qrt pcr reactions  (Thermo Fisher)


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    Structured Review

    Thermo Fisher qrt pcr reactions
    Coefficient analysis of gene expression levels obtained from RNA-Seq and <t>qRT-PCR</t> data. Log 2 fold change: log 2 fold-change in gene expression between Si-treated and non-Si-treated samples.
    Qrt Pcr Reactions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1212 article reviews
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    qrt pcr reactions - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Transcriptome Analysis Reveals New Insights into the Bacterial Wilt Resistance Mechanism Mediated by Silicon in Tomato"

    Article Title: Transcriptome Analysis Reveals New Insights into the Bacterial Wilt Resistance Mechanism Mediated by Silicon in Tomato

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20030761

    Coefficient analysis of gene expression levels obtained from RNA-Seq and qRT-PCR data. Log 2 fold change: log 2 fold-change in gene expression between Si-treated and non-Si-treated samples.
    Figure Legend Snippet: Coefficient analysis of gene expression levels obtained from RNA-Seq and qRT-PCR data. Log 2 fold change: log 2 fold-change in gene expression between Si-treated and non-Si-treated samples.

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

    2) Product Images from "Protein Kinase C ? Regulates the Phenotype of Murine CD4+ Th17 Cells"

    Article Title: Protein Kinase C ? Regulates the Phenotype of Murine CD4+ Th17 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096401

    PKCθ-deficiency does not alter expression of IFN-γ and T-bet under neutral or Th1-polarizing conditions. Naïve CD4 + T cells were cultured under neutral (Th0) and Th1-promoting conditions for 4 days. The expression of IFN-γ (A, flow cytometric staining, Bioplex) and T-bet (B, flow cytometric staining, qRT-PCR) was analyzed. Graphs show combined data from two independent experiments, each with n = 3 per genotype; mean values with error bars indicating +/− SEM are presented. Statistical significance was assessed by a two-way ANOVA with a Bonferroni post hoc test. MFI - mean fluorescent intensity.
    Figure Legend Snippet: PKCθ-deficiency does not alter expression of IFN-γ and T-bet under neutral or Th1-polarizing conditions. Naïve CD4 + T cells were cultured under neutral (Th0) and Th1-promoting conditions for 4 days. The expression of IFN-γ (A, flow cytometric staining, Bioplex) and T-bet (B, flow cytometric staining, qRT-PCR) was analyzed. Graphs show combined data from two independent experiments, each with n = 3 per genotype; mean values with error bars indicating +/− SEM are presented. Statistical significance was assessed by a two-way ANOVA with a Bonferroni post hoc test. MFI - mean fluorescent intensity.

    Techniques Used: Expressing, Cell Culture, Flow Cytometry, Staining, Quantitative RT-PCR

    Regulation of STAT4 differs between wild-type and PKCθ −/− CD4 + T cells during the Th17-priming. A) Western blot analysis of phosphorylated (pSTAT4) and total STAT4 protein. Naïve CD4 + T cell were stimulated with indicated cytokines for 45 min (upper panel) or differentiated for 20 h in the presence of anti-CD3/anti-CD28 antibodies into Th17 (IL-6/TGF-β) or Th1 (IL-12) effector cells. One representative experiment is shown. B) Western blot analysis of pSTAT4 and STAT4 in naïve CD4 + T cells stimulated for 20 h under Th17-promoting conditions with and without IL-23. One representative experiment, with two biological replicates of each genotype, is shown. C) Stat4 mRNA expression was analyzed by qRT-PCR measurements (relative expression values normalized to TBP as a reference gene) during the Th17 priming of naïve CD4 + T cells. D) PKCθ mRNA expression was analyzed by qRT-PCR measurements (relative expression values normalized to TBP) during the Th17 priming of naïve CD4 + T cells. Graphs show representative data of one of three independent experiments, each with n = 3 per genotype. All graphs represent mean values and error bars indicate +/− SEM. Statistical significance was assessed by a two-way ANOVA with a Bonferroni post hoc test.
    Figure Legend Snippet: Regulation of STAT4 differs between wild-type and PKCθ −/− CD4 + T cells during the Th17-priming. A) Western blot analysis of phosphorylated (pSTAT4) and total STAT4 protein. Naïve CD4 + T cell were stimulated with indicated cytokines for 45 min (upper panel) or differentiated for 20 h in the presence of anti-CD3/anti-CD28 antibodies into Th17 (IL-6/TGF-β) or Th1 (IL-12) effector cells. One representative experiment is shown. B) Western blot analysis of pSTAT4 and STAT4 in naïve CD4 + T cells stimulated for 20 h under Th17-promoting conditions with and without IL-23. One representative experiment, with two biological replicates of each genotype, is shown. C) Stat4 mRNA expression was analyzed by qRT-PCR measurements (relative expression values normalized to TBP as a reference gene) during the Th17 priming of naïve CD4 + T cells. D) PKCθ mRNA expression was analyzed by qRT-PCR measurements (relative expression values normalized to TBP) during the Th17 priming of naïve CD4 + T cells. Graphs show representative data of one of three independent experiments, each with n = 3 per genotype. All graphs represent mean values and error bars indicate +/− SEM. Statistical significance was assessed by a two-way ANOVA with a Bonferroni post hoc test.

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

    Activation of IFN-γ/STAT1/T-bet axis is enhanced in the PKCθ −/− CD4 + T cells under Th17-polarizing conditions. Naïve CD4 + T cells were cultured under Th17-promoting conditions and analyzed. A) IL-17A, IFN-γ and T-bet expression was measured by intracellular flow cytometry staining on three subsequent days of Th17 differentiation. Graphs show combined data from two independent experiments, each with n = 3 per genotype. B) Early induction of IFN-γ and T-bet mRNA expression in Th17 differentiation cultures was analyzed by qRT-PCR (relative expression normalized to TBP as a reference gene). All graphs summarize data from three independent experiments, each with n = 3 per genotype. C) A representative western blot analysis of T-bet expression and total and phosphorylated STAT1 in Th17-differentiated CD4 + T cells (day 1 and 4). One out of three independent experiments, with two biological replicates per genotype, is shown. D) Naïve CD4 + T cells were cultured under Th17-promoting conditions in the presence (Th17 sd – Th17 standard conditions) or absence of anti-IFN-γ neutralizing antibodies for 4 days. IFN-γ and T-bet expression was analyzed by intracellular flow cytometric staining. Graphs show combined data from two independent experiments, each with n = 3 per genotype. All the graphs represent mean values with error bars indicating +/− SEM. Statistical significance was assessed by a two-way ANOVA with a Bonferroni post hoc test. MFI - mean fluorescent intensity.
    Figure Legend Snippet: Activation of IFN-γ/STAT1/T-bet axis is enhanced in the PKCθ −/− CD4 + T cells under Th17-polarizing conditions. Naïve CD4 + T cells were cultured under Th17-promoting conditions and analyzed. A) IL-17A, IFN-γ and T-bet expression was measured by intracellular flow cytometry staining on three subsequent days of Th17 differentiation. Graphs show combined data from two independent experiments, each with n = 3 per genotype. B) Early induction of IFN-γ and T-bet mRNA expression in Th17 differentiation cultures was analyzed by qRT-PCR (relative expression normalized to TBP as a reference gene). All graphs summarize data from three independent experiments, each with n = 3 per genotype. C) A representative western blot analysis of T-bet expression and total and phosphorylated STAT1 in Th17-differentiated CD4 + T cells (day 1 and 4). One out of three independent experiments, with two biological replicates per genotype, is shown. D) Naïve CD4 + T cells were cultured under Th17-promoting conditions in the presence (Th17 sd – Th17 standard conditions) or absence of anti-IFN-γ neutralizing antibodies for 4 days. IFN-γ and T-bet expression was analyzed by intracellular flow cytometric staining. Graphs show combined data from two independent experiments, each with n = 3 per genotype. All the graphs represent mean values with error bars indicating +/− SEM. Statistical significance was assessed by a two-way ANOVA with a Bonferroni post hoc test. MFI - mean fluorescent intensity.

    Techniques Used: Activation Assay, Cell Culture, Expressing, Flow Cytometry, Cytometry, Staining, Quantitative RT-PCR, Western Blot

    3) Product Images from "miR-103 Regulates Oxidative Stress by Targeting the BCL2/Adenovirus E1B 19 kDa Interacting Protein 3 in HUVECs"

    Article Title: miR-103 Regulates Oxidative Stress by Targeting the BCL2/Adenovirus E1B 19 kDa Interacting Protein 3 in HUVECs

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2015/489647

    Validation of BNIP3 as a miR-103 target gene. (a) The 3′-UTR of BNIP3 and mutant 3′-UTR sequences that abolished binding. (b) Luciferase activity was assessed in HUVECs transfected with BNIP3 3′-UTR-WT or BNIP3 3′-UTR-MUT and the mimic control or miR-103. (c) Luciferase activity was assessed in HUVECs transfected with BNIP3 3′-UTR-WT or BNIP3 3′-UTR-MUT and the inhibitor control or miR-103 inhibitor. (d) BNIP3 mRNA levels analyzed by qRT-PCR. (e) Western blot analysis of the endogenous expression of BNIP3 upon forced expression of miR-103. (f) The protein expression of BNIP3 in HUVECs transfected with the miR-103 inhibitor or inhibitor control was determined by western blotting. ∗ p
    Figure Legend Snippet: Validation of BNIP3 as a miR-103 target gene. (a) The 3′-UTR of BNIP3 and mutant 3′-UTR sequences that abolished binding. (b) Luciferase activity was assessed in HUVECs transfected with BNIP3 3′-UTR-WT or BNIP3 3′-UTR-MUT and the mimic control or miR-103. (c) Luciferase activity was assessed in HUVECs transfected with BNIP3 3′-UTR-WT or BNIP3 3′-UTR-MUT and the inhibitor control or miR-103 inhibitor. (d) BNIP3 mRNA levels analyzed by qRT-PCR. (e) Western blot analysis of the endogenous expression of BNIP3 upon forced expression of miR-103. (f) The protein expression of BNIP3 in HUVECs transfected with the miR-103 inhibitor or inhibitor control was determined by western blotting. ∗ p

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

    Salidroside mediated the expression of miR-103 in HUVECs induced by H 2 O 2 . (a) The expression of miR-103 was detected by qRT-PCR after H 2 O 2 treatment at the indicated time points. (b) The expression of miR-103 was determined in HUVECs treated with the indicated concentration of H 2 O 2 for 6 h. (c) HUVECs were treated with H 2 O 2 alone or in combination with salidroside. The relative expression of miR-103 was detected by qRT-PCR. (d) qRT-PCR was used to assess the relative expression of miR-103 in HUVECs treated with or without salidroside. Data are shown as the mean ± SD from three independent experiments. ∗ p
    Figure Legend Snippet: Salidroside mediated the expression of miR-103 in HUVECs induced by H 2 O 2 . (a) The expression of miR-103 was detected by qRT-PCR after H 2 O 2 treatment at the indicated time points. (b) The expression of miR-103 was determined in HUVECs treated with the indicated concentration of H 2 O 2 for 6 h. (c) HUVECs were treated with H 2 O 2 alone or in combination with salidroside. The relative expression of miR-103 was detected by qRT-PCR. (d) qRT-PCR was used to assess the relative expression of miR-103 in HUVECs treated with or without salidroside. Data are shown as the mean ± SD from three independent experiments. ∗ p

