR values were obtained in the absence of LPS and samples. The experiment was repeated three times and similar results were obtained. Values represent means ± S.D. of three independent experiments. ## p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    8) Product Images from "DDX3 regulates endoplasmic reticulum stress-induced ATF4 expression"

    Article Title: DDX3 regulates endoplasmic reticulum stress-induced ATF4 expression

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14262-7

    DDX3 is required for ER stress-induced ATF4 expression. ( A – D ) Hep3B were treated with either DDX3-a, DDX3-b or control (Ct) siRNAs for ninety-six hours and then incubated with either 10 μM Sor or 100 nM Thap for two hours, as previously described 15 . ( A ) Left panels: Cells were harvested, lysed and proteins materials were analyzed by western blot for the expression of DDX3, ATF4, PeIF2α, the pan-eIF2α, and tubulin (Tub; loading control) using the corresponding antibodies. Right panel: The expression level of ATF4 was estimated by densitometry quantification of the film signal using Image Studio™ Lite Software and standardized against total tubulin. * P ≤ 0.05; **** P ≤ 0.0001 (Student’s t -test). The results are representative of at least 3 different experiments. ( B ) Total RNA was isolated and the level of ATF4 mRNA relative to GAPDH mRNA was quantified by real-time q(RT)-PCR using the ΔΔCt method. The presented results are the mean of triplicate measurements, with error bars corresponding to the S.D. ( C , D ) Hep3B treated with either DDX3-a, DDX3-b or control (Ct) siRNA were incubated with Sor. ( C ) Cells were processed for immunofluorescence to detect SG using anti-FMRP (green signal) and anti-DDX3 (red signal) antibodies. DAPI stains nuclei. Bars correspond to 20 μm. Representative results from 6 different fields and 3 different experiments containing a total of 1000 cells are shown. Right panel represents the percentage of cells harboring SG ( > 3 granules/cell). Error bars correspond to the S.D with variations not statistically significant. ( D ) Clonogenic survival assays. Hep3B were incubated with either DDX3-a, -b or control (Ct) siRNAs, then treated with Sor. After trypsinisation, equal numbers of cells were seeded in the absence of drug, and incubated for 10 days. Populations > 20 cells were counted as one surviving colony. Data were calculated as the percentage of surviving colonies relative to the number found in plates corresponding to mock-depleted cells plates. ** P ≤ 0.01; *** P ≤ 0.001.
    Figure Legend Snippet: DDX3 is required for ER stress-induced ATF4 expression. ( A – D ) Hep3B were treated with either DDX3-a, DDX3-b or control (Ct) siRNAs for ninety-six hours and then incubated with either 10 μM Sor or 100 nM Thap for two hours, as previously described 15 . ( A ) Left panels: Cells were harvested, lysed and proteins materials were analyzed by western blot for the expression of DDX3, ATF4, PeIF2α, the pan-eIF2α, and tubulin (Tub; loading control) using the corresponding antibodies. Right panel: The expression level of ATF4 was estimated by densitometry quantification of the film signal using Image Studio™ Lite Software and standardized against total tubulin. * P ≤ 0.05; **** P ≤ 0.0001 (Student’s t -test). The results are representative of at least 3 different experiments. ( B ) Total RNA was isolated and the level of ATF4 mRNA relative to GAPDH mRNA was quantified by real-time q(RT)-PCR using the ΔΔCt method. The presented results are the mean of triplicate measurements, with error bars corresponding to the S.D. ( C , D ) Hep3B treated with either DDX3-a, DDX3-b or control (Ct) siRNA were incubated with Sor. ( C ) Cells were processed for immunofluorescence to detect SG using anti-FMRP (green signal) and anti-DDX3 (red signal) antibodies. DAPI stains nuclei. Bars correspond to 20 μm. Representative results from 6 different fields and 3 different experiments containing a total of 1000 cells are shown. Right panel represents the percentage of cells harboring SG ( > 3 granules/cell). Error bars correspond to the S.D with variations not statistically significant. ( D ) Clonogenic survival assays. Hep3B were incubated with either DDX3-a, -b or control (Ct) siRNAs, then treated with Sor. After trypsinisation, equal numbers of cells were seeded in the absence of drug, and incubated for 10 days. Populations > 20 cells were counted as one surviving colony. Data were calculated as the percentage of surviving colonies relative to the number found in plates corresponding to mock-depleted cells plates. ** P ≤ 0.01; *** P ≤ 0.001.

    Techniques Used: Expressing, Incubation, Western Blot, Software, Isolation, Polymerase Chain Reaction, Immunofluorescence

    DDX3 regulates ATF4 expression at the posttranscriptional level. ( A ) Immunoprecipitation coupled to qRT-PCR. Hep3B were treated with 10 μM Sor for 2 hours, lysed and their extracts were used to immunoprecipitate DDX3 with anti-DDX3 antibodies and with IgG as a control. mRNAs were isolated from each immunoprecipitate and quantified by qRT-PCR. Left panel: Western blot analysis of total (T) and immunoprecipitated (Ip) proteins using antibodies specific to DDX3. The results are representative of two independent experiments. Right panel: The amounts of ATF4 mRNA and actin mRNA (as control) present in DDX3 precipitate are calculated relative to those in IgG precipitate, then were normalized against GAPDH mRNA. ( B , C ) Thap induces ATF4 expression more efficiently than Sor. ( B ) Hep3B were treated with either Sor (10 μM) or Thap (100 nM) then lysed and their protein contents were analyzed by western blot for the expression of ATF4 and tubulin (Tub; loading control) using the corresponding antibodies. P-eIF2α serves a positive control for drugs treatment and the pan-eIF2α is used as control for P-eIF2α. *** P ≤ 0.001; **** P ≤ 0.0001. ( C , D ) Translational luciferase assays. ( C ) Hep3B cells are co-transfected with a luciferase expressing vector containing the human ATF4 5′-UTR fused to Firefly luciferase (FLuc) gene, and the control plasmid expressing Renilla luciferase (RLuc). Cells are then treated with either Sor or Thap to induce translation of FLuc whose activity is measured in the cell extracts and expressed relative to RLuc. Error bars correspond to the S.D. ( D ) Hep3B expressing DDX3-a or control (Ct) siRNAs are co-transfected with a luciferase expressing vector containing the human ATF4 5′-UTR fused to FLuc gene, and the control plasmid expressing RLuc. Cells are then treated with Thap to induce translation of FLuc whose activity is measured as above. ** P ≤ 0.01. ( E , F ) Analysis of the ATF4 mRNA association with polyribosomes. Hep3B were incubated with either DDX3 or control (Ct) siRNAs, then treated with Sor. Cytoplasmic extracts of Sor-treated Hep3B cells were fractionated onto 15% (Top) and 55% (Bot: Bottom) sucrose gradients and their polyribosomes profiles ( E ) were recorded by measuring the OD 254 , as described 27 . LP: light polyribosomes. HP: heavy polyribosomes. ( F ) RNA content was isolated from pooled LP and HP fractions and associated ATF4 mRNA was quantified by qRT-PCR using the ΔΔCt method. ATF4 mRNA levels were normalized against 18 s ribosomal RNA and expressed as indicated (left panels). DDX3 mRNA present in LP and HP was similarly quantified and serves as an additional control for DDX3 mRNA knockdown (right panel). The results are representative of two independent experiments. ( G ) Top panels: cytoplasmic extracts prepared from either untreated or Sor-treated Hep3B were fractionated through 15–55% (w/v) sucrose density gradients and their polyribosomes profiles were monitored as above. Bottom panels: Western blot analysis of the collected fractions for the distribution of DDX3 and as a control S6 protein using specific antibodies. The results are representative of two independent experiments.
    Figure Legend Snippet: DDX3 regulates ATF4 expression at the posttranscriptional level. ( A ) Immunoprecipitation coupled to qRT-PCR. Hep3B were treated with 10 μM Sor for 2 hours, lysed and their extracts were used to immunoprecipitate DDX3 with anti-DDX3 antibodies and with IgG as a control. mRNAs were isolated from each immunoprecipitate and quantified by qRT-PCR. Left panel: Western blot analysis of total (T) and immunoprecipitated (Ip) proteins using antibodies specific to DDX3. The results are representative of two independent experiments. Right panel: The amounts of ATF4 mRNA and actin mRNA (as control) present in DDX3 precipitate are calculated relative to those in IgG precipitate, then were normalized against GAPDH mRNA. ( B , C ) Thap induces ATF4 expression more efficiently than Sor. ( B ) Hep3B were treated with either Sor (10 μM) or Thap (100 nM) then lysed and their protein contents were analyzed by western blot for the expression of ATF4 and tubulin (Tub; loading control) using the corresponding antibodies. P-eIF2α serves a positive control for drugs treatment and the pan-eIF2α is used as control for P-eIF2α. *** P ≤ 0.001; **** P ≤ 0.0001. ( C , D ) Translational luciferase assays. ( C ) Hep3B cells are co-transfected with a luciferase expressing vector containing the human ATF4 5′-UTR fused to Firefly luciferase (FLuc) gene, and the control plasmid expressing Renilla luciferase (RLuc). Cells are then treated with either Sor or Thap to induce translation of FLuc whose activity is measured in the cell extracts and expressed relative to RLuc. Error bars correspond to the S.D. ( D ) Hep3B expressing DDX3-a or control (Ct) siRNAs are co-transfected with a luciferase expressing vector containing the human ATF4 5′-UTR fused to FLuc gene, and the control plasmid expressing RLuc. Cells are then treated with Thap to induce translation of FLuc whose activity is measured as above. ** P ≤ 0.01. ( E , F ) Analysis of the ATF4 mRNA association with polyribosomes. Hep3B were incubated with either DDX3 or control (Ct) siRNAs, then treated with Sor. Cytoplasmic extracts of Sor-treated Hep3B cells were fractionated onto 15% (Top) and 55% (Bot: Bottom) sucrose gradients and their polyribosomes profiles ( E ) were recorded by measuring the OD 254 , as described 27 . LP: light polyribosomes. HP: heavy polyribosomes. ( F ) RNA content was isolated from pooled LP and HP fractions and associated ATF4 mRNA was quantified by qRT-PCR using the ΔΔCt method. ATF4 mRNA levels were normalized against 18 s ribosomal RNA and expressed as indicated (left panels). DDX3 mRNA present in LP and HP was similarly quantified and serves as an additional control for DDX3 mRNA knockdown (right panel). The results are representative of two independent experiments. ( G ) Top panels: cytoplasmic extracts prepared from either untreated or Sor-treated Hep3B were fractionated through 15–55% (w/v) sucrose density gradients and their polyribosomes profiles were monitored as above. Bottom panels: Western blot analysis of the collected fractions for the distribution of DDX3 and as a control S6 protein using specific antibodies. The results are representative of two independent experiments.

    Techniques Used: Expressing, Immunoprecipitation, Quantitative RT-PCR, Isolation, Western Blot, Positive Control, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Incubation

    9) Product Images from "Non-targeting siRNA induces NPGPx expression to cooperate with exoribonuclease XRN2 for releasing the stress"

    Article Title: Non-targeting siRNA induces NPGPx expression to cooperate with exoribonuclease XRN2 for releasing the stress

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr714

    NPGPx expression was induced by non-targeting shRNA/RNAi . ( a and b ) Western blot analysis of NPGPx, p84 and α-tubulin proteins from WI38 cells infected with lentivirus carrying shLuc, shRFP, shLacZ, GFP, shEmpty (shRNA cloning vector) or uninfected as control. Specific antibodies against each protein were used as probes. p84 and α-tubulin served as internal loading controls. ( c ) Western blot analysis of NPGPx protein level in WI38 cells transfected with various amount of non-targeting siRNA (CTRL siRNA). Relative NPGPx/p84 ratio was shown in below. ( d ) NPGPx mRNA expression analyzed by RT–qPCR from WI38 cells infected with lentivirus carrying with shLuc, GFP or control vector (shEmpty) or uninfected as control (mock). Relative NPGPx mRNA level (normalized with β-actin and compare with Mock) was shown. ( e and f ) RT–qPCR analysis. WI38 cells transfected with non-targeting siRNA (Ctrl siRNA, 160 nM) or PPIB-1 siRNA were used in this assay. The PPIB-1 mRNA amount (e) and NPGPx mRNA expression level (f) were shown. Mock: WI38 cells without siRNA transfection. ( g ) Western blot analysis of WI38 cells infected with vector control (shEmpty) or shLuc, and then transfected with either pBSK or Luciferase expression vector (pGL3-Luc). Cell lysates were harvested 48 h after transfection for western blot analysis. Relative expression ratio of NPGPx/α-tubulin (Rel. NP/α-tu) was calculated. ( h ) Expressions of stress-related proteins including NPGPx, Calnexin, GRP78, eIF2α, phospho-eIF2α (p-eIF2α) in WI38 cells infected with lentivirus carrying shLuc, GFP, shRNA cloning vector (shEmpty) or uninfected as control (mock). α-Tubulin served as an internal loading control. ( i ) Expression of unspliced (uXBP1) or spliced (sXBP1) XBP1 mRNA analyzed by RT–PCR from WI38 cells infected with lentivirus carrying with shLuc, shRFP, shEmpty or uninfected as the control (mock). Cells treated with tunicamycin (2 µg/ml for 8 h) served as a positive control, where spliced XBP1 (sXBP1) was detected. Ribosomal RNA S26 served as an internal loading control. Each experiment has been repeated at least twice, and the representative data from one of these experiments were shown.
    Figure Legend Snippet: NPGPx expression was induced by non-targeting shRNA/RNAi . ( a and b ) Western blot analysis of NPGPx, p84 and α-tubulin proteins from WI38 cells infected with lentivirus carrying shLuc, shRFP, shLacZ, GFP, shEmpty (shRNA cloning vector) or uninfected as control. Specific antibodies against each protein were used as probes. p84 and α-tubulin served as internal loading controls. ( c ) Western blot analysis of NPGPx protein level in WI38 cells transfected with various amount of non-targeting siRNA (CTRL siRNA). Relative NPGPx/p84 ratio was shown in below. ( d ) NPGPx mRNA expression analyzed by RT–qPCR from WI38 cells infected with lentivirus carrying with shLuc, GFP or control vector (shEmpty) or uninfected as control (mock). Relative NPGPx mRNA level (normalized with β-actin and compare with Mock) was shown. ( e and f ) RT–qPCR analysis. WI38 cells transfected with non-targeting siRNA (Ctrl siRNA, 160 nM) or PPIB-1 siRNA were used in this assay. The PPIB-1 mRNA amount (e) and NPGPx mRNA expression level (f) were shown. Mock: WI38 cells without siRNA transfection. ( g ) Western blot analysis of WI38 cells infected with vector control (shEmpty) or shLuc, and then transfected with either pBSK or Luciferase expression vector (pGL3-Luc). Cell lysates were harvested 48 h after transfection for western blot analysis. Relative expression ratio of NPGPx/α-tubulin (Rel. NP/α-tu) was calculated. ( h ) Expressions of stress-related proteins including NPGPx, Calnexin, GRP78, eIF2α, phospho-eIF2α (p-eIF2α) in WI38 cells infected with lentivirus carrying shLuc, GFP, shRNA cloning vector (shEmpty) or uninfected as control (mock). α-Tubulin served as an internal loading control. ( i ) Expression of unspliced (uXBP1) or spliced (sXBP1) XBP1 mRNA analyzed by RT–PCR from WI38 cells infected with lentivirus carrying with shLuc, shRFP, shEmpty or uninfected as the control (mock). Cells treated with tunicamycin (2 µg/ml for 8 h) served as a positive control, where spliced XBP1 (sXBP1) was detected. Ribosomal RNA S26 served as an internal loading control. Each experiment has been repeated at least twice, and the representative data from one of these experiments were shown.