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

    Effect of miR-103 on HUVECs. (a) qRT-PCR was performed to verify the expression of miR-103 in HUVECs transfected with miR-103. (b) Effect of miR-103 on HUVEC viability induced by H 2 O 2 was measured with the CCK-8 assay. (c) An apoptosis assay was performed to assess the apoptosis levels of HUVECs treated as indicated. (d) Effects of miR-103 on the intracellular formation of ROS triggered by preincubation exposure of HUVECs to H 2 O 2 assessed by DCF assays. ∗∗ p
    Figure Legend Snippet: Effect of miR-103 on HUVECs. (a) qRT-PCR was performed to verify the expression of miR-103 in HUVECs transfected with miR-103. (b) Effect of miR-103 on HUVEC viability induced by H 2 O 2 was measured with the CCK-8 assay. (c) An apoptosis assay was performed to assess the apoptosis levels of HUVECs treated as indicated. (d) Effects of miR-103 on the intracellular formation of ROS triggered by preincubation exposure of HUVECs to H 2 O 2 assessed by DCF assays. ∗∗ p

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Apoptosis Assay

    4) Product Images from "Tuba1a is uniquely important for axon guidance through midline commissural structures"

    Article Title: Tuba1a is uniquely important for axon guidance through midline commissural structures

    Journal: bioRxiv

    doi: 10.1101/2020.05.05.079376

    TUBA1A ND impairs incorporation into cellular microtubules and reduces α-tubulin protein abundance. A. Schematic of TUBA1A -His6 tag and experimental design. His6 epitope tag was added to an internal loop on TUBA1A (top), and TUBA1A-His6 plasmid DNA was transfected into Cos-7 cells to label cellular microtubules in the presence or absence of proteasome inhibitor treatment (bottom). B. Images showing Cos-7 cells transfected with wild-type, TUBA1A ND , TUBA1A TE (polymerization-incompetent mutant), and TUBA1A EA (highly polymer stable mutant) TUBA1A -His6 plasmid DNA. Cells were immunolabeled with α-His6 antibodies to reveal microtubule incorporation of wild-type and mutant TUBA1A-His6 protein. TUBA1A-His6 images on the left are compared to traditional immunolabeling of α-tubulin using DM1A antibody in untransfected Cos-7 cells on right. C. Western blot for His6 in TUBA1A-His6 transfected Cos-7 cell lysates. A subset of transfected cells were treated with 5μM Lactacystin A (LactA) for 24 hours to block proteasomal degradation. His6 signal was normalized to GFP, which was expressed from the same plasmid. D. Quantification of band density for His6 western blot shown in C . His6 band density was normalized to GFP-expressing cells in control-treated (left), and cells treated with 5μM LactA for 24-hours (right). Data were analyzed by t test. N=3 transfections; n=3 technical replicates; p=0.04 for all comparisons marked with asterisks; p=0.83 for control vs LactA-treated TUBA1A ND . E. Bar graph representing TUBA1A mRNA expression in Cos-7 cells transfected with TUBA1A-His6. TUBA1A mRNA expression was normalized to GFP mRNA expression. Data were normalized to TUBA1A expression in wild-type (WT) TUBA1A-His6-transfected cells and represent 3 separate transfection experiments and 3 qRT-PCR replicates. Differences between groups were assessed by t test (p=0.97). All images were taken at 40X magnification, scale bars are 10μm. All graphs show mean of data ± SEM, *p
    Figure Legend Snippet: TUBA1A ND impairs incorporation into cellular microtubules and reduces α-tubulin protein abundance. A. Schematic of TUBA1A -His6 tag and experimental design. His6 epitope tag was added to an internal loop on TUBA1A (top), and TUBA1A-His6 plasmid DNA was transfected into Cos-7 cells to label cellular microtubules in the presence or absence of proteasome inhibitor treatment (bottom). B. Images showing Cos-7 cells transfected with wild-type, TUBA1A ND , TUBA1A TE (polymerization-incompetent mutant), and TUBA1A EA (highly polymer stable mutant) TUBA1A -His6 plasmid DNA. Cells were immunolabeled with α-His6 antibodies to reveal microtubule incorporation of wild-type and mutant TUBA1A-His6 protein. TUBA1A-His6 images on the left are compared to traditional immunolabeling of α-tubulin using DM1A antibody in untransfected Cos-7 cells on right. C. Western blot for His6 in TUBA1A-His6 transfected Cos-7 cell lysates. A subset of transfected cells were treated with 5μM Lactacystin A (LactA) for 24 hours to block proteasomal degradation. His6 signal was normalized to GFP, which was expressed from the same plasmid. D. Quantification of band density for His6 western blot shown in C . His6 band density was normalized to GFP-expressing cells in control-treated (left), and cells treated with 5μM LactA for 24-hours (right). Data were analyzed by t test. N=3 transfections; n=3 technical replicates; p=0.04 for all comparisons marked with asterisks; p=0.83 for control vs LactA-treated TUBA1A ND . E. Bar graph representing TUBA1A mRNA expression in Cos-7 cells transfected with TUBA1A-His6. TUBA1A mRNA expression was normalized to GFP mRNA expression. Data were normalized to TUBA1A expression in wild-type (WT) TUBA1A-His6-transfected cells and represent 3 separate transfection experiments and 3 qRT-PCR replicates. Differences between groups were assessed by t test (p=0.97). All images were taken at 40X magnification, scale bars are 10μm. All graphs show mean of data ± SEM, *p

    Techniques Used: Plasmid Preparation, Transfection, Mutagenesis, Immunolabeling, Western Blot, Blocking Assay, Expressing, Quantitative RT-PCR

    5) Product Images from "Broad anti-coronaviral activity of FDA approved drugs against SARS-CoV-2 in vitro and SARS-CoV in vivo"

    Article Title: Broad anti-coronaviral activity of FDA approved drugs against SARS-CoV-2 in vitro and SARS-CoV in vivo

    Journal: bioRxiv

    doi: 10.1101/2020.03.25.008482

    Hydroxychloroquine and chloroquine inhibit production of SARS-CoV-2 N and RdRp mRNA. Vero cells were pre-treated with hydroxychloroquine sulfate (A, C and E) or chloroquine phosphate (B, D and F) at the indicated concentration (or 0.1% water as vehicle control) for 2 h prior to infection with SARS-CoV-2 (WA-1 strain) at MOI 0.1. 24 h post-infection cells were collected in TRIzol. RNA was extracted from TRIzol sample and qRT-PCR was performed for viral RdRp (A and B) or N (C and D) mRNA using WHO primers. RNA levels were normalized with 18S RNA and fold change for drug treated to vehicle control was calculated (dotted line to denote a fold change of 1 which is no change over control). Data are from 3 independent infections performed on triplicate wells, the fold change was calculated in each independent experiment and the mean fold change is plotted with error bars displaying standard deviation. Along with TRIzol samples for RNA supernatant was collected from cells and used for TCID 50 assays to determine infectious virus production following treatment with HCQ (E) or CQ (F) Data are from 3 independent infections performed on triplicate wells with the TCID 50 /ml being averaged across all wells. Error bars are the standard deviation. G) Cells were treated with 50 μM HCQ or 0.1% water as control. Drug was either added 2 h prior to infection, at the time of infection or 2 h after infection with MOI 0.1 SARS-CoV-2. After 24 h infection, supernatant was collected and used for TCID 50 assays to determine infectious virus production. Data are from 3 independent infections performed on triplicate wells with the TCID 50 /ml being averaged across all wells. Error bars are the standard deviation. H) SARS-CoV spike psuedoviruses (PV) were used for infection of BSC1 cells. The cells were treated with 10 μM of CQ or CPZ for 1 h prior to infection with PV for 3 h. The PV carry BlaM and cells were loaded with CCF2 to monitor cleavage and shift in fluorescence output for evidence of S-mediated entry into cells. Data are normalised to PV alone and are from 3 independent experiments with error bars representing standard deviation.
    Figure Legend Snippet: Hydroxychloroquine and chloroquine inhibit production of SARS-CoV-2 N and RdRp mRNA. Vero cells were pre-treated with hydroxychloroquine sulfate (A, C and E) or chloroquine phosphate (B, D and F) at the indicated concentration (or 0.1% water as vehicle control) for 2 h prior to infection with SARS-CoV-2 (WA-1 strain) at MOI 0.1. 24 h post-infection cells were collected in TRIzol. RNA was extracted from TRIzol sample and qRT-PCR was performed for viral RdRp (A and B) or N (C and D) mRNA using WHO primers. RNA levels were normalized with 18S RNA and fold change for drug treated to vehicle control was calculated (dotted line to denote a fold change of 1 which is no change over control). Data are from 3 independent infections performed on triplicate wells, the fold change was calculated in each independent experiment and the mean fold change is plotted with error bars displaying standard deviation. Along with TRIzol samples for RNA supernatant was collected from cells and used for TCID 50 assays to determine infectious virus production following treatment with HCQ (E) or CQ (F) Data are from 3 independent infections performed on triplicate wells with the TCID 50 /ml being averaged across all wells. Error bars are the standard deviation. G) Cells were treated with 50 μM HCQ or 0.1% water as control. Drug was either added 2 h prior to infection, at the time of infection or 2 h after infection with MOI 0.1 SARS-CoV-2. After 24 h infection, supernatant was collected and used for TCID 50 assays to determine infectious virus production. Data are from 3 independent infections performed on triplicate wells with the TCID 50 /ml being averaged across all wells. Error bars are the standard deviation. H) SARS-CoV spike psuedoviruses (PV) were used for infection of BSC1 cells. The cells were treated with 10 μM of CQ or CPZ for 1 h prior to infection with PV for 3 h. The PV carry BlaM and cells were loaded with CCF2 to monitor cleavage and shift in fluorescence output for evidence of S-mediated entry into cells. Data are normalised to PV alone and are from 3 independent experiments with error bars representing standard deviation.

    Techniques Used: Concentration Assay, Infection, Quantitative RT-PCR, Standard Deviation, Fluorescence

    6) Product Images from "A Dominant-Negative Isoform of IKAROS Expands Primitive Normal Human Hematopoietic Cells"

    Article Title: A Dominant-Negative Isoform of IKAROS Expands Primitive Normal Human Hematopoietic Cells

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2014.09.006

    IKAROS Expression and Inhibition by IK6 in Human CB Cells (A) Human IKAROS exon structure. (B) Isoform-specific IKZF1 transcripts determined by qRT-PCR in IK6- and control-transduced CD34 + CB cells and their clonally derived erythroid (from BFU-E) and GM progeny (from CFC-GM; mean ± SEM; three experiments). (C) IKAROS protein isoforms in CD34 + CB cells analyzed by western blotting with histone H3 as the loading control. (D) Representative flow cytometric profiles of intracellular IKAROS in normal human CB cell subsets (from one of three experiments). (E) Confocal microscopy images of representative single untransduced or IK6-transduced CD34 + CB cells stained with DAPI (blue) and an antibody reactive with both full-length IKAROS and IK6 (red). Scale bar, 10 μm.
    Figure Legend Snippet: IKAROS Expression and Inhibition by IK6 in Human CB Cells (A) Human IKAROS exon structure. (B) Isoform-specific IKZF1 transcripts determined by qRT-PCR in IK6- and control-transduced CD34 + CB cells and their clonally derived erythroid (from BFU-E) and GM progeny (from CFC-GM; mean ± SEM; three experiments). (C) IKAROS protein isoforms in CD34 + CB cells analyzed by western blotting with histone H3 as the loading control. (D) Representative flow cytometric profiles of intracellular IKAROS in normal human CB cell subsets (from one of three experiments). (E) Confocal microscopy images of representative single untransduced or IK6-transduced CD34 + CB cells stained with DAPI (blue) and an antibody reactive with both full-length IKAROS and IK6 (red). Scale bar, 10 μm.

    Techniques Used: Expressing, Inhibition, Quantitative RT-PCR, Derivative Assay, Western Blot, Flow Cytometry, Confocal Microscopy, Staining

    7) Product Images from "Epigenetic silencing of miR-145-5p contributes to brain metastasis"

    Article Title: Epigenetic silencing of miR-145-5p contributes to brain metastasis

    Journal: Oncotarget

    doi:

    miR-145-5p impairs the expression of MUC-1 and MYC proteins A. MUC-1 immunocitochemistry in H1299 cells upon miR-145-5p over-expression. B. MUC-1 immunocitochemistry in H1299 cells upon 5 uM vorinostat (SAHA) treatment. C. MUC-1 immunocitochemistry in H1299 cells upon MUC-1 RNA interference (left panel) and transwell migration assay in the same conditions (right panel). D. Western-blot analysis of MYC protein expression levels in H1299 cells upon miR-145-5p over-expression. E. qRT-PCR analysis of MYC mRNA levels in H1299 cells upon 24 hours of 5 uM vorinostat (SAHA) treatment. F. Western-blot analysis of MYC in H1299, MDA-231 and M14 cells upon 24 hours of vorinostat (SAHA) treatments. G. MYC protein levels in H1299 cells upon MYC RNA interference and transwell migration assay in the same conditions.
    Figure Legend Snippet: miR-145-5p impairs the expression of MUC-1 and MYC proteins A. MUC-1 immunocitochemistry in H1299 cells upon miR-145-5p over-expression. B. MUC-1 immunocitochemistry in H1299 cells upon 5 uM vorinostat (SAHA) treatment. C. MUC-1 immunocitochemistry in H1299 cells upon MUC-1 RNA interference (left panel) and transwell migration assay in the same conditions (right panel). D. Western-blot analysis of MYC protein expression levels in H1299 cells upon miR-145-5p over-expression. E. qRT-PCR analysis of MYC mRNA levels in H1299 cells upon 24 hours of 5 uM vorinostat (SAHA) treatment. F. Western-blot analysis of MYC in H1299, MDA-231 and M14 cells upon 24 hours of vorinostat (SAHA) treatments. G. MYC protein levels in H1299 cells upon MYC RNA interference and transwell migration assay in the same conditions.

    Techniques Used: Expressing, Over Expression, Transwell Migration Assay, Western Blot, Quantitative RT-PCR, Multiple Displacement Amplification

    miR-145-5p impairs the expression of EGFR protein A. Immunohistochemistry for EGFR in 2 representative patients slides (pt=patient). B. Quantification of positive EGFR staining in tissues. C. Western-blot analysis of EGFR protein expression in H1299 cells upon miR-145-5p over-expression. D. Renilla luciferase activity of EGFR-3′UTR reporter gene in H1299 transiently transfected with miR-145-5p mimic or control mimic. E. qRT-PCR analysis of EGFR mRNA levels in H1299 cells upon 5 uM vorinostat (SAHA) treatment. F. Western-blot analysis of EGFR in H1299, MDA-231 and M14 cells upon 24 hours of vorinostat (SAHA) treatments. G. Quantification of immunofluorescence assay to analyze EGFR localization in H1299 cells treated with EGF (20 ng/mL) upon miR-145-5p over-expression. H. Analysis of pEGFR protein expression levels in H1299 cells treated with EGF (20 ng/mL) or maintained in serum free (Sf) medium upon miR-145-5p over-expression. I. c-Met protein levels in H1299 cells treated with EGF (20 ng/mL) or maintained in serum free (Sf) medium upon miR-145-5p over-expression. L. Quantification of immunofluorescence assay to analyze EGFR localization in H1299 cells treated with EGF (20 ng/mL) upon MUC-1 RNA interference.
    Figure Legend Snippet: miR-145-5p impairs the expression of EGFR protein A. Immunohistochemistry for EGFR in 2 representative patients slides (pt=patient). B. Quantification of positive EGFR staining in tissues. C. Western-blot analysis of EGFR protein expression in H1299 cells upon miR-145-5p over-expression. D. Renilla luciferase activity of EGFR-3′UTR reporter gene in H1299 transiently transfected with miR-145-5p mimic or control mimic. E. qRT-PCR analysis of EGFR mRNA levels in H1299 cells upon 5 uM vorinostat (SAHA) treatment. F. Western-blot analysis of EGFR in H1299, MDA-231 and M14 cells upon 24 hours of vorinostat (SAHA) treatments. G. Quantification of immunofluorescence assay to analyze EGFR localization in H1299 cells treated with EGF (20 ng/mL) upon miR-145-5p over-expression. H. Analysis of pEGFR protein expression levels in H1299 cells treated with EGF (20 ng/mL) or maintained in serum free (Sf) medium upon miR-145-5p over-expression. I. c-Met protein levels in H1299 cells treated with EGF (20 ng/mL) or maintained in serum free (Sf) medium upon miR-145-5p over-expression. L. Quantification of immunofluorescence assay to analyze EGFR localization in H1299 cells treated with EGF (20 ng/mL) upon MUC-1 RNA interference.

    Techniques Used: Expressing, Immunohistochemistry, Staining, Western Blot, Over Expression, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Multiple Displacement Amplification, Immunofluorescence

    miR-145-5p impairs the expression of TPD52 protein A. List of the most significant miR-145-5p putative target genes with a known role in metastasis pathways, that resulted to be inversely correlated to miR-145-5p expression in the TCGA casuistry of 526 lung adenocarcinoma tissues. Genes were ranked by the number of the softwares that predicted miR-145-5p binding on the 3′UTR. B. Heat map of TPD52 and miR-145-5p genes expression in 526 tumoral samples of lung adenocarcinoma TCGA casuistry. C. miR-145-5p and TPD52 genes expression in matched samples of normal (N) and tumoral (T) tissues of lung adenocarcinoma TCGA casuistry. D. Immunohistochemistry for TPD52 in 2 representative patients slides (pt=patient). E. Western-blot analysis of TPD52 protein expression in H1299 cells upon miR-145-5p and miR-145-3p mimics over-expression. F. qRT-PCR analysis of TPD52 mRNA levels in H1299 cells upon 24 hours of 5 uM vorinostat (SAHA) treatment. G. Western-blot analysis of TPD52 in H1299 upon vorinostat (SAHA) treatments. H. TPD52 protein levels in H1299 cells upon TPD52 RNA interference (left panel) and transwell migration assay in the same conditions (right panel).
    Figure Legend Snippet: miR-145-5p impairs the expression of TPD52 protein A. List of the most significant miR-145-5p putative target genes with a known role in metastasis pathways, that resulted to be inversely correlated to miR-145-5p expression in the TCGA casuistry of 526 lung adenocarcinoma tissues. Genes were ranked by the number of the softwares that predicted miR-145-5p binding on the 3′UTR. B. Heat map of TPD52 and miR-145-5p genes expression in 526 tumoral samples of lung adenocarcinoma TCGA casuistry. C. miR-145-5p and TPD52 genes expression in matched samples of normal (N) and tumoral (T) tissues of lung adenocarcinoma TCGA casuistry. D. Immunohistochemistry for TPD52 in 2 representative patients slides (pt=patient). E. Western-blot analysis of TPD52 protein expression in H1299 cells upon miR-145-5p and miR-145-3p mimics over-expression. F. qRT-PCR analysis of TPD52 mRNA levels in H1299 cells upon 24 hours of 5 uM vorinostat (SAHA) treatment. G. Western-blot analysis of TPD52 in H1299 upon vorinostat (SAHA) treatments. H. TPD52 protein levels in H1299 cells upon TPD52 RNA interference (left panel) and transwell migration assay in the same conditions (right panel).

    Techniques Used: Expressing, Binding Assay, Immunohistochemistry, Western Blot, Over Expression, Quantitative RT-PCR, Transwell Migration Assay

    miR-145-5p expression is down-regulated in brain metastases A. Heat map of the identified signature of 8 miRs differentially expressed in 13 brain metastasis (BM) versus 13 matched primary lung cancer (PLC). B. Unsupervised principal component analysis (PCA). C. qRT-PCR analysis of miR-145-5p expression levels in 10 normal lung (NL), 13 matched primary lung cancer (PLC), and 29 brain metastases from lung (13 matched and 16 unmatched) (BML). D. qRT-PCR analysis of miR-145-5p expression levels in 6 normal brain (NB), 29 brain metastases from lung (BML) and 15 brain metastases from melanoma (6) and breast (9) (BM). E. Schematic representation of miR-145-5p CpG island genomic localization. F. - G. Pyrosequencing analysis of miR-145-5p CpG island methylation status in a representative patient of the casuistry (NL= normal lung; PLC= primitive lung cancer; BM= brain metastases; p= patient; reg1= region 1; reg2= region 2).
    Figure Legend Snippet: miR-145-5p expression is down-regulated in brain metastases A. Heat map of the identified signature of 8 miRs differentially expressed in 13 brain metastasis (BM) versus 13 matched primary lung cancer (PLC). B. Unsupervised principal component analysis (PCA). C. qRT-PCR analysis of miR-145-5p expression levels in 10 normal lung (NL), 13 matched primary lung cancer (PLC), and 29 brain metastases from lung (13 matched and 16 unmatched) (BML). D. qRT-PCR analysis of miR-145-5p expression levels in 6 normal brain (NB), 29 brain metastases from lung (BML) and 15 brain metastases from melanoma (6) and breast (9) (BM). E. Schematic representation of miR-145-5p CpG island genomic localization. F. - G. Pyrosequencing analysis of miR-145-5p CpG island methylation status in a representative patient of the casuistry (NL= normal lung; PLC= primitive lung cancer; BM= brain metastases; p= patient; reg1= region 1; reg2= region 2).

    Techniques Used: Expressing, Planar Chromatography, Quantitative RT-PCR, Methylation

    8) Product Images from "Long Non-Coding RNA (LncRNA) Urothelial Carcinoma Associated 1 (UCA1) Increases Multi-Drug Resistance of Gastric Cancer via Downregulating miR-27b"

    Article Title: Long Non-Coding RNA (LncRNA) Urothelial Carcinoma Associated 1 (UCA1) Increases Multi-Drug Resistance of Gastric Cancer via Downregulating miR-27b

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.900688

    UCA1 is negatively correlated with miR-27b in gastric cancer. ( A ) Microarray data of the most upregulated lncRNAs in six gastric cancer tissues and six paired peritumoral tissues. Data was retrieved from GEO dataset, with the accession No. GSE53137. ( B, C ) QRT-PCR analysis of UCA1 expression ( B ) and miR-27b ( C ) expression in 28 gastric cancer tissues and paired peritumoral tissues. ( D ) Linear regression analysis of the correlation between UCA1 and miR-27b.
    Figure Legend Snippet: UCA1 is negatively correlated with miR-27b in gastric cancer. ( A ) Microarray data of the most upregulated lncRNAs in six gastric cancer tissues and six paired peritumoral tissues. Data was retrieved from GEO dataset, with the accession No. GSE53137. ( B, C ) QRT-PCR analysis of UCA1 expression ( B ) and miR-27b ( C ) expression in 28 gastric cancer tissues and paired peritumoral tissues. ( D ) Linear regression analysis of the correlation between UCA1 and miR-27b.