    Techniques Used: Expressing, shRNA, Western Blot, Infection, Clone Assay, Plasmid Preparation, Transfection, Quantitative RT-PCR, Luciferase, Reverse Transcription Polymerase Chain Reaction, Positive Control

    10) Product Images from "Role of endothelial‐to‐mesenchymal transition induced by TGF‐β1 in transplant kidney interstitial fibrosis"

    Article Title: Role of endothelial‐to‐mesenchymal transition induced by TGF‐β1 in transplant kidney interstitial fibrosis

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13157

    TGF‐β1 promotes α‐SMA and collagen I expression and suppresses VE‐cadherin and CD31 expression in the HUVECs. ( A and B ) Equal amounts of protein from whole cell lysates were analysed by western blotting with antibodies against α‐SMA, collagen I, VE‐cadherin, CD31 and GAPDH after incubation of HUVECs with 5 ng/ml TGF‐β1 for the indicated time points ( A ) or stimulation of HUVECs with various concentrations of TGF‐β1 for 48 hrs ( B ). As can be seen, TGF‐β1 promoted the expression of α‐SMA and collagen I and suppressed the expression of VE‐cadherin and CD31 in a time‐ and dose‐dependent manner. ( C–J ) HUVECs were stimulated with TGF‐β1 (5 ng/ml) for the indicated time points (C, E, G and I) or stimulated with various concentrations of TGF‐β1 for 48 hrs ( D, F, H and J ). Total RNA was isolated and reverse transcribed, and the resultant RNA was subjected to quantitative real‐time PCR to detect the gene expression of α‐SMA, collagen I, VE‐cadherin and CD31. The results of quantitative real‐time PCR were normalized to β2‐macroglobulin expression and expressed as the fold‐change relative to unstimulated control cells. Relative abundance of mRNAs is presented as the mean ± S.D. value of three independent experiments. The PCR results were in agreement with the western blot results. * P
    Figure Legend Snippet: TGF‐β1 promotes α‐SMA and collagen I expression and suppresses VE‐cadherin and CD31 expression in the HUVECs. ( A and B ) Equal amounts of protein from whole cell lysates were analysed by western blotting with antibodies against α‐SMA, collagen I, VE‐cadherin, CD31 and GAPDH after incubation of HUVECs with 5 ng/ml TGF‐β1 for the indicated time points ( A ) or stimulation of HUVECs with various concentrations of TGF‐β1 for 48 hrs ( B ). As can be seen, TGF‐β1 promoted the expression of α‐SMA and collagen I and suppressed the expression of VE‐cadherin and CD31 in a time‐ and dose‐dependent manner. ( C–J ) HUVECs were stimulated with TGF‐β1 (5 ng/ml) for the indicated time points (C, E, G and I) or stimulated with various concentrations of TGF‐β1 for 48 hrs ( D, F, H and J ). Total RNA was isolated and reverse transcribed, and the resultant RNA was subjected to quantitative real‐time PCR to detect the gene expression of α‐SMA, collagen I, VE‐cadherin and CD31. The results of quantitative real‐time PCR were normalized to β2‐macroglobulin expression and expressed as the fold‐change relative to unstimulated control cells. Relative abundance of mRNAs is presented as the mean ± S.D. value of three independent experiments. The PCR results were in agreement with the western blot results. * P

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

    11) Product Images from "Transcriptome sequencing and analysis during seed growth and development in Euryale ferox Salisb"

    Article Title: Transcriptome sequencing and analysis during seed growth and development in Euryale ferox Salisb

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-4707-9

    Relative expression levels of six DEGs in four developmental stages of Euryale ferox Salisb. seed. We compared expression levels determined by RNA-seq and qRT-PCR analyses for each DEG
    Figure Legend Snippet: Relative expression levels of six DEGs in four developmental stages of Euryale ferox Salisb. seed. We compared expression levels determined by RNA-seq and qRT-PCR analyses for each DEG

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

    12) Product Images from "Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in HepG2.2.15 Cells via JAK/STAT Signaling"

    Article Title: Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in HepG2.2.15 Cells via JAK/STAT Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0156496

    HBV expression was inhibited in Hepg2.2.15 cells after transfected with shUSP18 or shRR lentiviruses. (A) HBV pgRNA and total RNA levels were measured by quantitative real-time PCR. HepG2.2.15 cells were transduced with shUSP18 or shRR lentiviral particles, and then the supernatant was collected at day 1, 2, 3, 5, 7. (B) The extracellular HBV DNA load in the culture was quantified using quantitative real-time PCR kits. The secretion of HBV DNA was suppressed after transduced with shUSP18. (C) and (D) The supernatant was collected for detection of HBsAg and HBeAg using ELISA kits at day 1, 2, 3, 5, 7. The secretion of HBsAg and HBeAg was suppressed after transduced with shUSP18. The results are presented as the means ± SD, n = 3, error bars indicate SD. *P
    Figure Legend Snippet: HBV expression was inhibited in Hepg2.2.15 cells after transfected with shUSP18 or shRR lentiviruses. (A) HBV pgRNA and total RNA levels were measured by quantitative real-time PCR. HepG2.2.15 cells were transduced with shUSP18 or shRR lentiviral particles, and then the supernatant was collected at day 1, 2, 3, 5, 7. (B) The extracellular HBV DNA load in the culture was quantified using quantitative real-time PCR kits. The secretion of HBV DNA was suppressed after transduced with shUSP18. (C) and (D) The supernatant was collected for detection of HBsAg and HBeAg using ELISA kits at day 1, 2, 3, 5, 7. The secretion of HBsAg and HBeAg was suppressed after transduced with shUSP18. The results are presented as the means ± SD, n = 3, error bars indicate SD. *P

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

    13) Product Images from "Prominent Bone Loss Mediated by RANKL and IL-17 Produced by CD4+ T Cells in TallyHo/JngJ Mice"

    Article Title: Prominent Bone Loss Mediated by RANKL and IL-17 Produced by CD4+ T Cells in TallyHo/JngJ Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018168

    Imbalance of osteoblasts and osteoclasts in bone marrow of TH mice. (A) Total RNA was isolated from bone marrow cells of B6 and TH mice at 8 weeks of age (n = 6) and used for real time-PCR analysis. Relative expression levels of OCN, OPG, RANKL, and IL-6 were calculated after normalization to the level of β-actin. (B) Culture supernatants were collected from bone marrow cells, and RANKL and IL-6 levels were determined by ELISA. (C) Bone marrow cells were isolated and cultured for 24 h. After removal of floating cells, cells were maintained in DMEM supplemented with ascorbic acid (50 µg/ml), β-glycerophosphate (2 mM) and dexamethasone (10 nM) for 8 days. Cells were fixed and stained with either ALP or TRAP.
    Figure Legend Snippet: Imbalance of osteoblasts and osteoclasts in bone marrow of TH mice. (A) Total RNA was isolated from bone marrow cells of B6 and TH mice at 8 weeks of age (n = 6) and used for real time-PCR analysis. Relative expression levels of OCN, OPG, RANKL, and IL-6 were calculated after normalization to the level of β-actin. (B) Culture supernatants were collected from bone marrow cells, and RANKL and IL-6 levels were determined by ELISA. (C) Bone marrow cells were isolated and cultured for 24 h. After removal of floating cells, cells were maintained in DMEM supplemented with ascorbic acid (50 µg/ml), β-glycerophosphate (2 mM) and dexamethasone (10 nM) for 8 days. Cells were fixed and stained with either ALP or TRAP.

    Techniques Used: Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining, ALP Assay

    14) Product Images from "Pathway of Toll-Like Receptor 7/B Cell Activating Factor/B Cell Activating Factor Receptor Plays a Role in Immune Thrombocytopenia In Vivo"

    Article Title: Pathway of Toll-Like Receptor 7/B Cell Activating Factor/B Cell Activating Factor Receptor Plays a Role in Immune Thrombocytopenia In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022708

    The platelet counts and levels of TLR7 in ITP mice and controls. (A) Changes in relative platelet counts of controls (n = 8) and experimental mice (n = 10). With rat platelet injection, experimental mice developed progressively thrombocytopenia. The relative platelet counts in experimental mice reached the minimum 3 weeks after the first immunization (0.49±0.15 vs 1, P = 0.000). There was no significant change in controls. SMCs were isolated from ITP mice (n = 10) and controls (n = 8) for RNA and protein extraction. (B) Results of real-time PCR analysis showed that the ratio of TLR7 mRNA in ITP mice compared to that of controls was 3.14 (P = 0.003). (C) Increased levels of TLR7 protein in ITP mice were revealed by western blot. The graph showed the densitometric quantitation of TLR7 to the housekeeping gene GAPDH. Bars represent SD, * represents P
    Figure Legend Snippet: The platelet counts and levels of TLR7 in ITP mice and controls. (A) Changes in relative platelet counts of controls (n = 8) and experimental mice (n = 10). With rat platelet injection, experimental mice developed progressively thrombocytopenia. The relative platelet counts in experimental mice reached the minimum 3 weeks after the first immunization (0.49±0.15 vs 1, P = 0.000). There was no significant change in controls. SMCs were isolated from ITP mice (n = 10) and controls (n = 8) for RNA and protein extraction. (B) Results of real-time PCR analysis showed that the ratio of TLR7 mRNA in ITP mice compared to that of controls was 3.14 (P = 0.003). (C) Increased levels of TLR7 protein in ITP mice were revealed by western blot. The graph showed the densitometric quantitation of TLR7 to the housekeeping gene GAPDH. Bars represent SD, * represents P

    Techniques Used: Mouse Assay, Injection, Isolation, Protein Extraction, Real-time Polymerase Chain Reaction, Western Blot, Quantitation Assay

    15) Product Images from "Hepatoprotective Effect of Polysaccharides Isolated from Dendrobium officinale against Acetaminophen-Induced Liver Injury in Mice via Regulation of the Nrf2-Keap1 Signaling Pathway"

    Article Title: Hepatoprotective Effect of Polysaccharides Isolated from Dendrobium officinale against Acetaminophen-Induced Liver Injury in Mice via Regulation of the Nrf2-Keap1 Signaling Pathway

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2018/6962439

    Effects of polysaccharides isolated from Dendrobium officinale (DOP) on the mRNA expression levels of GCLC (a), GCLM (b), NQO1 (c), and HO-1 (d). Total RNA from liver tissues was extracted and reverse transcribed into cDNA prior to quantitative real-time PCR analysis to detect mRNA expression of GCLC, GCLM, NQO1, and HO-1. Data are presented as mean ± SEM ( n = 3). ## P
    Figure Legend Snippet: Effects of polysaccharides isolated from Dendrobium officinale (DOP) on the mRNA expression levels of GCLC (a), GCLM (b), NQO1 (c), and HO-1 (d). Total RNA from liver tissues was extracted and reverse transcribed into cDNA prior to quantitative real-time PCR analysis to detect mRNA expression of GCLC, GCLM, NQO1, and HO-1. Data are presented as mean ± SEM ( n = 3). ## P

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

    16) Product Images from "A novel chalcone derivative has antitumor activity in melanoma by inducing DNA damage through the upregulation of ROS products"

    Article Title: A novel chalcone derivative has antitumor activity in melanoma by inducing DNA damage through the upregulation of ROS products

    Journal: Cancer Cell International

    doi: 10.1186/s12935-020-1114-5

    RNA-seq analyses of the effect of lj-1-59 on the gene expression profile. a The heatmap of SK-Mel-28 after lj-1-59 treatment. b Top 20 enriched KEGG pathways after lj-1-59 treated. c GSEA enrichment plots after lj-1-59 treated, and Normalized enrichment score (NES) and Normalized p -value (P) are shown in each plot. d SK-Mel-28 cells were treated with 5 µM lj-1-59 for 48 h. Then extract total RNA to Q-RT-PCR analysis as described in “ Methods ”. The results are expressed as the mean (n = 6) ± SD. Significant differences were evaluated using Student’s t-test, and an asterisk (*) indicates a significant difference ( p
    Figure Legend Snippet: RNA-seq analyses of the effect of lj-1-59 on the gene expression profile. a The heatmap of SK-Mel-28 after lj-1-59 treatment. b Top 20 enriched KEGG pathways after lj-1-59 treated. c GSEA enrichment plots after lj-1-59 treated, and Normalized enrichment score (NES) and Normalized p -value (P) are shown in each plot. d SK-Mel-28 cells were treated with 5 µM lj-1-59 for 48 h. Then extract total RNA to Q-RT-PCR analysis as described in “ Methods ”. The results are expressed as the mean (n = 6) ± SD. Significant differences were evaluated using Student’s t-test, and an asterisk (*) indicates a significant difference ( p

    Techniques Used: RNA Sequencing Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

    17) Product Images from "Molecular and functional characterization of a novel chromoplast-specific lycopene ?-cyclase from Citrus and its relation to lycopene accumulation"

    Article Title: Molecular and functional characterization of a novel chromoplast-specific lycopene ?-cyclase from Citrus and its relation to lycopene accumulation

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erp048

    Quantitative RT-PCR analysis of the expression of β-LCY1 , β-LCY2 , and β-CHX genes in the flavedo and the pulp of Navel orange ( C. sinensis ) (black bars) and Star Ruby grapefruit ( C. paradisi ) (white bars) at the IG (immature green), MG (mature green), B (breaker), and FC (full-colour) stages. The levels of expression were normalized to the amount of RNA and the value of Navel flavedo at the FC stage was set to 100. The data are means ±SD of three experimental replicates.
    Figure Legend Snippet: Quantitative RT-PCR analysis of the expression of β-LCY1 , β-LCY2 , and β-CHX genes in the flavedo and the pulp of Navel orange ( C. sinensis ) (black bars) and Star Ruby grapefruit ( C. paradisi ) (white bars) at the IG (immature green), MG (mature green), B (breaker), and FC (full-colour) stages. The levels of expression were normalized to the amount of RNA and the value of Navel flavedo at the FC stage was set to 100. The data are means ±SD of three experimental replicates.

    Techniques Used: Quantitative RT-PCR, Expressing

    18) Product Images from "Myeloid Sirtuin 2 Expression Does Not Impact Long-Term Mycobacterium tuberculosis Control"

    Article Title: Myeloid Sirtuin 2 Expression Does Not Impact Long-Term Mycobacterium tuberculosis Control

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0131904

    The expression of inflammatory mediators in infected lungs is not altered in the absence of myeloid Sirt2. RNA was extracted from the lung tissue after 30 days of infection and the expression of Ifn γ, Il17 , Tnf , Il6 and Nos2 was analyzed by real-time PCR and normalized to the expression of ubiquitin. Data shows the mean ± SEM value (n = 5–6) and the significance was determined by the Student’s t -test. The data are representative of two independent experiments.
    Figure Legend Snippet: The expression of inflammatory mediators in infected lungs is not altered in the absence of myeloid Sirt2. RNA was extracted from the lung tissue after 30 days of infection and the expression of Ifn γ, Il17 , Tnf , Il6 and Nos2 was analyzed by real-time PCR and normalized to the expression of ubiquitin. Data shows the mean ± SEM value (n = 5–6) and the significance was determined by the Student’s t -test. The data are representative of two independent experiments.

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

    19) Product Images from "Mycobacterium tuberculosis Strains Are Differentially Recognized by TLRs with an Impact on the Immune Response"

    Article Title: Mycobacterium tuberculosis Strains Are Differentially Recognized by TLRs with an Impact on the Immune Response

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067277

    Mtb Bj strain 02-171 is more virulent during the innate immune response than H37Rv. (A) WT mice were intranasally infected with Mtb strains H37Rv (white bars) or 02-171 (black bars) and at the indicated time points post-infection, lung cell suspensions were prepared, diluted and plated into 7H11 agar to determine the number of mycobacterial CFUs in the lung. The bacterial burden 24 h post-infection was Log10(CFU) 2.67±0.57 and Log10(CFU) 2.45±0.76 (Mean±SEM for 6 animals per strain) for Mtb strains H37Rv or 02-171 infected mice, respectively. (B) WT mice were infected as before and, at the indicated time points post-infection, sections were prepared from formalin-fixed lungs. HE stained tissue was blindly analysed and lung inflammation scored for each animal within the group (6 animals). Inflammation scores were: absent = 0; mild = 1; abundant = 2; severe = 3; exacerbated = 4. (C) WT mice were infected with Mtb strains H37Rv (open circles) or 02-171 (close circles), RNA extracted from the lung tissue and the expression of TNF, IFN-β, IFN-γ and iNOS analyzed by quantitative real-time PCR and normalized to the expression of ubiquitin. Data represented for day 0 correspond to uninfected animals. For A, B and C data points show the Mean±SEM for 5 mice per group and the significance was determined by the Student’s t-test (*,p≤0.05; **,p≤0.01; ***,p≤0.001) for each time point, between Mtb strains 02-171 and H37Rv. The data are representative of three (A and B) or two (C) independent experiments. (D) Kaplan-Meier survival analysis of WT (solid lines) or Rag2 −/− (dashed lines) mice infected intranasally with Mtb strains H37Rv (open circles) or 02-171 (close circles). H37Rv-infected mice showed improved survival (49 days vs 33 days with 02-171-infected mice), as calculated by log-rank test (***,p≤0.0001). WT animals infected in parallel survived up to day 200 post-infection. The data are representative of two independent experiments. (E) WT or Rag2 −/− mice were infected with Mtb strains H37Rv or 02-171 and lung CFU numbers determined on days 18 and 30 post-infection as indicated before. Data points show the Mean±SEM for 6 mice per group. The statistics analysis between the different groups was determined by the Student’s t-test (**,p≤0.01; ***,p≤0.001).
    Figure Legend Snippet: Mtb Bj strain 02-171 is more virulent during the innate immune response than H37Rv. (A) WT mice were intranasally infected with Mtb strains H37Rv (white bars) or 02-171 (black bars) and at the indicated time points post-infection, lung cell suspensions were prepared, diluted and plated into 7H11 agar to determine the number of mycobacterial CFUs in the lung. The bacterial burden 24 h post-infection was Log10(CFU) 2.67±0.57 and Log10(CFU) 2.45±0.76 (Mean±SEM for 6 animals per strain) for Mtb strains H37Rv or 02-171 infected mice, respectively. (B) WT mice were infected as before and, at the indicated time points post-infection, sections were prepared from formalin-fixed lungs. HE stained tissue was blindly analysed and lung inflammation scored for each animal within the group (6 animals). Inflammation scores were: absent = 0; mild = 1; abundant = 2; severe = 3; exacerbated = 4. (C) WT mice were infected with Mtb strains H37Rv (open circles) or 02-171 (close circles), RNA extracted from the lung tissue and the expression of TNF, IFN-β, IFN-γ and iNOS analyzed by quantitative real-time PCR and normalized to the expression of ubiquitin. Data represented for day 0 correspond to uninfected animals. For A, B and C data points show the Mean±SEM for 5 mice per group and the significance was determined by the Student’s t-test (*,p≤0.05; **,p≤0.01; ***,p≤0.001) for each time point, between Mtb strains 02-171 and H37Rv. The data are representative of three (A and B) or two (C) independent experiments. (D) Kaplan-Meier survival analysis of WT (solid lines) or Rag2 −/− (dashed lines) mice infected intranasally with Mtb strains H37Rv (open circles) or 02-171 (close circles). H37Rv-infected mice showed improved survival (49 days vs 33 days with 02-171-infected mice), as calculated by log-rank test (***,p≤0.0001). WT animals infected in parallel survived up to day 200 post-infection. The data are representative of two independent experiments. (E) WT or Rag2 −/− mice were infected with Mtb strains H37Rv or 02-171 and lung CFU numbers determined on days 18 and 30 post-infection as indicated before. Data points show the Mean±SEM for 6 mice per group. The statistics analysis between the different groups was determined by the Student’s t-test (**,p≤0.01; ***,p≤0.001).

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

    20) Product Images from "Knockdown of Midgut Genes by dsRNA-Transgenic Plant-Mediated RNA Interference in the Hemipteran Insect Nilaparvata lugens"

    Article Title: Knockdown of Midgut Genes by dsRNA-Transgenic Plant-Mediated RNA Interference in the Hemipteran Insect Nilaparvata lugens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020504

    Expression of Nlsid-1 and Nlaub in N. lugens. (A) Developmental expression of Nlsid-1 and Nlaub in N. lugens from 1 st nymph to male adult (Ma) and female adult (Fa). (B) Tissue distribution of Nlsid-1 and Nlaub in N. lugens 3 rd instar nymph. qRT-PCR analyses were performed using total RNA from midgut (Mi), salivary gland (Sg), fat body (Fb), cuticle (Cu), leg (Le), and head (He). Data shown are means ± standard errors (N = 3).
    Figure Legend Snippet: Expression of Nlsid-1 and Nlaub in N. lugens. (A) Developmental expression of Nlsid-1 and Nlaub in N. lugens from 1 st nymph to male adult (Ma) and female adult (Fa). (B) Tissue distribution of Nlsid-1 and Nlaub in N. lugens 3 rd instar nymph. qRT-PCR analyses were performed using total RNA from midgut (Mi), salivary gland (Sg), fat body (Fb), cuticle (Cu), leg (Le), and head (He). Data shown are means ± standard errors (N = 3).

    Techniques Used: Expressing, Quantitative RT-PCR

    21) Product Images from "Mutual regulation between GDF11 and TET2 prevents senescence of mesenchymal stem cells"

    Article Title: Mutual regulation between GDF11 and TET2 prevents senescence of mesenchymal stem cells

    Journal: bioRxiv

    doi: 10.1101/2020.03.30.008722

    Influences of GDF11 on replicative senescence of hMSC. (A and B) Oil Red O staining to measure adipogenic ability of hMSC at an earlier passage (p13), a later passage (p22) and a later passage (p22) treated with GDF11 (40ng/ml). Scale bar, 100μm. (C and D) Alizarin Red S staining to determine osteogenic potential of hMSC at an earlier passage (P11), a later passage (p24) and a later passage (p24) treated with GDF11 (40ng/ml). Scale bar, 100μm. (E and F) SA-β-gal staining of hMSC treated with GDF11 (40ng/ml) for 48h at a late passage (p23). Scale bar, 100μm. (G and H) Immunofluorescence staining and quantification of Ki67 in hMSC treated with GDF11 (40ng/ml) for 48h at a later passage (p23). Scale bar, 1000μm. (I) Cell cycle phase distributions of hMSC treated with GDF11 (40ng/ml) for 48h at passage (p23) were determined using flow cytometry. (J) Mitochondrial Reactive oxygen species (ROS) in hMSC with or without GDF11 treatment for 48h at passage 24 (p24). (K) qPCR detection of relative telomere lengths of hMSC treated with GDF11 (40ng/ml) for 48h at passage 23 (p23). (L-M) qRT-PCR detection of a telomerase RNA component (TERC) and GDF11 expression. Data are shown as mean ± SEM; statistical analysis was performed with t test in (B, F, H to M); *P
    Figure Legend Snippet: Influences of GDF11 on replicative senescence of hMSC. (A and B) Oil Red O staining to measure adipogenic ability of hMSC at an earlier passage (p13), a later passage (p22) and a later passage (p22) treated with GDF11 (40ng/ml). Scale bar, 100μm. (C and D) Alizarin Red S staining to determine osteogenic potential of hMSC at an earlier passage (P11), a later passage (p24) and a later passage (p24) treated with GDF11 (40ng/ml). Scale bar, 100μm. (E and F) SA-β-gal staining of hMSC treated with GDF11 (40ng/ml) for 48h at a late passage (p23). Scale bar, 100μm. (G and H) Immunofluorescence staining and quantification of Ki67 in hMSC treated with GDF11 (40ng/ml) for 48h at a later passage (p23). Scale bar, 1000μm. (I) Cell cycle phase distributions of hMSC treated with GDF11 (40ng/ml) for 48h at passage (p23) were determined using flow cytometry. (J) Mitochondrial Reactive oxygen species (ROS) in hMSC with or without GDF11 treatment for 48h at passage 24 (p24). (K) qPCR detection of relative telomere lengths of hMSC treated with GDF11 (40ng/ml) for 48h at passage 23 (p23). (L-M) qRT-PCR detection of a telomerase RNA component (TERC) and GDF11 expression. Data are shown as mean ± SEM; statistical analysis was performed with t test in (B, F, H to M); *P

    Techniques Used: Staining, Immunofluorescence, Flow Cytometry, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    22) Product Images from "Type I IFN Inhibits Alternative Macrophage Activation during Mycobacterium tuberculosis Infection and Leads to Enhanced Protection in the Absence of IFN-γ Signaling"

    Article Title: Type I IFN Inhibits Alternative Macrophage Activation during Mycobacterium tuberculosis Infection and Leads to Enhanced Protection in the Absence of IFN-γ Signaling

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1600584

    Concomitant loss of type I and type II IFN signaling increases the expression of genes associated with alternatively activated macrophages in M. tuberculosis –infected lungs. WT, Ifnar −/− , Ifngr −/− , and Ifngr −/− × Ifnar −/− (dKO) mice were infected with the M. tuberculosis strain BTB 02-171. At day 20 postinfection, RNA was extracted from infected lungs and Nos2 ( A ), Arg1 ( B ), Ym1 ( C ), Fizz1 ( D ), Tnf ( E ), Il4 ( F ), Il5 ( G ), Il13 ( H ), and Il10 ( I ) mRNA expression was analyzed by quantitative real-time PCR and normalized to the expression of Hprt1 , with the exception of Tnf , which was normalized to Ubiquitin expression. Each bar represents mean ± SEM for five mice per group. Data are representative of two independent experiments. Significance is shown relative to WT (*), Ifnar −/− ( # ), or Ifngr −/− ( § ). * , # , § p
    Figure Legend Snippet: Concomitant loss of type I and type II IFN signaling increases the expression of genes associated with alternatively activated macrophages in M. tuberculosis –infected lungs. WT, Ifnar −/− , Ifngr −/− , and Ifngr −/− × Ifnar −/− (dKO) mice were infected with the M. tuberculosis strain BTB 02-171. At day 20 postinfection, RNA was extracted from infected lungs and Nos2 ( A ), Arg1 ( B ), Ym1 ( C ), Fizz1 ( D ), Tnf ( E ), Il4 ( F ), Il5 ( G ), Il13 ( H ), and Il10 ( I ) mRNA expression was analyzed by quantitative real-time PCR and normalized to the expression of Hprt1 , with the exception of Tnf , which was normalized to Ubiquitin expression. Each bar represents mean ± SEM for five mice per group. Data are representative of two independent experiments. Significance is shown relative to WT (*), Ifnar −/− ( # ), or Ifngr −/− ( § ). * , # , § p

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

    23) Product Images from "Targeting skeletal endothelium to ameliorate bone loss"