    Techniques Used: Microarray, Quantitative RT-PCR, Expressing

    Knockdown of UCA1 restores miR-27b expression in the MDR gastric cancer cells. ( A, B ) QRT-PCR analysis of UCA1 expression ( A ) and miR-27b ( B ) expression in SGC-7901 cells, and SGC-7901 derived SGC-7901/ADR, SGC-7901/DDP, and SGC-7901/5-FU cells. ( C ) Predicted binding sites between UCA1 and miR-27b. ( D ) Fluorescence in situ hybridization experiments show that UCA1 and miR-27b (red) display opposite expression levels in SGC-7901 and SGC-7901/ADR cells. ( E, F ) QRT-PCR analysis of UCA1 expression ( E ) and miR-27b ( F ) expression in SGC-7901/ADR, SGC-7901/DDP, and SGC-7901/5-FU cells with or without the transfection of UCA1 siRNA. ** p
    Figure Legend Snippet: Knockdown of UCA1 restores miR-27b expression in the MDR gastric cancer cells. ( A, B ) QRT-PCR analysis of UCA1 expression ( A ) and miR-27b ( B ) expression in SGC-7901 cells, and SGC-7901 derived SGC-7901/ADR, SGC-7901/DDP, and SGC-7901/5-FU cells. ( C ) Predicted binding sites between UCA1 and miR-27b. ( D ) Fluorescence in situ hybridization experiments show that UCA1 and miR-27b (red) display opposite expression levels in SGC-7901 and SGC-7901/ADR cells. ( E, F ) QRT-PCR analysis of UCA1 expression ( E ) and miR-27b ( F ) expression in SGC-7901/ADR, SGC-7901/DDP, and SGC-7901/5-FU cells with or without the transfection of UCA1 siRNA. ** p

    Techniques Used: Expressing, Quantitative RT-PCR, Derivative Assay, Binding Assay, Fluorescence, In Situ Hybridization, Transfection

    9) Product Images from "Characterization of the Deep-Sea Streptomyces sp. SCSIO 02999 Derived VapC/VapB Toxin-Antitoxin System in Escherichia coli"

    Article Title: Characterization of the Deep-Sea Streptomyces sp. SCSIO 02999 Derived VapC/VapB Toxin-Antitoxin System in Escherichia coli

    Journal: Toxins

    doi: 10.3390/toxins8070195

    VapC cross-activates the toxins in E. coli in a Lon-dependent manner. Fold changes of 14 TA transcripts and 1 RNase gene ( rbn ) in cells overexpressing VapC via pCA24N- vapC as compared to empty vector pCA24N in E. coli were quantified by qRT-PCR. The purA was used as negative control. Two independent cultures were used for the assay, and standard errors are indicated.
    Figure Legend Snippet: VapC cross-activates the toxins in E. coli in a Lon-dependent manner. Fold changes of 14 TA transcripts and 1 RNase gene ( rbn ) in cells overexpressing VapC via pCA24N- vapC as compared to empty vector pCA24N in E. coli were quantified by qRT-PCR. The purA was used as negative control. Two independent cultures were used for the assay, and standard errors are indicated.

    Techniques Used: Plasmid Preparation, Quantitative RT-PCR, Negative Control

    10) Product Images from "Exocarp Properties and Transcriptomic Analysis of Cucumber (Cucumis sativus) Fruit Expressing Age-Related Resistance to Phytophthora capsici"

    Article Title: Exocarp Properties and Transcriptomic Analysis of Cucumber (Cucumis sativus) Fruit Expressing Age-Related Resistance to Phytophthora capsici

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0142133

    Expression analysis of putative defense related genes. (A) qRT-PCR verification of potential pathogen defense-related genes with elevated expression in 16 days post pollination (dpp) peels. (B) Relative expression of SYP121/SNAP33 co-expressed genes in 8 and 16 dpp pericarp and peel samples as assessed by 454 pyrosequencing. Genes shown are in the order listed in Table 4 . (C) Expression of CsFM01 as assessed by 454 pyrosequencing (left) and qRT-PCR analysis (right). 8 = 8 dpp pericarp; 8p = 8 dpp peel; 16 = 16 dpp pericarp; 16p = 16 dpp peel.
    Figure Legend Snippet: Expression analysis of putative defense related genes. (A) qRT-PCR verification of potential pathogen defense-related genes with elevated expression in 16 days post pollination (dpp) peels. (B) Relative expression of SYP121/SNAP33 co-expressed genes in 8 and 16 dpp pericarp and peel samples as assessed by 454 pyrosequencing. Genes shown are in the order listed in Table 4 . (C) Expression of CsFM01 as assessed by 454 pyrosequencing (left) and qRT-PCR analysis (right). 8 = 8 dpp pericarp; 8p = 8 dpp peel; 16 = 16 dpp pericarp; 16p = 16 dpp peel.

    Techniques Used: Expressing, Quantitative RT-PCR

    11) Product Images from "AmyR Is a Novel Negative Regulator of Amylovoran Production in Erwinia amylovora"

    Article Title: AmyR Is a Novel Negative Regulator of Amylovoran Production in Erwinia amylovora

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045038

    Over-expression of the amyR gene suppresses amylovoran production in various amylovoran over-producing mutants. A . Amylovoran production of various mutant strains with or without pAmyR2 plasmid. Bacterial strains were grown in MBMA media with 1% sorbitol for 24 hours at 28°C with shaking. The amount of amylovoran was measured with the CPC assay and normalized to a cell density of 1. B . Relative quantification of amyR and amylovoran biosynthetic genes in various mutant strains with or without pAmyR2 plasmid by qRT-PCR. Bacterial strains were grown in MBMA media with 1% sorbitol for 18 hours at 28°C with shaking. 1: Δ rcsC , 2: Δ rcsC (pAmyR2), 3: Δ envZ , 4: Δ envZ (pAmyR2), 5: Δ ompR , 6: Δ ompR (pAmyR2), 7: Δ grrS , 8: Δ grrS (pAmyR2), 9: Δ grrA , 10: Δ grrA (pAmyR2), 11: Δ hns , 12: Δ hns (pAmyR2).
    Figure Legend Snippet: Over-expression of the amyR gene suppresses amylovoran production in various amylovoran over-producing mutants. A . Amylovoran production of various mutant strains with or without pAmyR2 plasmid. Bacterial strains were grown in MBMA media with 1% sorbitol for 24 hours at 28°C with shaking. The amount of amylovoran was measured with the CPC assay and normalized to a cell density of 1. B . Relative quantification of amyR and amylovoran biosynthetic genes in various mutant strains with or without pAmyR2 plasmid by qRT-PCR. Bacterial strains were grown in MBMA media with 1% sorbitol for 18 hours at 28°C with shaking. 1: Δ rcsC , 2: Δ rcsC (pAmyR2), 3: Δ envZ , 4: Δ envZ (pAmyR2), 5: Δ ompR , 6: Δ ompR (pAmyR2), 7: Δ grrS , 8: Δ grrS (pAmyR2), 9: Δ grrA , 10: Δ grrA (pAmyR2), 11: Δ hns , 12: Δ hns (pAmyR2).

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

    AmyR regulates amylovoran production and gene expression. A. Growth of Erwinia amylovora wild type (WT), amyR mutant, complementation strain and WT containing pAmyR2 plasmid on Luria-Bertani plates. Pictures were taken at 24 h post-inoculation. pAmyR2: plasmid containing 1.1-kb PCR fragment of Erwinia amyR gene and promoter in pGEM T-easy vector. B . Amylovoran production of E. amylovora wild type (WT), amyR mutant, complementation strain and WT containing pAmyR2 plasmid in vitro . Bacterial strains were grown in MBMA media with 1% sorbitol for 24 h at 28°C with shaking. The amount of amylovoran was measured with the CPC assay and normalized to a cell density of 1. Data points represent means of three replicates ± standard deviations. Similar results were obtained in three independent experiments. 1: Ea1189, 2: Δ amyR , 3: Δ amyR (pAmyR2), 4: Δ amyR (pAmyR3), 5: Ea273, 6: Ea273 (pAmyR2). C . Relative quantification of amylovoran biosynthetic genes in amyR mutant compared to WT strain by qRT-PCR. Bacterial cells were grown in MBMA with 1% sorbitol for 18 hours at 28°C with shaking. D . Relative quantification of amylovoran biosynthetic genes in WT containing pAmyR2 plasmid compared to WT strain by qRT-PCR. Bacterial cells were grown in MBMA with 1% sorbitol for 18 hours at 28°C with shaking.
    Figure Legend Snippet: AmyR regulates amylovoran production and gene expression. A. Growth of Erwinia amylovora wild type (WT), amyR mutant, complementation strain and WT containing pAmyR2 plasmid on Luria-Bertani plates. Pictures were taken at 24 h post-inoculation. pAmyR2: plasmid containing 1.1-kb PCR fragment of Erwinia amyR gene and promoter in pGEM T-easy vector. B . Amylovoran production of E. amylovora wild type (WT), amyR mutant, complementation strain and WT containing pAmyR2 plasmid in vitro . Bacterial strains were grown in MBMA media with 1% sorbitol for 24 h at 28°C with shaking. The amount of amylovoran was measured with the CPC assay and normalized to a cell density of 1. Data points represent means of three replicates ± standard deviations. Similar results were obtained in three independent experiments. 1: Ea1189, 2: Δ amyR , 3: Δ amyR (pAmyR2), 4: Δ amyR (pAmyR3), 5: Ea273, 6: Ea273 (pAmyR2). C . Relative quantification of amylovoran biosynthetic genes in amyR mutant compared to WT strain by qRT-PCR. Bacterial cells were grown in MBMA with 1% sorbitol for 18 hours at 28°C with shaking. D . Relative quantification of amylovoran biosynthetic genes in WT containing pAmyR2 plasmid compared to WT strain by qRT-PCR. Bacterial cells were grown in MBMA with 1% sorbitol for 18 hours at 28°C with shaking.

    Techniques Used: Expressing, Mutagenesis, Plasmid Preparation, Polymerase Chain Reaction, In Vitro, Quantitative RT-PCR

    12) Product Images from "MicroRNA-200 Family Modulation in Distinct Breast Cancer Phenotypes"

    Article Title: MicroRNA-200 Family Modulation in Distinct Breast Cancer Phenotypes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0047709

    Expression profile of miR-200f and EMT-transcriptional inducers in breast tumors. The expression levels of miR-200 family members ( A ) and EMT-transcriptional inducers ( B ) were quantified by qRT-PCR in 70 breast cancer samples. Data are depicted as box-and-whisker plots. Adjusted p -values are shown where significant differences were found (Wilcoxon rank-sum test). ER+ (estrogen receptor positive tumors), HER2+ (HER2-positive tumors), TN (triple negative; ER−, PR−, HER2−), MBC (metaplastic breast carcinomas). ( C ) Upper heatmap represents Pearson coefficient (R) correlation values between the expression of EMT-transcriptional inducers and miR200f members. Bottom heatmap depicts the level of statistical significance (P) of the correlations.
    Figure Legend Snippet: Expression profile of miR-200f and EMT-transcriptional inducers in breast tumors. The expression levels of miR-200 family members ( A ) and EMT-transcriptional inducers ( B ) were quantified by qRT-PCR in 70 breast cancer samples. Data are depicted as box-and-whisker plots. Adjusted p -values are shown where significant differences were found (Wilcoxon rank-sum test). ER+ (estrogen receptor positive tumors), HER2+ (HER2-positive tumors), TN (triple negative; ER−, PR−, HER2−), MBC (metaplastic breast carcinomas). ( C ) Upper heatmap represents Pearson coefficient (R) correlation values between the expression of EMT-transcriptional inducers and miR200f members. Bottom heatmap depicts the level of statistical significance (P) of the correlations.