    Article Title: Targeting skeletal endothelium to ameliorate bone loss

    Journal: Nature medicine

    doi: 10.1038/s41591-018-0020-z

    Inhibition of Shn3 enhances Slit3 expression in osteoblasts. ( a , b ) Representative images ( a ) and relative quantification ( b ) of a transwell migration assay of BM-derived endothelial progenitor outgrowth cells (EPOCs). Basal medium, (BM); Conditioned medium, (CM). n = 5 per group. ( c , d ) Representative images ( c ) and relative quantification of tube branch numbers ( d ) of a Matrigel tube formation assay with EPOCs. n = 5 per group. ( e ) Gene ontology (GO) enrichment analysis of genes differentially expressed in Shn3 −/− osteoblasts relative to Shn3 +/+ osteoblasts ( f ) Proangiogenic gene expression in primary Shn3 +/+ and Shn3 −/− osteoblasts. ( g ) Real-time PCR of Slit3 expression in Shn3 +/+ and Shn3 −/− osteoblasts. n = 4 per group. ( h ) Messenger RNA (mRNA) (left) and protein (right) levels of Slit3 in human mesenchymal stromal cells (hMSCs) expressing a GFP targeting control or Shn3 shRNAs cultured under osteogenic conditions. n = 4 per group. ( i ) mRNA (left) and protein (right) levels of Slit3 in hMSCs overexpressing a vector control or Shn3 cultured under osteogenic conditions.. n = 4 per group. ( j ) ELISA for SLIT3 secretion by Shn3 +/+ and Shn3 −/− osteoblasts. n = 6 per group. ( k ) Real-time PCR analysis of Slit3 in Shn3 +/+ and Shn3 KI/KI osteoblasts ( n = 4). ( l ) Real-time PCR analysis of Slit3 in hMSCs treated with trametinib (TTNB). n = 6 per group. ( m ) Representative confocal images ( n = 3 total images per group) of CD31 (green) and EMCN (red) immunostained sections from the femurs of 2-week-old Shn3 +/+ and Shn3 KI/KI male mice. Growth plate is marked with a dashed line. Scale bars, 100μm. Values represent mean ± s.e.m.; * P
    Figure Legend Snippet: Inhibition of Shn3 enhances Slit3 expression in osteoblasts. ( a , b ) Representative images ( a ) and relative quantification ( b ) of a transwell migration assay of BM-derived endothelial progenitor outgrowth cells (EPOCs). Basal medium, (BM); Conditioned medium, (CM). n = 5 per group. ( c , d ) Representative images ( c ) and relative quantification of tube branch numbers ( d ) of a Matrigel tube formation assay with EPOCs. n = 5 per group. ( e ) Gene ontology (GO) enrichment analysis of genes differentially expressed in Shn3 −/− osteoblasts relative to Shn3 +/+ osteoblasts ( f ) Proangiogenic gene expression in primary Shn3 +/+ and Shn3 −/− osteoblasts. ( g ) Real-time PCR of Slit3 expression in Shn3 +/+ and Shn3 −/− osteoblasts. n = 4 per group. ( h ) Messenger RNA (mRNA) (left) and protein (right) levels of Slit3 in human mesenchymal stromal cells (hMSCs) expressing a GFP targeting control or Shn3 shRNAs cultured under osteogenic conditions. n = 4 per group. ( i ) mRNA (left) and protein (right) levels of Slit3 in hMSCs overexpressing a vector control or Shn3 cultured under osteogenic conditions.. n = 4 per group. ( j ) ELISA for SLIT3 secretion by Shn3 +/+ and Shn3 −/− osteoblasts. n = 6 per group. ( k ) Real-time PCR analysis of Slit3 in Shn3 +/+ and Shn3 KI/KI osteoblasts ( n = 4). ( l ) Real-time PCR analysis of Slit3 in hMSCs treated with trametinib (TTNB). n = 6 per group. ( m ) Representative confocal images ( n = 3 total images per group) of CD31 (green) and EMCN (red) immunostained sections from the femurs of 2-week-old Shn3 +/+ and Shn3 KI/KI male mice. Growth plate is marked with a dashed line. Scale bars, 100μm. Values represent mean ± s.e.m.; * P

    Techniques Used: Inhibition, Expressing, Transwell Migration Assay, Derivative Assay, Tube Formation Assay, Real-time Polymerase Chain Reaction, Cell Culture, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Mouse Assay

    24) Product Images from "Transcriptional corepressor SHP recruits SIRT1 histone deacetylase to inhibit LRH-1 transactivation"

    Article Title: Transcriptional corepressor SHP recruits SIRT1 histone deacetylase to inhibit LRH-1 transactivation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq227

    Utilization of differential repressive mechanism by SHP. Reporter assays ( A–C ) were performed as described in ‘Materials and Methods’ section. HepG2 cells were cotransfected with indicated reporter luciferase vectors (100 ng each) and expression vectors (200 ng each). β-gal expression vector (100 ng) was used as an internal control for each transfected well. Cells were treated with RA (1 µM; panel A, right) or E2 (10 nM; panel B, right) for 12 h followed by TSA (100 nM) or NAM (20 µM) treatments for further 12 h in the absence or presence of RA or E2. Cells were treated only with TSA or NAM (panel A, B; left, middle) for 12 h prior to the measurement of luciferase activity. ( D ) Effect of siRNAs of siSIRT1 and siHDAC1 on the expression of SIRT1 and HDAC1 respectively. HepG2 cells were transfected with pSuper- siSIRT1 or siHDAC1 or pSuper [control (–)] and cells were collected for RNA isolation, by RT–PCR analysis, 72 h post-transfection. β-actin (ACTB) gene expression was shown as control. Data is representative of at least three independently performed experiments.
    Figure Legend Snippet: Utilization of differential repressive mechanism by SHP. Reporter assays ( A–C ) were performed as described in ‘Materials and Methods’ section. HepG2 cells were cotransfected with indicated reporter luciferase vectors (100 ng each) and expression vectors (200 ng each). β-gal expression vector (100 ng) was used as an internal control for each transfected well. Cells were treated with RA (1 µM; panel A, right) or E2 (10 nM; panel B, right) for 12 h followed by TSA (100 nM) or NAM (20 µM) treatments for further 12 h in the absence or presence of RA or E2. Cells were treated only with TSA or NAM (panel A, B; left, middle) for 12 h prior to the measurement of luciferase activity. ( D ) Effect of siRNAs of siSIRT1 and siHDAC1 on the expression of SIRT1 and HDAC1 respectively. HepG2 cells were transfected with pSuper- siSIRT1 or siHDAC1 or pSuper [control (–)] and cells were collected for RNA isolation, by RT–PCR analysis, 72 h post-transfection. β-actin (ACTB) gene expression was shown as control. Data is representative of at least three independently performed experiments.

    Techniques Used: Luciferase, Expressing, Plasmid Preparation, Transfection, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    Involvement of SHP and SIRT1 in the repression of LRH1 target genes. Reporter assay ( A and B ) was performed as described in ‘Materials and Methods’ section. HepG2 cells were cotransfected with luciferase constructs of human CYP7A1 (hCYP7A1–Luc) and human SHP (hSHP–Luc) gene promoters (100 ng each) and indicated expression vectors (200 ng each). β-gal expression vector (100 ng) was used as an internal control for each transfected well. Cells were treated with NAM (20 µM) or resveratrol (Resv, 100 nM) for 12 h prior to the measurement of luficerase activity. ( C ) HepG2 cells were transfected with pcDNA3 only (–) or pcDNA3–HA–LRH1 (400 ng each) or infected with indicated adenovirus vectors (50 MOI) for 36–72 h and cells extracts were for western blot analysis using indicated antibodies. ( D and E ) HepG2 cells were treated with indicated adenovirus vectors (50 MOI) for 36–72 h. Twelve hours prior to RNA extraction cells were treated with NAM (20 µM, lane 3 and 6) and total RNA isolated were used for RT–PCR (top) and qPCR (bottom) analysis of CYP7A1 and SHP gene expression. β-actin (ACTB) gene expression was shown as control. Data is representative of at least three independently performed experiments and shown as mean ± SD; * P
    Figure Legend Snippet: Involvement of SHP and SIRT1 in the repression of LRH1 target genes. Reporter assay ( A and B ) was performed as described in ‘Materials and Methods’ section. HepG2 cells were cotransfected with luciferase constructs of human CYP7A1 (hCYP7A1–Luc) and human SHP (hSHP–Luc) gene promoters (100 ng each) and indicated expression vectors (200 ng each). β-gal expression vector (100 ng) was used as an internal control for each transfected well. Cells were treated with NAM (20 µM) or resveratrol (Resv, 100 nM) for 12 h prior to the measurement of luficerase activity. ( C ) HepG2 cells were transfected with pcDNA3 only (–) or pcDNA3–HA–LRH1 (400 ng each) or infected with indicated adenovirus vectors (50 MOI) for 36–72 h and cells extracts were for western blot analysis using indicated antibodies. ( D and E ) HepG2 cells were treated with indicated adenovirus vectors (50 MOI) for 36–72 h. Twelve hours prior to RNA extraction cells were treated with NAM (20 µM, lane 3 and 6) and total RNA isolated were used for RT–PCR (top) and qPCR (bottom) analysis of CYP7A1 and SHP gene expression. β-actin (ACTB) gene expression was shown as control. Data is representative of at least three independently performed experiments and shown as mean ± SD; * P

    Techniques Used: Reporter Assay, Luciferase, Construct, Expressing, Plasmid Preparation, Transfection, Activity Assay, Infection, Western Blot, RNA Extraction, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    25) Product Images from "The NOTCH1-HEY1 pathway regulates self-renewal and epithelial-mesenchymal transition of salivary adenoid cystic carcinoma cells"

    Article Title: The NOTCH1-HEY1 pathway regulates self-renewal and epithelial-mesenchymal transition of salivary adenoid cystic carcinoma cells

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.36407

    The NOTCH1-HEY1 pathway is a positive feedback loop in SACC cells. (A) The expression of NOTCH1, HEY1, TNNT3, IFI6, RELB, KRT-17, MMP7, TPD52 and ABHD3 genes, which were selected from the RNA-Seq results, was measured by qRT-PCR. (B) Linear regression analysis was used to compare the expression levels of the abovementioned genes between the RNA-Seq and qRT-PCR results. (C, D) Western blot analysis and the quantification of NOTCH1 and HEY1 expression in SACC cells after NOTCH1 upregulation by pcDNA3.1-NICD1 plasmid transfection. (E) The mRNA expression of NOTCH1 and HEY1 after HEY1 downregulation by siRNAs was detected by RT-PCR. (F, G) The protein expression of NOTCH1 and HEY1 when HEY1 inhibition was measured by western blot analysis. *P
    Figure Legend Snippet: The NOTCH1-HEY1 pathway is a positive feedback loop in SACC cells. (A) The expression of NOTCH1, HEY1, TNNT3, IFI6, RELB, KRT-17, MMP7, TPD52 and ABHD3 genes, which were selected from the RNA-Seq results, was measured by qRT-PCR. (B) Linear regression analysis was used to compare the expression levels of the abovementioned genes between the RNA-Seq and qRT-PCR results. (C, D) Western blot analysis and the quantification of NOTCH1 and HEY1 expression in SACC cells after NOTCH1 upregulation by pcDNA3.1-NICD1 plasmid transfection. (E) The mRNA expression of NOTCH1 and HEY1 after HEY1 downregulation by siRNAs was detected by RT-PCR. (F, G) The protein expression of NOTCH1 and HEY1 when HEY1 inhibition was measured by western blot analysis. *P

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction, Inhibition

    26) Product Images from "Lignin Induces ES Cells to Differentiate into Neuroectodermal Cells through Mediation of the Wnt Signaling Pathway"

    Article Title: Lignin Induces ES Cells to Differentiate into Neuroectodermal Cells through Mediation of the Wnt Signaling Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0066376

    Analysis of the effects of lignin on Wnt/β catenin signaling of ES cells. Gene expression analysis by real-time PCR. ES cells cultured in an undifferentiated state were separated from MEF and we added lignin (50 µg/mL) to ES cells. After lignin was added to ES cells, total RNA were collected after 0, 0.5, 1 and 3 hours, and gene expression analysis was conducted by real-time PCR. The expression level of each marker gene was normalized to time 0. Data are expressed as the mean ± SD of the three pone.0066376.g005.tifexperiments. *P
    Figure Legend Snippet: Analysis of the effects of lignin on Wnt/β catenin signaling of ES cells. Gene expression analysis by real-time PCR. ES cells cultured in an undifferentiated state were separated from MEF and we added lignin (50 µg/mL) to ES cells. After lignin was added to ES cells, total RNA were collected after 0, 0.5, 1 and 3 hours, and gene expression analysis was conducted by real-time PCR. The expression level of each marker gene was normalized to time 0. Data are expressed as the mean ± SD of the three pone.0066376.g005.tifexperiments. *P

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

    27) Product Images from "Dexamethasone-induced Intra-Uterine Growth Restriction impacts NOSTRIN and its downstream effector genes in the rat mesometrial uterus"

    Article Title: Dexamethasone-induced Intra-Uterine Growth Restriction impacts NOSTRIN and its downstream effector genes in the rat mesometrial uterus

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26590-3

    Induction of IUGR impairs metrial gland expression of genes that are known to be regulated by NOSTRIN. Real time PCR analysis of transcripts in the metrial gland. The amount of a specific mRNA was normalized relative to the amount of rpL7 (∆Ct = Ct gene − Ct rpL7 ). Fold change of gene expression was measured by using 2 −∆∆Ct , where ∆∆Ct denoted the change in ∆Ct values between Dex and control metrial glands RNA. Error bars represents standard error of mean from three different biological replicates. ( A ) Transcripts of the receptor tyrosine kinases including Flt-1, Kdr, Tek and the pro-angiogeneic ligand Pgf but not Kit was down regulated significantly in IUGR samples. ( B ) IUGR led to decrease in transcript levels of the genes involved in adhesion and invasion such as Itgα5, Itgβ3, Fn1 and Col18a1 . ( C ) Proteases, such as Mmp2, Plau and Adam17 transcripts were decreased in IUGR metrial gland. (D) Pro-inflammatory cytokines such as Il6, Ccl2, Cxcl1 and Cxcl2 were down regulated in IUGR metrial glands. (n = 3, *p
    Figure Legend Snippet: Induction of IUGR impairs metrial gland expression of genes that are known to be regulated by NOSTRIN. Real time PCR analysis of transcripts in the metrial gland. The amount of a specific mRNA was normalized relative to the amount of rpL7 (∆Ct = Ct gene − Ct rpL7 ). Fold change of gene expression was measured by using 2 −∆∆Ct , where ∆∆Ct denoted the change in ∆Ct values between Dex and control metrial glands RNA. Error bars represents standard error of mean from three different biological replicates. ( A ) Transcripts of the receptor tyrosine kinases including Flt-1, Kdr, Tek and the pro-angiogeneic ligand Pgf but not Kit was down regulated significantly in IUGR samples. ( B ) IUGR led to decrease in transcript levels of the genes involved in adhesion and invasion such as Itgα5, Itgβ3, Fn1 and Col18a1 . ( C ) Proteases, such as Mmp2, Plau and Adam17 transcripts were decreased in IUGR metrial gland. (D) Pro-inflammatory cytokines such as Il6, Ccl2, Cxcl1 and Cxcl2 were down regulated in IUGR metrial glands. (n = 3, *p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Effect of Dexamethasone (Dex) induced IUGR on NOSTRIN expression at gestation Day 20.5 in utero-placental tissues. ( A ) Quantitative real time PCR analysis of Nostrin transcript using RNA from three distinct utero-placental zones, Metrial gland (MG), Junctional zone (JZ) and Labyrinth zone (LZ) ofDex-treated and Control animals. The transcript levels were normalized relative to Rpl7 . Experiments were repeated using five independent biological replicates. Error bars represent standard errors of mean. ( B ) Western blot analysis of NOSTRIN using the same tissue samples as described in ( A ). RpL7 was used as a loading control. Experiments were repeated using five independent biological replicates. ( C ) Quantification of NOSTRIN levels in Control and Dex treatment (from B) normalized to the loading control using ImageJ software. A significant (p
    Figure Legend Snippet: Effect of Dexamethasone (Dex) induced IUGR on NOSTRIN expression at gestation Day 20.5 in utero-placental tissues. ( A ) Quantitative real time PCR analysis of Nostrin transcript using RNA from three distinct utero-placental zones, Metrial gland (MG), Junctional zone (JZ) and Labyrinth zone (LZ) ofDex-treated and Control animals. The transcript levels were normalized relative to Rpl7 . Experiments were repeated using five independent biological replicates. Error bars represent standard errors of mean. ( B ) Western blot analysis of NOSTRIN using the same tissue samples as described in ( A ). RpL7 was used as a loading control. Experiments were repeated using five independent biological replicates. ( C ) Quantification of NOSTRIN levels in Control and Dex treatment (from B) normalized to the loading control using ImageJ software. A significant (p

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

    28) Product Images from "Hepatic SMARCA4 predicts HCC recurrence and promotes tumour cell proliferation by regulating SMAD6 expression"

    Article Title: Hepatic SMARCA4 predicts HCC recurrence and promotes tumour cell proliferation by regulating SMAD6 expression

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0090-8

    SMAD6 expression is regulated by BRG1 a Schematic representation of human SMAD6 promoter reporter constructs. Fragments of various lengths between −2155 and −1540 bp of SMAD6 were cloned downstream of the firefly luciferase reporter. b Luciferase activity in HEK293T cells transfected with firefly luciferase reporter plasmids containing various upstream regions of SMAD6. A Renilla luciferase reporter was cotransfected with a pGL3-basic or plasmid reporter. c Fragments of various lengths between −1961 and −1730 bp of SMAD6 were cloned downstream of the firefly luciferase reporter. d Luciferase activity in HEK293T cells transfected with firefly luciferase reporter plasmids containing various upstream regions of SMAD6. e Putative transcription factor binding sites in the region between −1860 and −1730 bp upstream of SMAD6 (upper panel). Luciferase activity associated with the region between −1860 and −1730 bp of SMAD6 in HEK293T cells transfected with small interfering RNA (siRNA) against HIC2, NR4A2 or with a negative control (NC) (lower panel). f HIC and SMAD6 expression in SK-Hep 1 and Huh-7 cells after HIC2 was knocked down as assessed with real-time PCR (upper panel). NR4A2 and SMAD6 expression in SK-Hep 1 and Huh-7 cells after NR4A22 was knocked down (lower panel). Data represent the mean ± SEM. ** P
    Figure Legend Snippet: SMAD6 expression is regulated by BRG1 a Schematic representation of human SMAD6 promoter reporter constructs. Fragments of various lengths between −2155 and −1540 bp of SMAD6 were cloned downstream of the firefly luciferase reporter. b Luciferase activity in HEK293T cells transfected with firefly luciferase reporter plasmids containing various upstream regions of SMAD6. A Renilla luciferase reporter was cotransfected with a pGL3-basic or plasmid reporter. c Fragments of various lengths between −1961 and −1730 bp of SMAD6 were cloned downstream of the firefly luciferase reporter. d Luciferase activity in HEK293T cells transfected with firefly luciferase reporter plasmids containing various upstream regions of SMAD6. e Putative transcription factor binding sites in the region between −1860 and −1730 bp upstream of SMAD6 (upper panel). Luciferase activity associated with the region between −1860 and −1730 bp of SMAD6 in HEK293T cells transfected with small interfering RNA (siRNA) against HIC2, NR4A2 or with a negative control (NC) (lower panel). f HIC and SMAD6 expression in SK-Hep 1 and Huh-7 cells after HIC2 was knocked down as assessed with real-time PCR (upper panel). NR4A2 and SMAD6 expression in SK-Hep 1 and Huh-7 cells after NR4A22 was knocked down (lower panel). Data represent the mean ± SEM. ** P

    Techniques Used: Expressing, Construct, Clone Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Binding Assay, Small Interfering RNA, Negative Control, Hydrophobic Interaction Chromatography, Real-time Polymerase Chain Reaction

    29) Product Images from "Differential transcription profiles of long non-coding RNAs in primary human brain microvascular endothelial cells in response to meningitic Escherichia coli"

    Article Title: Differential transcription profiles of long non-coding RNAs in primary human brain microvascular endothelial cells in response to meningitic Escherichia coli

    Journal: Scientific Reports

    doi: 10.1038/srep38903

    Quantitative PCR verification of the transcription of lncRNAs in primary hBMECs after meningitic E. coli infection. The relative expression levels of the most highly differentially expressed lncRNAs as indicated were examined using qPCR in primary hBMECs that were treated with meningitic E. coli for 3 h. Data from RNA-seq were highly consistent with the qPCR results. Data are expressed as mean ± SEM from three separate experiments.
    Figure Legend Snippet: Quantitative PCR verification of the transcription of lncRNAs in primary hBMECs after meningitic E. coli infection. The relative expression levels of the most highly differentially expressed lncRNAs as indicated were examined using qPCR in primary hBMECs that were treated with meningitic E. coli for 3 h. Data from RNA-seq were highly consistent with the qPCR results. Data are expressed as mean ± SEM from three separate experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Infection, Expressing, RNA Sequencing Assay

    30) Product Images from "Bisphenol A Increases Atherosclerosis in Pregnane X Receptor‐Humanized ApoE Deficient Mice"

    Article Title: Bisphenol A Increases Atherosclerosis in Pregnane X Receptor‐Humanized ApoE Deficient Mice

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

    doi: 10.1161/JAHA.113.000492

    Generation of huPXR•ApoE −/− mice. A, Genotype analysis of huPXR•ApoE −/− and PXR −/− ApoE −/− mice by 3 different PCR assays. The presence of the human (h) PXR transgene was determined by hPXR primers (576 bp). Mouse (m) PXR primers were used to identify WT allele (348 bp) and PXR null allele (265 bp). Mouse ApoE primers were used to identify WT allele (155 bp) and ApoE null allele (245 bp). Mouse no. 1 is huPXR•ApoE −/− , no. 2 is PXR −/− ApoE −/− , and no. 3 is WT control. B, Six‐week‐old male huPXR•ApoE −/− and PXR −/− ApoE −/− mice were treated with DMSO vehicle control, mPXR‐specific ligand pregnenolone 16α‐carbonitrile (PCN), or hPXR‐specific ligand rifampicin (RIF) by intraperitoneal injection at the dose of 10 mg/kg per day for 3 days. Total RNA was extracted from the liver, and the mRNA levels of prototypic PXR activated gene CYP3A11 were measured by QPCR (n=5 per group, ** P
    Figure Legend Snippet: Generation of huPXR•ApoE −/− mice. A, Genotype analysis of huPXR•ApoE −/− and PXR −/− ApoE −/− mice by 3 different PCR assays. The presence of the human (h) PXR transgene was determined by hPXR primers (576 bp). Mouse (m) PXR primers were used to identify WT allele (348 bp) and PXR null allele (265 bp). Mouse ApoE primers were used to identify WT allele (155 bp) and ApoE null allele (245 bp). Mouse no. 1 is huPXR•ApoE −/− , no. 2 is PXR −/− ApoE −/− , and no. 3 is WT control. B, Six‐week‐old male huPXR•ApoE −/− and PXR −/− ApoE −/− mice were treated with DMSO vehicle control, mPXR‐specific ligand pregnenolone 16α‐carbonitrile (PCN), or hPXR‐specific ligand rifampicin (RIF) by intraperitoneal injection at the dose of 10 mg/kg per day for 3 days. Total RNA was extracted from the liver, and the mRNA levels of prototypic PXR activated gene CYP3A11 were measured by QPCR (n=5 per group, ** P

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Injection, Real-time Polymerase Chain Reaction

    31) Product Images from "Transcriptomic analysis of Litchi chinensis pericarp during maturation with a focus on chlorophyll degradation and flavonoid biosynthesis"

    Article Title: Transcriptomic analysis of Litchi chinensis pericarp during maturation with a focus on chlorophyll degradation and flavonoid biosynthesis

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1433-4

    Coefficient analysis of gene expression levels obtained from RNA-Seq and quantitative real-time PCR data. The real-time PCR log 2 values (x-axis) were plotted against coloration stages (y-axis). **indicates a significant difference at p ≤ 0.01.
    Figure Legend Snippet: Coefficient analysis of gene expression levels obtained from RNA-Seq and quantitative real-time PCR data. The real-time PCR log 2 values (x-axis) were plotted against coloration stages (y-axis). **indicates a significant difference at p ≤ 0.01.

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

    32) Product Images from "Notch1 pathway-mediated microRNA-151-5p promotes gastric cancer progression"

    Article Title: Notch1 pathway-mediated microRNA-151-5p promotes gastric cancer progression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9342

    Activated Notch1 pathway enhanced miR-151 and FAK expressions in gastric cancer cells A. The relative levels of miR-151-3p and miR-151-5p in N1IC-expressing SC-M1/HA-N1IC ( left ), K562/HA-N1IC ( middle ), and HEK293/myc-N1IC ( right ) cells and their control cells (SC-M1/pcDNA3, K562/pcDNA3, and HEK293/pcDNA3 cells, respectively) were measured by miRNA quantitative real-time PCR. Levels of miR-151-3p and miR-151-5p in control cells were set to unity. B. The relative levels of miR-151-3p and miR-151-5p in SC-M1, AZ521, and NUGC-3 cells were determined using miRNA quantitative real-time PCR after treatment with 50 mM DAPT for 24 hours. C. Whole-cell extracts of N1IC-expressing SC-M1/HA-N1IC ( left ), K562/HA-N1IC ( middle ), and HEK293/myc-N1IC ( right ) cells and their control cells were prepared and then analyzed by Western blot analysis using anti-Notch1 C-terminal (C-ter), anti-FAK, anti-pFAK Y397, and anti-GAPDH antibodies. D. After treated with 50 mM DAPT for 24 hours, whole-cell extracts of SC-M1 ( left ), AZ521 ( middle ), and NUGC-3 ( right ) cells were prepared for Western blot analysis using anti-cleaved Notch1, anti-FAK, anti-pFAK Y397, and anti-GAPDH antibodies. E. Tumor and the adjacent non-tumor tissue sample pairs from gastric cancer patients (n=40) were examined using miRNA quantitative real-time PCR analysis. Levels of miR-151-5p and miR-151-3p in the gastric cancer tissues were compared with those of the corresponding adjacent normal tissues. F. Data of level 3 of mRNA and miRNA expressions from stomach adenocarcinoma samples and normal counterparts were downloaded from the TCGA and Broad GDAC Firehose data portal. Both mRNA RPKM (Reads per Kilobase of exon model per Million) and microRNA reads per million mappable reads of all samples were selected and analyzed to compare abundances using GraphPad Prism 5 software. The transcript levels of miR-151, Notch1 receptor, and FAK in stomach adenocarcinoma samples (miR-151, n=323; Notch1 receptor and FAK, n=274) and normal tissue samples (n=33) were measured by RNA sequencing in TCGA data. *, P
    Figure Legend Snippet: Activated Notch1 pathway enhanced miR-151 and FAK expressions in gastric cancer cells A. The relative levels of miR-151-3p and miR-151-5p in N1IC-expressing SC-M1/HA-N1IC ( left ), K562/HA-N1IC ( middle ), and HEK293/myc-N1IC ( right ) cells and their control cells (SC-M1/pcDNA3, K562/pcDNA3, and HEK293/pcDNA3 cells, respectively) were measured by miRNA quantitative real-time PCR. Levels of miR-151-3p and miR-151-5p in control cells were set to unity. B. The relative levels of miR-151-3p and miR-151-5p in SC-M1, AZ521, and NUGC-3 cells were determined using miRNA quantitative real-time PCR after treatment with 50 mM DAPT for 24 hours. C. Whole-cell extracts of N1IC-expressing SC-M1/HA-N1IC ( left ), K562/HA-N1IC ( middle ), and HEK293/myc-N1IC ( right ) cells and their control cells were prepared and then analyzed by Western blot analysis using anti-Notch1 C-terminal (C-ter), anti-FAK, anti-pFAK Y397, and anti-GAPDH antibodies. D. After treated with 50 mM DAPT for 24 hours, whole-cell extracts of SC-M1 ( left ), AZ521 ( middle ), and NUGC-3 ( right ) cells were prepared for Western blot analysis using anti-cleaved Notch1, anti-FAK, anti-pFAK Y397, and anti-GAPDH antibodies. E. Tumor and the adjacent non-tumor tissue sample pairs from gastric cancer patients (n=40) were examined using miRNA quantitative real-time PCR analysis. Levels of miR-151-5p and miR-151-3p in the gastric cancer tissues were compared with those of the corresponding adjacent normal tissues. F. Data of level 3 of mRNA and miRNA expressions from stomach adenocarcinoma samples and normal counterparts were downloaded from the TCGA and Broad GDAC Firehose data portal. Both mRNA RPKM (Reads per Kilobase of exon model per Million) and microRNA reads per million mappable reads of all samples were selected and analyzed to compare abundances using GraphPad Prism 5 software. The transcript levels of miR-151, Notch1 receptor, and FAK in stomach adenocarcinoma samples (miR-151, n=323; Notch1 receptor and FAK, n=274) and normal tissue samples (n=33) were measured by RNA sequencing in TCGA data. *, P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Software, RNA Sequencing Assay

    33) Product Images from "miR-139-5p inhibits isoproterenol-induced cardiac hypertrophy by targetting c-Jun"

    Article Title: miR-139-5p inhibits isoproterenol-induced cardiac hypertrophy by targetting c-Jun

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171430

    miR-139-5p targets c-Jun expression in cardiomyocytes ( A – D ) NRCMs were treated as indicated. Levels of c-Jun, IGF-1R, MyoCD, Wnt1, and β-catenin (A) and the phosphorylation status of Akt (B) were detected using Western blotting. The relative expression intensity (B,D) was obtained from three independent experiments were performed. ( E – I ) siRNAs for knocking down (KD) c-Jun expression were co-transfected into to NRCMs with the miR-139-5p inhibitor or the inhibitor control RNA for 48 h. Expression of c-Jun was determined using Western blotting (E) and the relative expression of c-Jun was quantitated (F). F-actin and nuclei were stained with Texas Red-Phalloidin and DAPI, respectively. Scale bars: 100 μm (G). Cell surface area was quantitated from at least 30 cells in three independent experiments (H). Expression of ANP was quantitated by using real-time PCR and normalized to the level of 18S rRNA ( n =3 per group) (I). Data are indicated as the mean ± S.E.M., * P
    Figure Legend Snippet: miR-139-5p targets c-Jun expression in cardiomyocytes ( A – D ) NRCMs were treated as indicated. Levels of c-Jun, IGF-1R, MyoCD, Wnt1, and β-catenin (A) and the phosphorylation status of Akt (B) were detected using Western blotting. The relative expression intensity (B,D) was obtained from three independent experiments were performed. ( E – I ) siRNAs for knocking down (KD) c-Jun expression were co-transfected into to NRCMs with the miR-139-5p inhibitor or the inhibitor control RNA for 48 h. Expression of c-Jun was determined using Western blotting (E) and the relative expression of c-Jun was quantitated (F). F-actin and nuclei were stained with Texas Red-Phalloidin and DAPI, respectively. Scale bars: 100 μm (G). Cell surface area was quantitated from at least 30 cells in three independent experiments (H). Expression of ANP was quantitated by using real-time PCR and normalized to the level of 18S rRNA ( n =3 per group) (I). Data are indicated as the mean ± S.E.M., * P