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

    miR-200f and EMT-transcriptional inducers are modulated during in vitro EMT in breast basal cell lines. Expression of ( A ) miR-200f and ( B ) EMT-transcriptional inducers was evaluated by qRT-PCR in the sorted epithelial (EpCAM+) and mesenchymal (Fibros) subpopulations within MCF12A and Myo1089 cell lines. Data are normalized to the expression of RNU48 and 18S respectively. Bars represent mean expression changes ±SE in Fibros subpopulation relative to EpCAM+ cells (baseline). Three biological replicates were measured. Moderated t-test was performed to evaluate statistical significance ( p
    Figure Legend Snippet: miR-200f and EMT-transcriptional inducers are modulated during in vitro EMT in breast basal cell lines. Expression of ( A ) miR-200f and ( B ) EMT-transcriptional inducers was evaluated by qRT-PCR in the sorted epithelial (EpCAM+) and mesenchymal (Fibros) subpopulations within MCF12A and Myo1089 cell lines. Data are normalized to the expression of RNU48 and 18S respectively. Bars represent mean expression changes ±SE in Fibros subpopulation relative to EpCAM+ cells (baseline). Three biological replicates were measured. Moderated t-test was performed to evaluate statistical significance ( p

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

    13) Product Images from "10-Hydroxy-2-decenoic acid, a natural product, improves hyperglycemia and insulin resistance in obese/diabetic KK-Ay mice, but does not prevent obesity"

    Article Title: 10-Hydroxy-2-decenoic acid, a natural product, improves hyperglycemia and insulin resistance in obese/diabetic KK-Ay mice, but does not prevent obesity

    Journal: The Journal of Veterinary Medical Science

    doi: 10.1292/jvms.17-0348

    KK-Ay mice fasted overnight were sacrificed the next day after completing 10H2DA or vehicle administration. A: Relative pAMPK protein levels in skeletal muscles were determined by Western blotting (n=6). B: Relative mRNA expression levels of Pgc-1α in skeletal muscles (n=7–8) were quantified by qRT-PCR and normalized to the levels of Gapdh mRNA. C: Relative translocated GLUT4 protein levels in skeletal muscles were determined by Western blotting (n=6). Data are presented as the mean ± SEM. * P
    Figure Legend Snippet: KK-Ay mice fasted overnight were sacrificed the next day after completing 10H2DA or vehicle administration. A: Relative pAMPK protein levels in skeletal muscles were determined by Western blotting (n=6). B: Relative mRNA expression levels of Pgc-1α in skeletal muscles (n=7–8) were quantified by qRT-PCR and normalized to the levels of Gapdh mRNA. C: Relative translocated GLUT4 protein levels in skeletal muscles were determined by Western blotting (n=6). Data are presented as the mean ± SEM. * P

    Techniques Used: Mouse Assay, Western Blot, Expressing, Pyrolysis Gas Chromatography, Quantitative RT-PCR

    KK-Ay mice fasted overnight were sacrificed the next day after completing 10H2DA or vehicle administration. Relative mRNA expression levels of Pgc-1α in liver (n=7–8) (A), G6Pase in liver (n=7–8) (B) and Pck1 in liver (n=7–8) (C) were quantified by qRT-PCR and normalized to the levels of Gapdh mRNA. D: Relative pAMPK protein levels in liver (n=6) were determined by Western blotting. Data are presented as the mean ± SEM. * P
    Figure Legend Snippet: KK-Ay mice fasted overnight were sacrificed the next day after completing 10H2DA or vehicle administration. Relative mRNA expression levels of Pgc-1α in liver (n=7–8) (A), G6Pase in liver (n=7–8) (B) and Pck1 in liver (n=7–8) (C) were quantified by qRT-PCR and normalized to the levels of Gapdh mRNA. D: Relative pAMPK protein levels in liver (n=6) were determined by Western blotting. Data are presented as the mean ± SEM. * P

    Techniques Used: Mouse Assay, Expressing, Pyrolysis Gas Chromatography, Quantitative RT-PCR, Western Blot

    14) Product Images from "Ocular Albinism Type 1 Regulates Melanogenesis in Mouse Melanocytes"

    Article Title: Ocular Albinism Type 1 Regulates Melanogenesis in Mouse Melanocytes

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17101596

    The overexpression of OA1 affects melanosomal proteins in mouse melanocytes. ( A ) The pigmentation-related gene mRNA levels were measured by qRT-PCR; ( B , C ) Western blot of cell lysates probed with primary antibody against the TYR, TRP1, TRP2 and GPNMB. β-actin was used as a protein loading control. Data are shown as the mean ± standard errors ( n = 3 each), * p
    Figure Legend Snippet: The overexpression of OA1 affects melanosomal proteins in mouse melanocytes. ( A ) The pigmentation-related gene mRNA levels were measured by qRT-PCR; ( B , C ) Western blot of cell lysates probed with primary antibody against the TYR, TRP1, TRP2 and GPNMB. β-actin was used as a protein loading control. Data are shown as the mean ± standard errors ( n = 3 each), * p

    Techniques Used: Over Expression, Quantitative RT-PCR, Western Blot

    The overexpression of OA1 affects the melanin content in an MITF-dependent fashion. Mice OA1 was PCR amplified and then subcloned into the pMSCV PIG vector with Xho I and EcoR I restriction sites. The pMSCV-OA1-GFP plasmid or empty vector was transfected into the mouse melanocytes. The cells were harvested to extract total RNA and total protein. The expression levels of OA1 were qualitatively and quantitatively analyzed by qRT-PCR and Western blot. ( A ) Melanin contents in melanocytes transfected with OA1 , as well as the control; ( B – D ) transfection efficiency of OA1 in mouse melanocytes; ( E – G ) OA1 overexpression influences microphthalmia-associated transcription factor ( MITF ) mRNA and protein levels in mouse melanocytes. Data are shown as the mean ± standard errors ( n = 3 each), * p
    Figure Legend Snippet: The overexpression of OA1 affects the melanin content in an MITF-dependent fashion. Mice OA1 was PCR amplified and then subcloned into the pMSCV PIG vector with Xho I and EcoR I restriction sites. The pMSCV-OA1-GFP plasmid or empty vector was transfected into the mouse melanocytes. The cells were harvested to extract total RNA and total protein. The expression levels of OA1 were qualitatively and quantitatively analyzed by qRT-PCR and Western blot. ( A ) Melanin contents in melanocytes transfected with OA1 , as well as the control; ( B – D ) transfection efficiency of OA1 in mouse melanocytes; ( E – G ) OA1 overexpression influences microphthalmia-associated transcription factor ( MITF ) mRNA and protein levels in mouse melanocytes. Data are shown as the mean ± standard errors ( n = 3 each), * p

    Techniques Used: Over Expression, Mouse Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Transfection, Expressing, Quantitative RT-PCR, Western Blot

    15) Product Images from "Early responses to dehydration in contrasting wild Arachis species"

    Article Title: Early responses to dehydration in contrasting wild Arachis species

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198191

    Expression of DEGs as determined by qRT-PCR. Relative quantification of mRNA levels of 15 candidate genes in A . duranensis roots during the dehydration treatment and collected after 25 (T25); 50 (T50); 75 (T75); 100 (T100); 125 (T125) and 150 (T150) min, relative to control (T0). Bars represent the standard deviation of three biological replicates. Significantly (P
    Figure Legend Snippet: Expression of DEGs as determined by qRT-PCR. Relative quantification of mRNA levels of 15 candidate genes in A . duranensis roots during the dehydration treatment and collected after 25 (T25); 50 (T50); 75 (T75); 100 (T100); 125 (T125) and 150 (T150) min, relative to control (T0). Bars represent the standard deviation of three biological replicates. Significantly (P

    Techniques Used: Expressing, Quantitative RT-PCR, Standard Deviation

    16) Product Images from "Large and small extracellular vesicles released by glioma cells in vitro and in vivo"

    Article Title: Large and small extracellular vesicles released by glioma cells in vitro and in vivo

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2019.1689784

    Characterization of large and small EV cargo released from GBM U87 EGFRvIII cells. (a) Western blot analysis of β-actin, HSPA5, CD81 and CD63 in whole cell lysate (WCL), large and small EV fractions isolated from U87 EGFRvIII cells demonstrated WCL enriched in β-actin, HSPA5, CD81; large EVs enriched in HSPA5and small EVs enriched in CD81, CD63. (b) Quantitative analysis of large blebs released per cell in U87 EGFRvIII cells treated with blebbistatin (a known inhibitor of bleb formation) or vehicle (mock) yielded a significant decrease in large EV release. (c) EGFRwt and EGFRvIII mRNA quantification using qRT-PCR demonstrated significantly higher amounts of EGFRwt mRNA in small EVs and similar amounts of EGFRvIII mRNA in both large and small EVs. (d) Nanoparticle tracking analysis (NTA) of particle counts of small EVs derived from HUVEC and U87 EGFRvIII cells represent a similar number of small EVs released from both the cell lines. (e) Size distribution of small EVs derived from HUVEC and U87 EGFRvIII cells depicted a similar size distribution of small EVs released from both the cell lines. (f) Flow-cytometry analysis of large EVs derived from HUVEC and U87 EGFRvIII cells depicting higher large EV release from U87 EGFRvIII cells. The results are presented as the mean ± SD ((f) n = 3; (d) n = 2).
    Figure Legend Snippet: Characterization of large and small EV cargo released from GBM U87 EGFRvIII cells. (a) Western blot analysis of β-actin, HSPA5, CD81 and CD63 in whole cell lysate (WCL), large and small EV fractions isolated from U87 EGFRvIII cells demonstrated WCL enriched in β-actin, HSPA5, CD81; large EVs enriched in HSPA5and small EVs enriched in CD81, CD63. (b) Quantitative analysis of large blebs released per cell in U87 EGFRvIII cells treated with blebbistatin (a known inhibitor of bleb formation) or vehicle (mock) yielded a significant decrease in large EV release. (c) EGFRwt and EGFRvIII mRNA quantification using qRT-PCR demonstrated significantly higher amounts of EGFRwt mRNA in small EVs and similar amounts of EGFRvIII mRNA in both large and small EVs. (d) Nanoparticle tracking analysis (NTA) of particle counts of small EVs derived from HUVEC and U87 EGFRvIII cells represent a similar number of small EVs released from both the cell lines. (e) Size distribution of small EVs derived from HUVEC and U87 EGFRvIII cells depicted a similar size distribution of small EVs released from both the cell lines. (f) Flow-cytometry analysis of large EVs derived from HUVEC and U87 EGFRvIII cells depicting higher large EV release from U87 EGFRvIII cells. The results are presented as the mean ± SD ((f) n = 3; (d) n = 2).

    Techniques Used: Western Blot, Isolation, Quantitative RT-PCR, Derivative Assay, Flow Cytometry, Cytometry

    17) Product Images from "Evolutionary Conservation and Expression Patterns of Neutral/Alkaline Invertases in Solanum"

    Article Title: Evolutionary Conservation and Expression Patterns of Neutral/Alkaline Invertases in Solanum

    Journal: Biomolecules

    doi: 10.3390/biom9120763

    Expression profiles of tomato CIN genes in response to phytohormone treatments. Expression levels of these CINs under various phytohormone treatments were detected using qRT-PCR. Bar graphs indicate relative expression values of each CIN gene after phytohormone treatment. Leaf samples were collected at 0, 1.5, 3, 6, 12 and 24 h after treatment, respectively. Each time point of sample collection represented by A, B, C, D, E, and F, respectively. The mean of three biological replicates was selected for analysis.
    Figure Legend Snippet: Expression profiles of tomato CIN genes in response to phytohormone treatments. Expression levels of these CINs under various phytohormone treatments were detected using qRT-PCR. Bar graphs indicate relative expression values of each CIN gene after phytohormone treatment. Leaf samples were collected at 0, 1.5, 3, 6, 12 and 24 h after treatment, respectively. Each time point of sample collection represented by A, B, C, D, E, and F, respectively. The mean of three biological replicates was selected for analysis.

    Techniques Used: Expressing, Quantitative RT-PCR

    18) Product Images from "Upregulated lncRNA SNHG1 contributes to progression of non-small cell lung cancer through inhibition of miR-101-3p and activation of Wnt/β-catenin signaling pathway"

    Article Title: Upregulated lncRNA SNHG1 contributes to progression of non-small cell lung cancer through inhibition of miR-101-3p and activation of Wnt/β-catenin signaling pathway

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14854

    LncRNA SNHG1 directly interacted with miR-101-3p ( A ) Sequence alignment of miR-101-3p with the putative binding sites with in the wild-type regions of SNHG1. ( B ) Dual-luciferase reporter assay showed that miR-101-3p mimics reduced the intensity of fluorescence in HEK293T cells transfected with SNHG1-Wt, while had no effect on the SNHG1-mut vector. ( C ) The correlation between SNHG1 mRNA and miR-101-3p expression in NSCLC tissues. ( D ) qRT-PCR analysis of miR-101-3p expression in A549 and SPC-A1 cells transfected with si-SNHG1 or si-NC. ( E ) qRT-PCR analysis of SNHG1 expression in A549 and SPC-A1 cells transfected with miR-101-3p or miR-NC. * P
    Figure Legend Snippet: LncRNA SNHG1 directly interacted with miR-101-3p ( A ) Sequence alignment of miR-101-3p with the putative binding sites with in the wild-type regions of SNHG1. ( B ) Dual-luciferase reporter assay showed that miR-101-3p mimics reduced the intensity of fluorescence in HEK293T cells transfected with SNHG1-Wt, while had no effect on the SNHG1-mut vector. ( C ) The correlation between SNHG1 mRNA and miR-101-3p expression in NSCLC tissues. ( D ) qRT-PCR analysis of miR-101-3p expression in A549 and SPC-A1 cells transfected with si-SNHG1 or si-NC. ( E ) qRT-PCR analysis of SNHG1 expression in A549 and SPC-A1 cells transfected with miR-101-3p or miR-NC. * P

    Techniques Used: Sequencing, Binding Assay, Luciferase, Reporter Assay, Fluorescence, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR

    LncRNA SNHG1 was up-regulated in NSCLC ( A ) The expression of SNHG1 among Pan-Cancer including 14 cancer types from The Cancer Genome Atlas (TCGA) Data Portal from starBASE v2.0. ( B ) The expression of SNHG1 in normal or NSCLC (LUAC and LUSC) from TCGA Data Portal. ( C ) The expression of SNHG1 was determined by qRT-PCR in 68 pairs of NSCLC tissues (T) compared with adjacent non-tumour tissues (N). ( D ) The expression of SNHG1 was examined by qRT-PCR in four NSCLC cell lines (A549, SPC-A1, H23 and NCI-H520) and human bronchial epithelial cell line 16HBE. ( E ) The Kaplan-Meier analysis showed that NSCLC patients with high SNHG1 expression had a poor overall survival compared to patients with low SNHG1 expression. LUAC: lung adenocarcinoma; LUSC: lung squamous cell carcinoma. * P
    Figure Legend Snippet: LncRNA SNHG1 was up-regulated in NSCLC ( A ) The expression of SNHG1 among Pan-Cancer including 14 cancer types from The Cancer Genome Atlas (TCGA) Data Portal from starBASE v2.0. ( B ) The expression of SNHG1 in normal or NSCLC (LUAC and LUSC) from TCGA Data Portal. ( C ) The expression of SNHG1 was determined by qRT-PCR in 68 pairs of NSCLC tissues (T) compared with adjacent non-tumour tissues (N). ( D ) The expression of SNHG1 was examined by qRT-PCR in four NSCLC cell lines (A549, SPC-A1, H23 and NCI-H520) and human bronchial epithelial cell line 16HBE. ( E ) The Kaplan-Meier analysis showed that NSCLC patients with high SNHG1 expression had a poor overall survival compared to patients with low SNHG1 expression. LUAC: lung adenocarcinoma; LUSC: lung squamous cell carcinoma. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    19) Product Images from "Effects of Doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo"

    Article Title: Effects of Doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-19-21

    Correlation between expression values for Wolbachia and Brugia malayi genes obtained by microarray (y-axis) and qRT-PCR (x-axis) . Normalized fold change values of microarray experiment and 2 -ΔΔ Ct values of qRT-PCR obtained for the same genes were Log transformed and analysed by the nonparametric Spearman rank correlation test (R = 0.4352; P = 0.007).
    Figure Legend Snippet: Correlation between expression values for Wolbachia and Brugia malayi genes obtained by microarray (y-axis) and qRT-PCR (x-axis) . Normalized fold change values of microarray experiment and 2 -ΔΔ Ct values of qRT-PCR obtained for the same genes were Log transformed and analysed by the nonparametric Spearman rank correlation test (R = 0.4352; P = 0.007).

    Techniques Used: Expressing, Microarray, Quantitative RT-PCR, Transformation Assay

    20) Product Images from "Promoter of CaZF, a Chickpea Gene That Positively Regulates Growth and Stress Tolerance, Is Activated by an AP2-Family Transcription Factor CAP2"

    Article Title: Promoter of CaZF, a Chickpea Gene That Positively Regulates Growth and Stress Tolerance, Is Activated by an AP2-Family Transcription Factor CAP2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056737

    Activation of CaZF -promoter by CAP2 in plant cell. (A, B) Enhancement of CaZF expression by transient overexpression of CAP2 in chickpea. Young leaves of 10-day-old chickpea seedlings were transformed with CaMV35S:c-Myc-CAP2 by particle bombardment. Chickpea leaves were harvested after 48 h of incubation. Leaves transformed with empty vector (pCAMBIA1302) were taken as control (Con). 2 µg of total RNA was reverse transcribed for cDNA preparation. CAP2 (A) and CaZF (B) expression was analyzed in the control and experimental tissues by semi-quantitative RT-PCR (27 cycles) and qReal-Time PCR. The expression level of Actin gene was taken as an internal control. Results from two biological replicates (Expt-1, Expt-2) are shown. (C) Schematic diagram of the effector and reporter constructs used in the co-transfection experiments. Full-length CAP2 cDNA was fused with 2X c-Myc at N-terminus and cloned under CaMV-35S promoter in pCAMBIA1302 to construct effector plasmid. pro CaZF with three CRTs was fused with GUS gene in pBI101 to construct reporter plasmid. (D) Both the effector and reporter plasmids as mentioned in the figure were co-introduced in to tobacco leaf explants by Agrobacterium -mediated transformation and antibiotic-selected shootlets were used for the GUS assay. Expression of kanamycin resistance gene (NPT II) as assessed by qRT-PCR was used for normalization of results in the transformed shoot-lets. (E) The effector and reporter constructs were co-introduced into tobacco BY2 protoplasts as mentioned in the table. CAP2 stands for the effector plasmid and pro- CaZF stands for the reporter plasmid. pro- CaZF (M1–M3) stands for the reporter plasmids with mutations in CRT1-CRT3 in pro- CaZF . GUS activity was measured fluorometrically after 48 h of transformation. The empty vectors without CAP2 (pCAMBIA1302) or pro CaZF (pBI101) were used as controls. Transfection efficiency of the CaMV-35S-EYFP1 plasmid included in protoplast experiment was used for normalization. The error bars indicate the standard deviation (SD). * indicates significant differences in comparison to the controls at p
    Figure Legend Snippet: Activation of CaZF -promoter by CAP2 in plant cell. (A, B) Enhancement of CaZF expression by transient overexpression of CAP2 in chickpea. Young leaves of 10-day-old chickpea seedlings were transformed with CaMV35S:c-Myc-CAP2 by particle bombardment. Chickpea leaves were harvested after 48 h of incubation. Leaves transformed with empty vector (pCAMBIA1302) were taken as control (Con). 2 µg of total RNA was reverse transcribed for cDNA preparation. CAP2 (A) and CaZF (B) expression was analyzed in the control and experimental tissues by semi-quantitative RT-PCR (27 cycles) and qReal-Time PCR. The expression level of Actin gene was taken as an internal control. Results from two biological replicates (Expt-1, Expt-2) are shown. (C) Schematic diagram of the effector and reporter constructs used in the co-transfection experiments. Full-length CAP2 cDNA was fused with 2X c-Myc at N-terminus and cloned under CaMV-35S promoter in pCAMBIA1302 to construct effector plasmid. pro CaZF with three CRTs was fused with GUS gene in pBI101 to construct reporter plasmid. (D) Both the effector and reporter plasmids as mentioned in the figure were co-introduced in to tobacco leaf explants by Agrobacterium -mediated transformation and antibiotic-selected shootlets were used for the GUS assay. Expression of kanamycin resistance gene (NPT II) as assessed by qRT-PCR was used for normalization of results in the transformed shoot-lets. (E) The effector and reporter constructs were co-introduced into tobacco BY2 protoplasts as mentioned in the table. CAP2 stands for the effector plasmid and pro- CaZF stands for the reporter plasmid. pro- CaZF (M1–M3) stands for the reporter plasmids with mutations in CRT1-CRT3 in pro- CaZF . GUS activity was measured fluorometrically after 48 h of transformation. The empty vectors without CAP2 (pCAMBIA1302) or pro CaZF (pBI101) were used as controls. Transfection efficiency of the CaMV-35S-EYFP1 plasmid included in protoplast experiment was used for normalization. The error bars indicate the standard deviation (SD). * indicates significant differences in comparison to the controls at p

    Techniques Used: Activation Assay, Expressing, Over Expression, Transformation Assay, Incubation, Plasmid Preparation, Quantitative RT-PCR, Polymerase Chain Reaction, Construct, Cotransfection, Clone Assay, GUS Gene Assay, Activity Assay, Transfection, Standard Deviation

    21) Product Images from "In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGF?-signaling and WT1"

    Article Title: In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGF?-signaling and WT1