    Techniques Used: Expressing, Western Blot, Transfection, Staining, Aqueous Normal-phase Chromatography, Real-time Polymerase Chain Reaction

    34) Product Images from "The nucleoprotein of influenza A virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase RNF43"

    Article Title: The nucleoprotein of influenza A virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase RNF43

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.131

    Host factor RNF43 decreases IAV replication. ( a–c ) HEK293T cells were transiently transfected with plasmid pCDNA3.1 (Mock) or pCDNA3.1-RNF43-Flag-HA (RNF43-HA) (1 and 2 μ g) followed by PR8 infection and incubated for 24 h. Total RNA was isolated and NP mRNA ( a ), NP vRNA ( b ) and RNF43 mRNA( c ) levels were estimated through qRT PCR. ( d ) HEK293T cells were treated with NT siRNA or RNF43 siRNA for 24 h followed by PR8 IAV infection for next 24 h. Cells were processed for qRT-PCR analysis of mRNA levels of NP and RNF43. ( e ) A549 cells, pretreated with either NT or RNF43 siRNA were transfected with plasmids-encoding polymerase complex components (PA, PB1, PB2 and NP) derived from PR8 (H1N1 virus) were co-transfected alongside a reporter plasmid containing noncoding sequence from the NS1 segment of influenza A virus and luciferase gene driven by the Pol 1 promoter. The relative luciferase units were calculated after normalization with plasmid pRL-TK (Promega, Madison, WI, USA), which expresses Renilla luciferase that was transfected along. Results are shown as mean±S.D. of three independent experiments. * and # indicate statistically significant difference at P
    Figure Legend Snippet: Host factor RNF43 decreases IAV replication. ( a–c ) HEK293T cells were transiently transfected with plasmid pCDNA3.1 (Mock) or pCDNA3.1-RNF43-Flag-HA (RNF43-HA) (1 and 2 μ g) followed by PR8 infection and incubated for 24 h. Total RNA was isolated and NP mRNA ( a ), NP vRNA ( b ) and RNF43 mRNA( c ) levels were estimated through qRT PCR. ( d ) HEK293T cells were treated with NT siRNA or RNF43 siRNA for 24 h followed by PR8 IAV infection for next 24 h. Cells were processed for qRT-PCR analysis of mRNA levels of NP and RNF43. ( e ) A549 cells, pretreated with either NT or RNF43 siRNA were transfected with plasmids-encoding polymerase complex components (PA, PB1, PB2 and NP) derived from PR8 (H1N1 virus) were co-transfected alongside a reporter plasmid containing noncoding sequence from the NS1 segment of influenza A virus and luciferase gene driven by the Pol 1 promoter. The relative luciferase units were calculated after normalization with plasmid pRL-TK (Promega, Madison, WI, USA), which expresses Renilla luciferase that was transfected along. Results are shown as mean±S.D. of three independent experiments. * and # indicate statistically significant difference at P

    Techniques Used: Transfection, Plasmid Preparation, Infection, Incubation, Isolation, Quantitative RT-PCR, Derivative Assay, Sequencing, Luciferase

    IAV infection decreases abundance of RNF43 at both mRNA and protein levels. ( a ) Lung epithelial A549 cells were infected with PR8 virus at an MOI of 1 and cells were harvested at indicated time intervals post infection and the whole-cell lysate was resolved on SDS-PAGE for Western blot analysis of RNF43, NP and GAPDH. ( b ) A549 cells were infected with PR8 virus at indicated MOIs and harvested at 24-h post infection for Western blot analysis of RNF43, NP and GAPDH. Quantitative representation of the immunoblots of both the experiments is shown as the line diagram after normalization with GAPDH (extreme right). ( c ) A549 cells were mock infected or infected with PR8 virus for 24 h and total RNA was extracted f ollowed by rnf43 mRNA estimation with qRT-PCR. Results are shown as mean of ±S.D. of three independent experiments. * indicates statistically significant difference at P
    Figure Legend Snippet: IAV infection decreases abundance of RNF43 at both mRNA and protein levels. ( a ) Lung epithelial A549 cells were infected with PR8 virus at an MOI of 1 and cells were harvested at indicated time intervals post infection and the whole-cell lysate was resolved on SDS-PAGE for Western blot analysis of RNF43, NP and GAPDH. ( b ) A549 cells were infected with PR8 virus at indicated MOIs and harvested at 24-h post infection for Western blot analysis of RNF43, NP and GAPDH. Quantitative representation of the immunoblots of both the experiments is shown as the line diagram after normalization with GAPDH (extreme right). ( c ) A549 cells were mock infected or infected with PR8 virus for 24 h and total RNA was extracted f ollowed by rnf43 mRNA estimation with qRT-PCR. Results are shown as mean of ±S.D. of three independent experiments. * indicates statistically significant difference at P

    Techniques Used: Infection, SDS Page, Western Blot, Quantitative RT-PCR

    RNF43 decreases NP driven increased p53 transcriptional activity and signaling in the cells. ( a ) A549 cells were transfected with p21-Luc reporter plasmid, with or without growing amounts of pEGFP-NP (500 and 750 ng, and 1 μg) in conjunction with pcDNA-p53 and a control plasmid, Renilla luciferase pRL-TK. Luciferase activity of cell lysates of transfectants was analyzed. ( b ) A549 cells were transfected with p21-Luc reporter plasmid, pcDNA-p53 and a control plasmid, Renilla luciferase pRL-TK along with plasmids pEGFPN1 (mock), pEGFP-NP (NP-GFP) and pCDNA3.1-RNF43-Flag-HA (RNF43-HA) in the indicated combinations. Luciferase activity was measured. ( c ) HEK293T cells were treated with NT siRNA or RNF43 siRNA for 24 h followed by PR8 IAV infection at 1 MOI. Cells were harvested at 24-h post infection and total RNA was extracted followed by p21 mRNA estimation with qRT-PCR. ( d ) A549 cells were transiently transfected with pEGFP-NP with or without pCDNA3.1-RNF43-Flag-HA and were harvested at 48 h followed by SDS-PAGE. Western blotting was done using indicated antibodies. ( e ) A549 cells were seeded in a 6-well plate and were infected with PR8 virus at 1 MOI. Cells were harvested at 0, 4, 8, 16 and 24-h post infection, and subjected to Western blotting with indicated antibodies. ( f ) A549 cells were transfected with pCDNA3.1 (mock) and pCDNA3.1-RNF43-Flag-HA (RNF43-HA) plasmids followed by PR8 infection, 24-h post transfection. The cells were harvested after 24 h followed by Western blot analysis with anti-acetyl p53, HA, NP and β -actin antibodies. ( g ) A549 cells were transiently transfected with pCDNA3.1 (mock) or pCDNA3.1-RNF43-Flag-HA (RNF43-HA) plasmid constructs and after 24 h of incubation, cells were infected with PR8 virus at an MOI of 1 for 24 h. ( h ) Similarly, A549 cells were transiently transfected with pEGFP-NP with or without pCDNA3.1-RNF43-Flag-HA and were incubated for 48 h. ( g and h ) Cells were harvested and processed for Western blot analysis with indicated antibodies. Results in a – c are shown as mean±S.D. three independent experiments. * and # indicate statistically significant difference at P
    Figure Legend Snippet: RNF43 decreases NP driven increased p53 transcriptional activity and signaling in the cells. ( a ) A549 cells were transfected with p21-Luc reporter plasmid, with or without growing amounts of pEGFP-NP (500 and 750 ng, and 1 μg) in conjunction with pcDNA-p53 and a control plasmid, Renilla luciferase pRL-TK. Luciferase activity of cell lysates of transfectants was analyzed. ( b ) A549 cells were transfected with p21-Luc reporter plasmid, pcDNA-p53 and a control plasmid, Renilla luciferase pRL-TK along with plasmids pEGFPN1 (mock), pEGFP-NP (NP-GFP) and pCDNA3.1-RNF43-Flag-HA (RNF43-HA) in the indicated combinations. Luciferase activity was measured. ( c ) HEK293T cells were treated with NT siRNA or RNF43 siRNA for 24 h followed by PR8 IAV infection at 1 MOI. Cells were harvested at 24-h post infection and total RNA was extracted followed by p21 mRNA estimation with qRT-PCR. ( d ) A549 cells were transiently transfected with pEGFP-NP with or without pCDNA3.1-RNF43-Flag-HA and were harvested at 48 h followed by SDS-PAGE. Western blotting was done using indicated antibodies. ( e ) A549 cells were seeded in a 6-well plate and were infected with PR8 virus at 1 MOI. Cells were harvested at 0, 4, 8, 16 and 24-h post infection, and subjected to Western blotting with indicated antibodies. ( f ) A549 cells were transfected with pCDNA3.1 (mock) and pCDNA3.1-RNF43-Flag-HA (RNF43-HA) plasmids followed by PR8 infection, 24-h post transfection. The cells were harvested after 24 h followed by Western blot analysis with anti-acetyl p53, HA, NP and β -actin antibodies. ( g ) A549 cells were transiently transfected with pCDNA3.1 (mock) or pCDNA3.1-RNF43-Flag-HA (RNF43-HA) plasmid constructs and after 24 h of incubation, cells were infected with PR8 virus at an MOI of 1 for 24 h. ( h ) Similarly, A549 cells were transiently transfected with pEGFP-NP with or without pCDNA3.1-RNF43-Flag-HA and were incubated for 48 h. ( g and h ) Cells were harvested and processed for Western blot analysis with indicated antibodies. Results in a – c are shown as mean±S.D. three independent experiments. * and # indicate statistically significant difference at P

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Infection, Quantitative RT-PCR, SDS Page, Western Blot, Construct, Incubation

    35) Product Images from "Macrophages commit postnatal endothelium-derived progenitors to angiogenesis and restrict endothelial to mesenchymal transition during muscle regeneration"

    Article Title: Macrophages commit postnatal endothelium-derived progenitors to angiogenesis and restrict endothelial to mesenchymal transition during muscle regeneration

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.558

    Pro-angiogenic genes are upregulated during skeletal muscle regeneration. ( a ) IF on TA cross-sections from 2-month-old Cdh5-CreER T2 ; R26R-NGZ mice before and 5, 7 and 15 days after CTX damage. Representative images of sections immunostained for CD31 (red) overlayed to the nuclear X-Gal staining (converted in pseudo fluorescent green signal), showing colocalization of labeled cells with CD31. Magnification × 20. Scale bar: 50 μ m. ( b ) TA muscles from 2-month-old Cdh5-CreER T2 ; R26R-EYFP mice were collected immediately before and 1, 3, 7, 10 and 15 days after injection of CTX. Total muscle lysates were collected and processed for RNA and protein extraction. Real-time PCR analyses for HIF1- α , MMP-2, MMP-13, MMP-14, VEGF- β , PDGF- α , PDGF- β , TGF- β , Ang-I and Ang-II mRNA expression were performed. Results were normalized to 28S mRNA levels and expressed as relative fold changes compared with undamaged muscles (NT). The data are expressed as means±S.E.M. ( n =6). Statistically significant differences are indicated (* P
    Figure Legend Snippet: Pro-angiogenic genes are upregulated during skeletal muscle regeneration. ( a ) IF on TA cross-sections from 2-month-old Cdh5-CreER T2 ; R26R-NGZ mice before and 5, 7 and 15 days after CTX damage. Representative images of sections immunostained for CD31 (red) overlayed to the nuclear X-Gal staining (converted in pseudo fluorescent green signal), showing colocalization of labeled cells with CD31. Magnification × 20. Scale bar: 50 μ m. ( b ) TA muscles from 2-month-old Cdh5-CreER T2 ; R26R-EYFP mice were collected immediately before and 1, 3, 7, 10 and 15 days after injection of CTX. Total muscle lysates were collected and processed for RNA and protein extraction. Real-time PCR analyses for HIF1- α , MMP-2, MMP-13, MMP-14, VEGF- β , PDGF- α , PDGF- β , TGF- β , Ang-I and Ang-II mRNA expression were performed. Results were normalized to 28S mRNA levels and expressed as relative fold changes compared with undamaged muscles (NT). The data are expressed as means±S.E.M. ( n =6). Statistically significant differences are indicated (* P

    Techniques Used: Mouse Assay, Staining, Labeling, Injection, Protein Extraction, Real-time Polymerase Chain Reaction, Expressing

    MP depletion affects pro-angiogenic factor production and TGF- β /SMAD signaling during muscle healing. ( a – c ) TA muscles were collected from sham and Cll-injected mice 5, 7, 10 and 15 days after CTX injection. Muscles were totally lysated and processed for RNA extraction. Real-time PCR analyses for mRNA expression of HIF1- α , Ang-I, Ang-II, PDGF- α , PDGF- β , VEGF- β ( a ), MMP-2, MMP-13, MMP-14 ( b ), TGF- β and Snail ( c ) were performed. Results were normalized to 28S mRNA levels and expressed as relative fold changes compared with sham-treated mice 5 days after damage. The data are expressed as means±S.E.M. ( n =5). Statistically significant differences are indicated (sham versus Cll * P
    Figure Legend Snippet: MP depletion affects pro-angiogenic factor production and TGF- β /SMAD signaling during muscle healing. ( a – c ) TA muscles were collected from sham and Cll-injected mice 5, 7, 10 and 15 days after CTX injection. Muscles were totally lysated and processed for RNA extraction. Real-time PCR analyses for mRNA expression of HIF1- α , Ang-I, Ang-II, PDGF- α , PDGF- β , VEGF- β ( a ), MMP-2, MMP-13, MMP-14 ( b ), TGF- β and Snail ( c ) were performed. Results were normalized to 28S mRNA levels and expressed as relative fold changes compared with sham-treated mice 5 days after damage. The data are expressed as means±S.E.M. ( n =5). Statistically significant differences are indicated (sham versus Cll * P

    Techniques Used: Injection, Mouse Assay, RNA Extraction, Real-time Polymerase Chain Reaction, Expressing

    Gene expression of VE-Cad + derived cells after postnatal Cre activation. ( a ) IF performed on TA of Cdh5-CreER T2 ; R26R-EYFP mice activated with TAM injection at postnatal days (P) 6–8, at P11 and P60. Representative images of muscle cross-sections immunostained with antibodies specific for CD31 (in red), EYFP (in green), showing colocalization of labeled cells with CD31. Magnification × 20. Scale bar: 50 μ m. ( b ) Gating strategy for FACS sorting of EYFP + cells from muscle; 2D density plot showing FS versus SS, delimits a tight region around the live cell population (R1). A second density plot, gated only on events encircled within R1, shows EYFP + cells on the x-axis and SS on the y axis (R2). EYFP + cells are selected and sorted. Cells are used for RNA extraction, at P11 and P60. ( c ) Real-time PCR performed on cDNA obtained from RNA of EYFP + cells sorted from P11 and P60 Cdh5-CreER T2 ; R26R-EYFP mice activated with TAM injection at postnatal days (P) 6–8. The data are normalized on cyclophillin A expression
    Figure Legend Snippet: Gene expression of VE-Cad + derived cells after postnatal Cre activation. ( a ) IF performed on TA of Cdh5-CreER T2 ; R26R-EYFP mice activated with TAM injection at postnatal days (P) 6–8, at P11 and P60. Representative images of muscle cross-sections immunostained with antibodies specific for CD31 (in red), EYFP (in green), showing colocalization of labeled cells with CD31. Magnification × 20. Scale bar: 50 μ m. ( b ) Gating strategy for FACS sorting of EYFP + cells from muscle; 2D density plot showing FS versus SS, delimits a tight region around the live cell population (R1). A second density plot, gated only on events encircled within R1, shows EYFP + cells on the x-axis and SS on the y axis (R2). EYFP + cells are selected and sorted. Cells are used for RNA extraction, at P11 and P60. ( c ) Real-time PCR performed on cDNA obtained from RNA of EYFP + cells sorted from P11 and P60 Cdh5-CreER T2 ; R26R-EYFP mice activated with TAM injection at postnatal days (P) 6–8. The data are normalized on cyclophillin A expression

    Techniques Used: Expressing, Derivative Assay, Activation Assay, Mouse Assay, Injection, Labeling, FACS, RNA Extraction, Real-time Polymerase Chain Reaction

    36) Product Images from "MicroRNA-143 and -145 modulate the phenotype of synovial fibroblasts in rheumatoid arthritis"

    Article Title: MicroRNA-143 and -145 modulate the phenotype of synovial fibroblasts in rheumatoid arthritis

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2017.108

    microRNA profiling in RA-FLSs. ( a ) Heatmap of microarray data showing DEmiRs among RA-FLSs versus OA-FLSs, RA-FLSs with IL-1β versus RA-FLSs, OA-FLSs with IL-1β versus OA-FLSs and RA-FLSs with IL-1β versus OA-FLSs with IL-1β. Red and green denote high and low expression of DEmiRs, respectively; red and green denote high and low expression of DEGs, respectively. ( b ) Higher levels of miR-143 and miR-145 in RA-FLSs ( n =5) than in OA-FLSs ( n =5) as determined by quantitative real-time PCR; 5.6-fold, P =0.031 for miR-143; 2.6-fold, P =0.028 for miR-145. RUN6B_2 small nuclear RNA (snRNA) was used as an internal control. ( c ) Spearman correlation of relative expression of miR-143 and miR-145 in RA-FLSs ( n =8). ( d ) miR-143 and miR-145 levels in synovial fluids obtained from RA patients (RA-SF, n =4) and OA patients (OA-SF, n =5) as determined by real-time PCR. ( e ) Increase in miR-145 expression in FLSs by TGFβ stimulation. The OA-FLSs ( n =4) and RA-FLSs ( n =4) were treated with IL-1β (1 ng ml −1 ), TNFα (10 ng ml −1 ) and TGFβ (10 ng ml −1 ) for 48 h, and miR-145 expression was then determined by real-time PCR. The data show the mean±s.d. * P
    Figure Legend Snippet: microRNA profiling in RA-FLSs. ( a ) Heatmap of microarray data showing DEmiRs among RA-FLSs versus OA-FLSs, RA-FLSs with IL-1β versus RA-FLSs, OA-FLSs with IL-1β versus OA-FLSs and RA-FLSs with IL-1β versus OA-FLSs with IL-1β. Red and green denote high and low expression of DEmiRs, respectively; red and green denote high and low expression of DEGs, respectively. ( b ) Higher levels of miR-143 and miR-145 in RA-FLSs ( n =5) than in OA-FLSs ( n =5) as determined by quantitative real-time PCR; 5.6-fold, P =0.031 for miR-143; 2.6-fold, P =0.028 for miR-145. RUN6B_2 small nuclear RNA (snRNA) was used as an internal control. ( c ) Spearman correlation of relative expression of miR-143 and miR-145 in RA-FLSs ( n =8). ( d ) miR-143 and miR-145 levels in synovial fluids obtained from RA patients (RA-SF, n =4) and OA patients (OA-SF, n =5) as determined by real-time PCR. ( e ) Increase in miR-145 expression in FLSs by TGFβ stimulation. The OA-FLSs ( n =4) and RA-FLSs ( n =4) were treated with IL-1β (1 ng ml −1 ), TNFα (10 ng ml −1 ) and TGFβ (10 ng ml −1 ) for 48 h, and miR-145 expression was then determined by real-time PCR. The data show the mean±s.d. * P

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

    37) Product Images from "TNNI3K, a Cardiac-Specific Kinase, Promotes Physiological Cardiac Hypertrophy in Transgenic Mice"

    Article Title: TNNI3K, a Cardiac-Specific Kinase, Promotes Physiological Cardiac Hypertrophy in Transgenic Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058570

    Generation of cardiac-specific TNNI3K transgenic mice. (A), Schematic of the TNNI3K transgene that was constructed with the α-MHC mouse promoter. pA: human growth hormone polyA sequences The positions of the Southern probe and northern probe were shown below the construct; (B), PCR genotyping of TNNI3K transgenic mice. 1–5: transgenic mice. P: positive control, wild-type mouse genomic DNA mixed with linearized transgenic fragment. N: negative control, wild-type mouse genomic DNA. B: blank, none DNA template. FABPI gene was amplified as internal control. (C), Southern blot analysis of wild-type and TNNI3K transgenic mice. Tail genomic DNA was digested with EcoRI and probed with hGH polyA sequence. Hybridization signals were present only in transgenic positive mice. Transgenic copy number was determined from the gray density against standard curve. 1 copy -10 copies: transgenic copy standards. (D). Northern blot analysis of RNA isolated from multiple tissues of the transgenic TG-L and TG-H lines. Hybridization signals were present only in the heart of transgenic mice. The RNA isolated from the heart of wild-type mouse was used as a negative control.
    Figure Legend Snippet: Generation of cardiac-specific TNNI3K transgenic mice. (A), Schematic of the TNNI3K transgene that was constructed with the α-MHC mouse promoter. pA: human growth hormone polyA sequences The positions of the Southern probe and northern probe were shown below the construct; (B), PCR genotyping of TNNI3K transgenic mice. 1–5: transgenic mice. P: positive control, wild-type mouse genomic DNA mixed with linearized transgenic fragment. N: negative control, wild-type mouse genomic DNA. B: blank, none DNA template. FABPI gene was amplified as internal control. (C), Southern blot analysis of wild-type and TNNI3K transgenic mice. Tail genomic DNA was digested with EcoRI and probed with hGH polyA sequence. Hybridization signals were present only in transgenic positive mice. Transgenic copy number was determined from the gray density against standard curve. 1 copy -10 copies: transgenic copy standards. (D). Northern blot analysis of RNA isolated from multiple tissues of the transgenic TG-L and TG-H lines. Hybridization signals were present only in the heart of transgenic mice. The RNA isolated from the heart of wild-type mouse was used as a negative control.

    Techniques Used: Transgenic Assay, Mouse Assay, Construct, Northern Blot, Polymerase Chain Reaction, Positive Control, Negative Control, Amplification, Southern Blot, Sequencing, Hybridization, Isolation

    38) Product Images from "PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination"

    Article Title: PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination

    Journal: Science Advances

    doi: 10.1126/sciadv.1500615

    PRMT8 knockout mice display abnormal Purkinje cell dendrites and aberrant behavior. ( A ) Gene targeting strategy for the prmt8 locus: schematic representation of the prmt8 +/+ [wild-type (WT)], prmt8 flox (floxed), and prmt8 −/− (knockout) alleles. Arrows indicate the positions of the P1, P2, and P3 genotyping primers. ( B ) Allele-specific PCR analysis using tail genomic DNA. Products of 504 and 252 base pairs (bp) were generated from prmt8 +/+ and prmt8 −/− mice, respectively. ( C ) prmt8 mRNA levels in 12-week-old mice were examined in total cerebellar RNA by qPCR. Results are shown as fold change of prmt8 mRNA expression relative to the mean WT value. Data were obtained using the ΔΔ C t method with normalization to the reference glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA (mean ± SEM, n = 4 per genotype; ** P
    Figure Legend Snippet: PRMT8 knockout mice display abnormal Purkinje cell dendrites and aberrant behavior. ( A ) Gene targeting strategy for the prmt8 locus: schematic representation of the prmt8 +/+ [wild-type (WT)], prmt8 flox (floxed), and prmt8 −/− (knockout) alleles. Arrows indicate the positions of the P1, P2, and P3 genotyping primers. ( B ) Allele-specific PCR analysis using tail genomic DNA. Products of 504 and 252 base pairs (bp) were generated from prmt8 +/+ and prmt8 −/− mice, respectively. ( C ) prmt8 mRNA levels in 12-week-old mice were examined in total cerebellar RNA by qPCR. Results are shown as fold change of prmt8 mRNA expression relative to the mean WT value. Data were obtained using the ΔΔ C t method with normalization to the reference glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA (mean ± SEM, n = 4 per genotype; ** P

    Techniques Used: Knock-Out, Mouse Assay, Polymerase Chain Reaction, Generated, Real-time Polymerase Chain Reaction, Expressing

    39) Product Images from "GP73 represses host innate immune response to promote virus replication by facilitating MAVS and TRAF6 degradation"

    Article Title: GP73 represses host innate immune response to promote virus replication by facilitating MAVS and TRAF6 degradation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006321

    GP73 facilitates HCV infection and replication. ( A ) GP73 -RNAi-transduced stable Huh7 or Huh7-MAVSR cells were transfected with in vitro transcribed FL-J6/JFH5’C19Rluc2Aubi RNA (J6/JFH) for 48 h, and Renilla luciferase activities were measured. ( B , C ) GP73 -RNAi-transduced stable Huh7-MAVSR cells were infected with HCV at MOI = 2 for indicated times. Intracellular HCV RNA abundance was determined by qRT-PCR (B), HCV core protein at 3 days post infection (d.p.i) was detected by WB (C). ( D , E ) Huh7-MAVSR-GP73-RNAi cells were transfected with GP73 -rescue plasmid ( GP73m4 ) for 24 h, followed by HCV infection at MOI = 2 for 3 days. HCV RNAs were determined by RT-PCR (D) and HCV core protein was detected by WB (E). ( F ) PHHs were transduced with GP73 -shRNA lentivirus for 48 h, followed by HCV infection at MOI = 2 for 2 days. HCV RNA levels were determined by RT-PCR. Bar graphs represent means ± SD, ** P
    Figure Legend Snippet: GP73 facilitates HCV infection and replication. ( A ) GP73 -RNAi-transduced stable Huh7 or Huh7-MAVSR cells were transfected with in vitro transcribed FL-J6/JFH5’C19Rluc2Aubi RNA (J6/JFH) for 48 h, and Renilla luciferase activities were measured. ( B , C ) GP73 -RNAi-transduced stable Huh7-MAVSR cells were infected with HCV at MOI = 2 for indicated times. Intracellular HCV RNA abundance was determined by qRT-PCR (B), HCV core protein at 3 days post infection (d.p.i) was detected by WB (C). ( D , E ) Huh7-MAVSR-GP73-RNAi cells were transfected with GP73 -rescue plasmid ( GP73m4 ) for 24 h, followed by HCV infection at MOI = 2 for 3 days. HCV RNAs were determined by RT-PCR (D) and HCV core protein was detected by WB (E). ( F ) PHHs were transduced with GP73 -shRNA lentivirus for 48 h, followed by HCV infection at MOI = 2 for 2 days. HCV RNA levels were determined by RT-PCR. Bar graphs represent means ± SD, ** P

    Techniques Used: Infection, Transfection, In Vitro, Luciferase, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Transduction, shRNA

    GP73 represses host innate immunity during viral infection. ( A ) HEK293 cells (1×10 5 ) were transfected with the IFN-β , ISRE or NF-κB reporters (0.1 μg) and GP73 expressing plasmid at indicated concentrations for 24 h, and infected with SeV for 10 h before luciferase reporter assays were performed. ( B ) HEK293 cells (1×10 5 ) were transfected with IFN-β or NF-κB reporter (0.05 μg) and GP73 -shRNAs expressing plasmids (1 μg) for 48 h, and infected with SeV for 10 h before reporter assays were performed. ( C , D ) GP73 -shRNAs transduced stable Huh7-MAVSR cells were transfected with in vitro transcribed JFH1 genomic RNA for 16 h. IFN-β , IL-6 and ISG56 mRNAs were quantified by RT-PCR (C) and p-IRF3 and p-IκBα were detected by WB (D). ( E , F ) GP73 -shRNAs transduced stable Huh7 ( E ) and THP-1 ( F ) cells were infected with SeV for 12 h. IFN-β , IFN-λl , IL-6 and TNF-α mNRAs were quantified by RT-PCR. Bar graphs represent means ± SD, * P
    Figure Legend Snippet: GP73 represses host innate immunity during viral infection. ( A ) HEK293 cells (1×10 5 ) were transfected with the IFN-β , ISRE or NF-κB reporters (0.1 μg) and GP73 expressing plasmid at indicated concentrations for 24 h, and infected with SeV for 10 h before luciferase reporter assays were performed. ( B ) HEK293 cells (1×10 5 ) were transfected with IFN-β or NF-κB reporter (0.05 μg) and GP73 -shRNAs expressing plasmids (1 μg) for 48 h, and infected with SeV for 10 h before reporter assays were performed. ( C , D ) GP73 -shRNAs transduced stable Huh7-MAVSR cells were transfected with in vitro transcribed JFH1 genomic RNA for 16 h. IFN-β , IL-6 and ISG56 mRNAs were quantified by RT-PCR (C) and p-IRF3 and p-IκBα were detected by WB (D). ( E , F ) GP73 -shRNAs transduced stable Huh7 ( E ) and THP-1 ( F ) cells were infected with SeV for 12 h. IFN-β , IFN-λl , IL-6 and TNF-α mNRAs were quantified by RT-PCR. Bar graphs represent means ± SD, * P

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

    40) Product Images from "Roxatidine attenuates mast cell-mediated allergic inflammation via inhibition of NF-κB and p38 MAPK activation"

    Article Title: Roxatidine attenuates mast cell-mediated allergic inflammation via inhibition of NF-κB and p38 MAPK activation

    Journal: Scientific Reports

    doi: 10.1038/srep41721

    Effects of roxatidine on compound 48/80-induced cytokines and caspase-1 activation in anaphylactic animal models. Mice were administrated with roxatidine (20 mg/kg, p.o.), DSCG (25 mg/kg, p.o) and PBS as a vehicle (n = 6 per group) for 1 h before compound 48/80 injection (8 mg/kg i.p.). ( A ) Serum level of TNF-α, IL-6 and IL-1β were determined by using EIA kits. ( B ) Total RNA prepared from the liver tissue and the level of TNF-α, IL-6 and IL-1β were determined by quantitative real-time PCR. ( C ) Expression of procaspae-1 was determined by western blot analysis using specific antibodies. Densitometric analysis was performed using Bio-Rad Quantity One Software. The data shown represent mean ± SD of three independent experiments. # p
    Figure Legend Snippet: Effects of roxatidine on compound 48/80-induced cytokines and caspase-1 activation in anaphylactic animal models. Mice were administrated with roxatidine (20 mg/kg, p.o.), DSCG (25 mg/kg, p.o) and PBS as a vehicle (n = 6 per group) for 1 h before compound 48/80 injection (8 mg/kg i.p.). ( A ) Serum level of TNF-α, IL-6 and IL-1β were determined by using EIA kits. ( B ) Total RNA prepared from the liver tissue and the level of TNF-α, IL-6 and IL-1β were determined by quantitative real-time PCR. ( C ) Expression of procaspae-1 was determined by western blot analysis using specific antibodies. Densitometric analysis was performed using Bio-Rad Quantity One Software. The data shown represent mean ± SD of three independent experiments. # p

    Techniques Used: Activation Assay, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Software

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    High Throughput Screening Assay:

    Article Title: Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput sequencing method
    Article Snippet: .. Ten µg of RNA was treated with DNAse (DNA-free, Ambion), and 2 µg of the DNase-treated RNA was added to a reverse transcription reaction which was performed using the SuperscriptTM III RNase H Reverse Transcriptase kit (Invitrogen) with Oligo-(dT)12–18 primers (Invitrogen). cDNA was used as template to generate a double-stranded DNA fragment by PCR for both the low-throughput sequencing (LTS) and high-throughput sequencing (HTS) experiments. .. LTS A PCR fragment (containing the edited site) that was 327 bp in length was generated for each of three Pet-1 wild-type and three Pet-1 knockout animals (Forward primer: 5′ AAA GGATCC TGT GCT ATT TTC AAC TGC GTC CAT CAT G 3′; Reverse primer: 5′ AAA GAATTC CGG CGT AGG ACG TAG ATC GTTAAG 3′) ( ).