    Journal: Basic Research in Cardiology

    doi: 10.1007/s00395-011-0181-0

    Knockdown of endoglin expression cannot block the process of TGFβ-stimulated EMT. Transduction of epicardial cells with lentivirus expression shRNAs for control GFP virus (Control) and human endoglin (shEndoglin) was visualized by the expression of GFP ( a1 , b1 ) and did not affect the cell morphology ( a2 , b2 ). Addition of TGFβ caused morphological changes in both shEndoglin-transduced cEPDCs and control ( a3 , b3 ). qRT-PCR ( c ) and Western blot ( d ) analysis showed reduction of endogenous endoglin expression after transduction ( c , d ). Addition of TGFβ caused increase in endoglin expression in both transduced and control epicardial cells. Analysis of epithelial marker VCAM - 1 ( e ) and EMT marker Snai1 ( f ) showed that knockdown of endoglin could not prevent the effects of TGFβ on these markers. ×100 in a1 – c2 . * P
    Figure Legend Snippet: Knockdown of endoglin expression cannot block the process of TGFβ-stimulated EMT. Transduction of epicardial cells with lentivirus expression shRNAs for control GFP virus (Control) and human endoglin (shEndoglin) was visualized by the expression of GFP ( a1 , b1 ) and did not affect the cell morphology ( a2 , b2 ). Addition of TGFβ caused morphological changes in both shEndoglin-transduced cEPDCs and control ( a3 , b3 ). qRT-PCR ( c ) and Western blot ( d ) analysis showed reduction of endogenous endoglin expression after transduction ( c , d ). Addition of TGFβ caused increase in endoglin expression in both transduced and control epicardial cells. Analysis of epithelial marker VCAM - 1 ( e ) and EMT marker Snai1 ( f ) showed that knockdown of endoglin could not prevent the effects of TGFβ on these markers. ×100 in a1 – c2 . * P

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

    Western blot and qRT-PCR analysis. Gene expression of cEPDCs (control; C) and cEPDCs treated with 1 ng/ml TGFβ3 (T) showed that the expression of ALK5 was increased by TGFβ ( a ). Western blot analysis of protein samples isolated from cEPDCs stimulated in the presence or absence of 1 ng/ml TGFβ3 and α-Endoglin probed for Pai-1 ( b ). As a loading control, α/β-tubulin was used ( b ). Western blot analysis of αSMA and endoglin in epicardial cells stimulated by sVCAM-1 (100 ng/ml) and α-Endoglin (0.5 μg/ml) independent or simultaneously with TGFβ3 (1 ng/ml) ( c , d ). GAPDH was used as a loading control in this experiment ( c , d ). Gene expression of cEPDCs and cEPDCs treated with TGFβ3, iALK5 and both simultaneously. Treatment with TGFβ showed significant decrease in epithelial markers and increase in EMT marker Snai1 , which is dependent on ALK5 kinase activity ( e ). * P
    Figure Legend Snippet: Western blot and qRT-PCR analysis. Gene expression of cEPDCs (control; C) and cEPDCs treated with 1 ng/ml TGFβ3 (T) showed that the expression of ALK5 was increased by TGFβ ( a ). Western blot analysis of protein samples isolated from cEPDCs stimulated in the presence or absence of 1 ng/ml TGFβ3 and α-Endoglin probed for Pai-1 ( b ). As a loading control, α/β-tubulin was used ( b ). Western blot analysis of αSMA and endoglin in epicardial cells stimulated by sVCAM-1 (100 ng/ml) and α-Endoglin (0.5 μg/ml) independent or simultaneously with TGFβ3 (1 ng/ml) ( c , d ). GAPDH was used as a loading control in this experiment ( c , d ). Gene expression of cEPDCs and cEPDCs treated with TGFβ3, iALK5 and both simultaneously. Treatment with TGFβ showed significant decrease in epithelial markers and increase in EMT marker Snai1 , which is dependent on ALK5 kinase activity ( e ). * P

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

    sVCAM-1 inhibits cell shape changes in human adult epicardial cells treated with TGFβ3. Unstimulated cells ( a ) and stimulated with sVCAM-1 (100 ng/ml) ( b ) and simultaneously with TGFβ3 (1 ng/ml) and sVCAM-1 (100 ng/ml) ( c ) are stained for β-catenin ( a2 – c2 ) and with phalloidin to visualize filamentous actin ( a3 – c3 ). Onset of differentiation into smooth muscle cells was visualized by staining for αSMA ( a4 – c4 ) and the state of differentiation was visualized by WT1 ( a5 – c5 ). The process of EMT was confirmed by qRT-PCR ( d ) analysis for epithelial and EMT markers. ×100 in a1 – c1 . Scale bar 20 μm. * P
    Figure Legend Snippet: sVCAM-1 inhibits cell shape changes in human adult epicardial cells treated with TGFβ3. Unstimulated cells ( a ) and stimulated with sVCAM-1 (100 ng/ml) ( b ) and simultaneously with TGFβ3 (1 ng/ml) and sVCAM-1 (100 ng/ml) ( c ) are stained for β-catenin ( a2 – c2 ) and with phalloidin to visualize filamentous actin ( a3 – c3 ). Onset of differentiation into smooth muscle cells was visualized by staining for αSMA ( a4 – c4 ) and the state of differentiation was visualized by WT1 ( a5 – c5 ). The process of EMT was confirmed by qRT-PCR ( d ) analysis for epithelial and EMT markers. ×100 in a1 – c1 . Scale bar 20 μm. * P

    Techniques Used: Staining, Quantitative RT-PCR

    22) Product Images from "Vimentin coordinates fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-β–Slug signaling"

    Article Title: Vimentin coordinates fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-β–Slug signaling

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

    doi: 10.1073/pnas.1519197113

    Inflammatory cytokines and infiltrated inflammatory cells in wounds in WT and VIM −/− mice (related to ). ( A ) qRT-PCR analysis of transcripts for IL-6 (Il6), IL-1α (Il1a), IL-1β (Il1b), IL-23 (Il23), and TNF-α
    Figure Legend Snippet: Inflammatory cytokines and infiltrated inflammatory cells in wounds in WT and VIM −/− mice (related to ). ( A ) qRT-PCR analysis of transcripts for IL-6 (Il6), IL-1α (Il1a), IL-1β (Il1b), IL-23 (Il23), and TNF-α

    Techniques Used: Mouse Assay, Quantitative RT-PCR

    Vimentin promotes TGF-β production from fibroblasts driving EMT and migration of keratinocytes. ( A ) qRT-PCR analysis of transcripts for TGF-β1 ( Tgfb1 ) in VIM −/− and WT MDFs. Bars show mean fold changes ± SEM relative
    Figure Legend Snippet: Vimentin promotes TGF-β production from fibroblasts driving EMT and migration of keratinocytes. ( A ) qRT-PCR analysis of transcripts for TGF-β1 ( Tgfb1 ) in VIM −/− and WT MDFs. Bars show mean fold changes ± SEM relative

    Techniques Used: Migration, Quantitative RT-PCR

    TGF-β1–Slug signaling promotes keratinocyte differentiation and migration. ( A ) qRT-PCR analysis of transcripts for TGF-β1 ( Tgfb1 ) in VIM −/− and WT wounds on days 3, 9, and 15 after wounding. Bars show mean fold
    Figure Legend Snippet: TGF-β1–Slug signaling promotes keratinocyte differentiation and migration. ( A ) qRT-PCR analysis of transcripts for TGF-β1 ( Tgfb1 ) in VIM −/− and WT wounds on days 3, 9, and 15 after wounding. Bars show mean fold

    Techniques Used: Migration, Quantitative RT-PCR

    23) Product Images from "GmPRP2 promoter drives root-preferential expression in transgenic Arabidopsis and soybean hairy roots"

    Article Title: GmPRP2 promoter drives root-preferential expression in transgenic Arabidopsis and soybean hairy roots

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-014-0245-z

    The expression level of GmPRP2 by qRT-PCR in different tissues. The relative expression level of GmPRP2 from real-time PCR was in different tissues. R, root; S, stem; L, leaf; F, flower; Sd, seed; H, hypocotyl. Data are the means of three replicates with SE shown by vertical bars. The capitals differ significantly by one-sided paired t test at P
    Figure Legend Snippet: The expression level of GmPRP2 by qRT-PCR in different tissues. The relative expression level of GmPRP2 from real-time PCR was in different tissues. R, root; S, stem; L, leaf; F, flower; Sd, seed; H, hypocotyl. Data are the means of three replicates with SE shown by vertical bars. The capitals differ significantly by one-sided paired t test at P

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

    24) Product Images from "Exon Array Analysis using re-defined probe sets results in reliable identification of alternatively spliced genes in non-small cell lung cancer"

    Article Title: Exon Array Analysis using re-defined probe sets results in reliable identification of alternatively spliced genes in non-small cell lung cancer

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-676

    Quantification of FOX2 transcript variant expression . (a) Four of the annotated transcript variants of FOX2 (for complete set, see Figure 6a) and location of qRT-PCR assays that measure gene level expression and transcript variant expression, respectively. (b) Location of primers for transcript variant specific qRT-PCR assays in the C-terminal region of FOX2 (green: constitutive exons; orange: cassette exon; grey: cassette exons not expressed; green arrows: primers; dotted arrow: junction primer). (c) Quantification of gene expression (G) and transcript variant expression (CE: cassette exon; ES: exon skipping) in adenocarcinoma and squamous cell carcinoma of NSCLC (red bars), respectively, compared to NAT (blue bars). Bars indicate median Δ Ct values, dots represent values for individual samples (AdCa: n = 6; SCC: n = 4), error bars indicate one standard deviation. (d) Fold-change (FC) of over-expression in adenocarcinoma of NSCLC versus NAT and splicing index (SI). Median values based on six sample pairs are shown (values for each patient shown as dots), significance was determined using a paired t-test. (e) Fold-change of over-expression in squamous cell carcinoma of NSCLC versus NAT and splicing index. Median values based on four sample pairs are shown.
    Figure Legend Snippet: Quantification of FOX2 transcript variant expression . (a) Four of the annotated transcript variants of FOX2 (for complete set, see Figure 6a) and location of qRT-PCR assays that measure gene level expression and transcript variant expression, respectively. (b) Location of primers for transcript variant specific qRT-PCR assays in the C-terminal region of FOX2 (green: constitutive exons; orange: cassette exon; grey: cassette exons not expressed; green arrows: primers; dotted arrow: junction primer). (c) Quantification of gene expression (G) and transcript variant expression (CE: cassette exon; ES: exon skipping) in adenocarcinoma and squamous cell carcinoma of NSCLC (red bars), respectively, compared to NAT (blue bars). Bars indicate median Δ Ct values, dots represent values for individual samples (AdCa: n = 6; SCC: n = 4), error bars indicate one standard deviation. (d) Fold-change (FC) of over-expression in adenocarcinoma of NSCLC versus NAT and splicing index (SI). Median values based on six sample pairs are shown (values for each patient shown as dots), significance was determined using a paired t-test. (e) Fold-change of over-expression in squamous cell carcinoma of NSCLC versus NAT and splicing index. Median values based on four sample pairs are shown.