    Incubation:

    Article Title: The role of G-protein-coupled membrane estrogen receptor in mouse Leydig cell function—in vivo and in vitro evaluation
    Article Snippet: .. To remove contaminating DNA and DNase from RNA preparations, the RNA samples were incubated with reagents from the TURBO DNA-free™ Kit (Ambion, Austin, TX). .. The yield and quality of the RNA were assessed using a NanoDrop ND2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

    Polymerase Chain Reaction:

    Article Title: Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput sequencing method
    Article Snippet: .. Ten µg of RNA was treated with DNAse (DNA-free, Ambion), and 2 µg of the DNase-treated RNA was added to a reverse transcription reaction which was performed using the SuperscriptTM III RNase H Reverse Transcriptase kit (Invitrogen) with Oligo-(dT)12–18 primers (Invitrogen). cDNA was used as template to generate a double-stranded DNA fragment by PCR for both the low-throughput sequencing (LTS) and high-throughput sequencing (HTS) experiments. .. LTS A PCR fragment (containing the edited site) that was 327 bp in length was generated for each of three Pet-1 wild-type and three Pet-1 knockout animals (Forward primer: 5′ AAA GGATCC TGT GCT ATT TTC AAC TGC GTC CAT CAT G 3′; Reverse primer: 5′ AAA GAATTC CGG CGT AGG ACG TAG ATC GTTAAG 3′) ( ).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Physiological stressors and invasive plant infections alter the small RNA transcriptome of the rice blast fungus, Magnaporthe oryzae
    Article Snippet: .. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Genomic DNA was removed from total RNA by TURBO® DNase treatment (Ambion, New York, USA). .. Reverse transcription reactions were performed with 500 ng of RNA in 20 μl reaction using High Capacity RNA-to-cDNA Kit (Applied Biosystems, California, USA) by following the manufacturer’s instructions.

    Concentration Assay:

    Article Title: Identification of miR-29b targets using 3-cyanovinylcarbazole containing mimics
    Article Snippet: .. RNA samples were DNase treated using TURBO DNase (Ambion) and purified using the RNeasy MinElute Cleanup Kit (Qiagen), to retain small RNAs according to manufacturer's instructions. cDNA was synthesized from 500 ng of extracted RNA using random hexamers at a concentration of 250 ng per 5 μg of RNA with the SuperScript III Reverse Transcriptase Kit (Invitrogen) as per manufacturer's instructions. .. The synthesized cDNA was diluted four times in RNase-free water and subsequently used for qRT-PCR to quantify mouse miR-29b target genes Col3a1 , Col1a1 , Col5a3 , Adam12, and Dnmt3a demonstrates primer binding sites for NIH3T3 qRT-PCR positive and negative control genes.

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  • 98
    Thermo Fisher zinc based ultrasensitive microscopic barrier assay znumba
    Transient, localized leaks in the tight junction barrier are followed by flares of active Rho and restoration of barrier function. a . Schematic of Zinc-based <t>Ultrasensitive</t> Microscopic Barrier Assay <t>(ZnUMBA).</t> Exchange of zinc and FluoZin-3 (FZ3) through disrupted tight junctions yields a local increase in FZ3 fluorescence. b . Validation of ZnUMBA by laser injury of the junction (red bracket). FZ3 (green) increases in intensity (yellow arrowhead) following laser injury and persists for roughly 75 seconds. Note that F-actin (Lifeact-RFP, magenta) accumulates at the site of injury, and the junction contracts (black bracket vs. grey bracket). c . FIRE Lookup Table (LUT) applied to FZ3 in the unperturbed Xenopus laevis epithelium. Arrowheads indicate short-lived, localized barrier leaks. d . Leaks (FZ3, white arrow) occur at sites of local ZO-1 loss (white arrowhead), and Rho flares (yellow arrowhead) follow at these sites. FZ3 intensity decreases while ZO-1 intensity increases following Rho flares. e . Mean normalized intensity for active Rho (mCherry-2xrGBD), FZ3, and ZO-1 (BFP-ZO-1) at the site of the Rho flare over time quantified from d .
    Zinc Based Ultrasensitive Microscopic Barrier Assay Znumba, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/zinc based ultrasensitive microscopic barrier assay znumba/product/Thermo Fisher
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    zinc based ultrasensitive microscopic barrier assay znumba - by Bioz Stars, 2020-08
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    94
    Thermo Fisher quantitative rt pcr analysis total rna
    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger <t>RNA</t> (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time <t>(qRT)‐PCR.</t> ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P
    Quantitative Rt Pcr Analysis Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative rt pcr analysis total rna/product/Thermo Fisher
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    89
    Thermo Fisher real time polymerase chain reaction pcr analysis total rna
    Time-dependent effect of PA on NPY, AgRP, and POMC expression in N1E-115 cells. Quantitative real-time <t>PCR</t> analysis of NPY, AgRP, and POMC performed with <t>RNA</t> extracted from N1E-115 cells treated with 10 μmol/L PA for different durations ( A – C ), respectively. Quantified mRNA levels were normalized to GAPDH and presented relative to 10 μmol/L PA for 0 min. Data are expressed as means ± SEM of three different experiments. Groups sharing different letters above the bars indicate statistically significant differences ( p
    Real Time Polymerase Chain Reaction Pcr Analysis Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 89 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    real time polymerase chain reaction pcr analysis total rna - by Bioz Stars, 2020-08
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    Image Search Results


    Transient, localized leaks in the tight junction barrier are followed by flares of active Rho and restoration of barrier function. a . Schematic of Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA). Exchange of zinc and FluoZin-3 (FZ3) through disrupted tight junctions yields a local increase in FZ3 fluorescence. b . Validation of ZnUMBA by laser injury of the junction (red bracket). FZ3 (green) increases in intensity (yellow arrowhead) following laser injury and persists for roughly 75 seconds. Note that F-actin (Lifeact-RFP, magenta) accumulates at the site of injury, and the junction contracts (black bracket vs. grey bracket). c . FIRE Lookup Table (LUT) applied to FZ3 in the unperturbed Xenopus laevis epithelium. Arrowheads indicate short-lived, localized barrier leaks. d . Leaks (FZ3, white arrow) occur at sites of local ZO-1 loss (white arrowhead), and Rho flares (yellow arrowhead) follow at these sites. FZ3 intensity decreases while ZO-1 intensity increases following Rho flares. e . Mean normalized intensity for active Rho (mCherry-2xrGBD), FZ3, and ZO-1 (BFP-ZO-1) at the site of the Rho flare over time quantified from d .

    Journal: Developmental cell

    Article Title: Rho flares repair local tight junction leaks

    doi: 10.1016/j.devcel.2019.01.016

    Figure Lengend Snippet: Transient, localized leaks in the tight junction barrier are followed by flares of active Rho and restoration of barrier function. a . Schematic of Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA). Exchange of zinc and FluoZin-3 (FZ3) through disrupted tight junctions yields a local increase in FZ3 fluorescence. b . Validation of ZnUMBA by laser injury of the junction (red bracket). FZ3 (green) increases in intensity (yellow arrowhead) following laser injury and persists for roughly 75 seconds. Note that F-actin (Lifeact-RFP, magenta) accumulates at the site of injury, and the junction contracts (black bracket vs. grey bracket). c . FIRE Lookup Table (LUT) applied to FZ3 in the unperturbed Xenopus laevis epithelium. Arrowheads indicate short-lived, localized barrier leaks. d . Leaks (FZ3, white arrow) occur at sites of local ZO-1 loss (white arrowhead), and Rho flares (yellow arrowhead) follow at these sites. FZ3 intensity decreases while ZO-1 intensity increases following Rho flares. e . Mean normalized intensity for active Rho (mCherry-2xrGBD), FZ3, and ZO-1 (BFP-ZO-1) at the site of the Rho flare over time quantified from d .

    Article Snippet: To carry out the Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA), 5–10 nl of 1 mM FluoZin3 (Thermo Fisher Scientific), 100 μM CaCl2 , and 100 μM EDTA was microinjected into the blastocoel of stage 10–11 (Nieuwkoop and Faber) X. laevis embryos.

    Techniques: Fluorescence

    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Journal: Thoracic Cancer

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    doi: 10.1111/1759-7714.12895

    Figure Lengend Snippet: Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

    Techniques: Expressing, Migration, In Vitro, Quantitative RT-PCR, Western Blot

    Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Journal: Thoracic Cancer

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    doi: 10.1111/1759-7714.12895

    Figure Lengend Snippet: Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

    Techniques: Migration, In Vitro, Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot

    Time-dependent effect of PA on NPY, AgRP, and POMC expression in N1E-115 cells. Quantitative real-time PCR analysis of NPY, AgRP, and POMC performed with RNA extracted from N1E-115 cells treated with 10 μmol/L PA for different durations ( A – C ), respectively. Quantified mRNA levels were normalized to GAPDH and presented relative to 10 μmol/L PA for 0 min. Data are expressed as means ± SEM of three different experiments. Groups sharing different letters above the bars indicate statistically significant differences ( p

    Journal: Nutrients

    Article Title: Involvement of CD36 in Modulating the Decrease of NPY and AgRP Induced by Acute Palmitic Acid Stimulation in N1E-115 Cells

    doi: 10.3390/nu9060626

    Figure Lengend Snippet: Time-dependent effect of PA on NPY, AgRP, and POMC expression in N1E-115 cells. Quantitative real-time PCR analysis of NPY, AgRP, and POMC performed with RNA extracted from N1E-115 cells treated with 10 μmol/L PA for different durations ( A – C ), respectively. Quantified mRNA levels were normalized to GAPDH and presented relative to 10 μmol/L PA for 0 min. Data are expressed as means ± SEM of three different experiments. Groups sharing different letters above the bars indicate statistically significant differences ( p

    Article Snippet: RNA Extraction and Real-Time Polymerase Chain Reaction (PCR) Analysis Total RNA was extracted by using TRIZOL (Thermo Fisher, 15596-026, Carlsbad, CA, USA) according to the manufacturer’s manual.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Effect of PA on CD36 expression in N1E-115 cells. Quantitative real-time PCR amplification of CD36 performed with RNA extracted from N1E-115 cells treated with 10 μmol/L PA at different time points ( A ) or different concentrations of PA for 20 min ( B ), respectively. ( C ) Representative Western blot analysis of CD36 protein in N1E-115 cells induced by 10 μmol/L PA at 0, 10, and 20 min. Data are expressed as means ± SEM of three different experiments. Groups sharing different letters above the bars indicate statistically significant differences ( p

    Journal: Nutrients

    Article Title: Involvement of CD36 in Modulating the Decrease of NPY and AgRP Induced by Acute Palmitic Acid Stimulation in N1E-115 Cells

    doi: 10.3390/nu9060626

    Figure Lengend Snippet: Effect of PA on CD36 expression in N1E-115 cells. Quantitative real-time PCR amplification of CD36 performed with RNA extracted from N1E-115 cells treated with 10 μmol/L PA at different time points ( A ) or different concentrations of PA for 20 min ( B ), respectively. ( C ) Representative Western blot analysis of CD36 protein in N1E-115 cells induced by 10 μmol/L PA at 0, 10, and 20 min. Data are expressed as means ± SEM of three different experiments. Groups sharing different letters above the bars indicate statistically significant differences ( p

    Article Snippet: RNA Extraction and Real-Time Polymerase Chain Reaction (PCR) Analysis Total RNA was extracted by using TRIZOL (Thermo Fisher, 15596-026, Carlsbad, CA, USA) according to the manufacturer’s manual.

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

    Dose-dependent effect of PA on NPY, AgRP, and POMC expression in N1E-115 cells. Quantitative real-time PCR amplification of NPY, AgRP, and POMC performed with RNA extracted from N1E-115 cells following treatment with differing concentrations of PA for 20 min ( A – C ), respectively. Quantified mRNA levels were normalized to GAPDH and presented relative to 0 μmol/L PA for 20 min. Data are expressed as means ± SEM of three different experiments. Groups sharing different letters above the bars indicate statistically significant differences ( p

    Journal: Nutrients

    Article Title: Involvement of CD36 in Modulating the Decrease of NPY and AgRP Induced by Acute Palmitic Acid Stimulation in N1E-115 Cells

    doi: 10.3390/nu9060626

    Figure Lengend Snippet: Dose-dependent effect of PA on NPY, AgRP, and POMC expression in N1E-115 cells. Quantitative real-time PCR amplification of NPY, AgRP, and POMC performed with RNA extracted from N1E-115 cells following treatment with differing concentrations of PA for 20 min ( A – C ), respectively. Quantified mRNA levels were normalized to GAPDH and presented relative to 0 μmol/L PA for 20 min. Data are expressed as means ± SEM of three different experiments. Groups sharing different letters above the bars indicate statistically significant differences ( p

    Article Snippet: RNA Extraction and Real-Time Polymerase Chain Reaction (PCR) Analysis Total RNA was extracted by using TRIZOL (Thermo Fisher, 15596-026, Carlsbad, CA, USA) according to the manufacturer’s manual.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Amplification