    Techniques Used: Variant Assay, Expressing, Quantitative RT-PCR, Standard Deviation, Over Expression

    Details of the exon array results and the laboratory validation results for ADD3 . (a) Exon structure and known transcript variants of ADD3 (introns not to scale; green: Ensembl transcripts; red: RefSeq entries; purple: Genscan predictions). (b) Position of probe sets in the new exon array chip definition (grey: absent probe sets; blue: present probe sets). (c) Exon expression in the NSCLC data set suggests higher inclusion of a cassette exon (arrow) in tumour compared to normal adjacent tissue (NAT) (red graph: exon expression in NSCLC; blue graph: exon expression in NAT). (d) Splicing indices for exons in the NSCLC data set (logarithmic scale). (e) Verification of RT-PCR product sizes generated from paired samples of adenocarcinoma of NSCLC and NAT of six patients (ø: no template control). In tumour, exon inclusion was observed in all cases analysed. Sequencing of representative products confirmed the expected exon-exon junctions (data not shown). (f) Quantification of gene expression (G) and transcript variant expression (CE: cassette exon; ES: exon skipping) in adenocarcinoma of NSCLC compared to NAT as measured by qRT-PCR. Median values based on six sample pairs are shown (values for each patient shown as dots), error bars indicate one standard deviation, significance was determined using a paired t-test. FC: Fold-change of over-expression in adenocarcinoma of NSCLC versus NAT. SI: Splicing index. (g) Quantification of gene expression and transcript variant expression in squamous cell carcinoma of NSCLC compared to NAT as measured by qRT-PCR. Median values based on four sample pairs are shown.
    Figure Legend Snippet: Details of the exon array results and the laboratory validation results for ADD3 . (a) Exon structure and known transcript variants of ADD3 (introns not to scale; green: Ensembl transcripts; red: RefSeq entries; purple: Genscan predictions). (b) Position of probe sets in the new exon array chip definition (grey: absent probe sets; blue: present probe sets). (c) Exon expression in the NSCLC data set suggests higher inclusion of a cassette exon (arrow) in tumour compared to normal adjacent tissue (NAT) (red graph: exon expression in NSCLC; blue graph: exon expression in NAT). (d) Splicing indices for exons in the NSCLC data set (logarithmic scale). (e) Verification of RT-PCR product sizes generated from paired samples of adenocarcinoma of NSCLC and NAT of six patients (ø: no template control). In tumour, exon inclusion was observed in all cases analysed. Sequencing of representative products confirmed the expected exon-exon junctions (data not shown). (f) Quantification of gene expression (G) and transcript variant expression (CE: cassette exon; ES: exon skipping) in adenocarcinoma of NSCLC compared to NAT as measured by qRT-PCR. Median values based on six sample pairs are shown (values for each patient shown as dots), error bars indicate one standard deviation, significance was determined using a paired t-test. FC: Fold-change of over-expression in adenocarcinoma of NSCLC versus NAT. SI: Splicing index. (g) Quantification of gene expression and transcript variant expression in squamous cell carcinoma of NSCLC compared to NAT as measured by qRT-PCR. Median values based on four sample pairs are shown.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Sequencing, Variant Assay, Quantitative RT-PCR, Standard Deviation, Over Expression

    25) Product Images from "Fitness and Phenotypic Characterization of Miltefosine-Resistant Leishmania major"

    Article Title: Fitness and Phenotypic Characterization of Miltefosine-Resistant Leishmania major

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003948

    Metacyclogenesis in WT and MIL-resistant L . major . L . major promastigotes resistant to MIL exhibit increased metacyclogenesis as determined by qRT-PCR of SHERP expression relative to housekeeping gene GAPDH and normalized to WT expression levels (left). 5-day stationary parasites were subjected to peanut agglutination and Ficoll-400 gradients and percentage of metacyclics is shown (right). Results are the average of triplicate experiments ± SD. Statistical differences determined with a Student’s t test relative to control values (* p
    Figure Legend Snippet: Metacyclogenesis in WT and MIL-resistant L . major . L . major promastigotes resistant to MIL exhibit increased metacyclogenesis as determined by qRT-PCR of SHERP expression relative to housekeeping gene GAPDH and normalized to WT expression levels (left). 5-day stationary parasites were subjected to peanut agglutination and Ficoll-400 gradients and percentage of metacyclics is shown (right). Results are the average of triplicate experiments ± SD. Statistical differences determined with a Student’s t test relative to control values (* p

    Techniques Used: Quantitative RT-PCR, Expressing, Agglutination

    26) Product Images from "Replication Study: Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia"

    Article Title: Replication Study: Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia

    Journal: eLife

    doi: 10.7554/eLife.25306

    BCL2 expression in I-BET151 treated MV4;11 and K-562 cells. MV4;11 and K-562 cells were treated with 500 nM I-BET151, or an equivalent volume of DMSO. Total RNA was isolated 6 hr after treatment and qRT-PCR was used to detect BCL2 and B2M expression. Fold change in BCL2 expression normalized to B2M and relative to DMSO is presented for I-BET151 treated MV4;11 and K-562 cells. Expression level of BCL2 in DMSO was assigned a value of 1. Means reported and error bars represent SD from three independent biological repeats. Two-sample t -test comparing fold gene expression values from MV4;11 cells to K-562 cells; t (4) = 17.23, uncorrected p =6.66×10 −5 , a priori Bonferroni adjusted significance threshold = 0.0167; (Bonferroni corrected p =0.0004). One-sample t -test comparing fold gene expression from K-562 cells to a constant of 1 (DMSO treated cells); t (2) = 3.53, uncorrected p =0.0719, a priori Bonferroni adjusted significance threshold = 0.0167; (Bonferroni corrected p =0.216). One-sample t -test comparing fold gene expression from MV4;11 cells to a constant of 1 (DMSO treated cells); t (2) = 17.86, uncorrected p =0.003, a priori Bonferroni adjusted significance threshold = 0.0167; (Bonferroni corrected p =0.009). Additional details for this experiment can be found at https://osf.io/np6gq/ . DOI: http://dx.doi.org/10.7554/eLife.25306.005
    Figure Legend Snippet: BCL2 expression in I-BET151 treated MV4;11 and K-562 cells. MV4;11 and K-562 cells were treated with 500 nM I-BET151, or an equivalent volume of DMSO. Total RNA was isolated 6 hr after treatment and qRT-PCR was used to detect BCL2 and B2M expression. Fold change in BCL2 expression normalized to B2M and relative to DMSO is presented for I-BET151 treated MV4;11 and K-562 cells. Expression level of BCL2 in DMSO was assigned a value of 1. Means reported and error bars represent SD from three independent biological repeats. Two-sample t -test comparing fold gene expression values from MV4;11 cells to K-562 cells; t (4) = 17.23, uncorrected p =6.66×10 −5 , a priori Bonferroni adjusted significance threshold = 0.0167; (Bonferroni corrected p =0.0004). One-sample t -test comparing fold gene expression from K-562 cells to a constant of 1 (DMSO treated cells); t (2) = 3.53, uncorrected p =0.0719, a priori Bonferroni adjusted significance threshold = 0.0167; (Bonferroni corrected p =0.216). One-sample t -test comparing fold gene expression from MV4;11 cells to a constant of 1 (DMSO treated cells); t (2) = 17.86, uncorrected p =0.003, a priori Bonferroni adjusted significance threshold = 0.0167; (Bonferroni corrected p =0.009). Additional details for this experiment can be found at https://osf.io/np6gq/ . DOI: http://dx.doi.org/10.7554/eLife.25306.005

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR

    27) Product Images from "Using laser micro-dissection and qRT-PCR to analyze cell type-specific gene expression in Norway spruce phloem"

    Article Title: Using laser micro-dissection and qRT-PCR to analyze cell type-specific gene expression in Norway spruce phloem

    Journal: PeerJ

    doi: 10.7717/peerj.362

    High resolution characteristics of Norway spruce phloem 0–14 days after inoculation with the necrotroph Ceratocystis polonica . (A) Polyphenolic parenchyma cells (PPC) and ray cells (RC) in control tissue with turquois stained phenolics and unstained starch grains. (B) PPC and RC 3 days after infection, the time point at which cells and tissues were collected for laser micro-dissection and real-time qRT-PCR analysis. (C, D) Arrows indicate hyphae of C. polonica inside cells 7 and 14 days after inoculation. Bars, 50 µm. All cross-sections (1 µm thick) represent conducting secondary phloem sampled 5 mm above the inoculation site and embedded in acrylic resin.
    Figure Legend Snippet: High resolution characteristics of Norway spruce phloem 0–14 days after inoculation with the necrotroph Ceratocystis polonica . (A) Polyphenolic parenchyma cells (PPC) and ray cells (RC) in control tissue with turquois stained phenolics and unstained starch grains. (B) PPC and RC 3 days after infection, the time point at which cells and tissues were collected for laser micro-dissection and real-time qRT-PCR analysis. (C, D) Arrows indicate hyphae of C. polonica inside cells 7 and 14 days after inoculation. Bars, 50 µm. All cross-sections (1 µm thick) represent conducting secondary phloem sampled 5 mm above the inoculation site and embedded in acrylic resin.

    Techniques Used: Staining, Infection, Dissection, Quantitative RT-PCR

    28) Product Images from "Bacterial colonization factors control specificity and stability of the gut microbiota"

    Article Title: Bacterial colonization factors control specificity and stability of the gut microbiota

    Journal: Nature

    doi: 10.1038/nature12447

    B. fragilis colonization of the colonic crypts is mediated by the CCF system a , qRT-PCR of ccf gene expression levels normalized to 16S rRNA (n=3 animals, 2 trials). b, Mice were mono-associated with either WT B. fragilis or B. fragilis ΔCCF, and challenged with WT B. fragilis . The percentage of challenge strain was determined in the lumen (feces) and colon after 1 day (n=8 animals/group). c , Confocal micrographs of germ-free, WT B. fragilis or B. fragilis ΔCCF mono-associated mice colon whole-mount. Crypts are visualized by DAPI (nuclei, blue) and phalloidin (F-actin, green). Bacteria (red) are stained with IgY polyclonal antibody raised against B. fragilis . Images are representative of 7 different sites analyzed from at least 2 different colons. Scale bar: 5 μm. d , 3D reconstructions of colon crypts from WT B. fragilis or B. fragilis ΔCCF mono-associated mice. Bacteria are detected on the apical surface of the epithelium (arrows) and in the crypt space (arrowhead). Scale bar: 10 μm. e , Quantification of bacterial penetration, measured as distance from the epithelial surface per crypt. Error bars indicate standard error of the mean (SEM). NS: not significant. ND: not detected. ** p
    Figure Legend Snippet: B. fragilis colonization of the colonic crypts is mediated by the CCF system a , qRT-PCR of ccf gene expression levels normalized to 16S rRNA (n=3 animals, 2 trials). b, Mice were mono-associated with either WT B. fragilis or B. fragilis ΔCCF, and challenged with WT B. fragilis . The percentage of challenge strain was determined in the lumen (feces) and colon after 1 day (n=8 animals/group). c , Confocal micrographs of germ-free, WT B. fragilis or B. fragilis ΔCCF mono-associated mice colon whole-mount. Crypts are visualized by DAPI (nuclei, blue) and phalloidin (F-actin, green). Bacteria (red) are stained with IgY polyclonal antibody raised against B. fragilis . Images are representative of 7 different sites analyzed from at least 2 different colons. Scale bar: 5 μm. d , 3D reconstructions of colon crypts from WT B. fragilis or B. fragilis ΔCCF mono-associated mice. Bacteria are detected on the apical surface of the epithelium (arrows) and in the crypt space (arrowhead). Scale bar: 10 μm. e , Quantification of bacterial penetration, measured as distance from the epithelial surface per crypt. Error bars indicate standard error of the mean (SEM). NS: not significant. ND: not detected. ** p

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

    B. fragilis requires the ccf genes for stable and resilient colonization of mice a , Groups of SPF Rag-/- mice were gavaged with either WT B. fragilis or B. fragilis ΔCCF. b , SPF Rag-/- mice were given a 1:1 co-inoculum of WT B. fragilis and B. fragilis ΔCCF by single gavage. c , SPF NOD mice were gavaged with either WT B. fragilis or B. fragilis ΔCCF. d , SPF NOD mice were given a 1:1 co-inoculum of WT B. fragilis and B. fragilis ΔCCF by single gavage. e , SPF mice were co-associated with WT B. fragilis and B. fragilis ΔCCF, and infected with Citrobacter rodentium . f , SPF mice were co-associated with WT B. fragilis and B. fragilis ΔCCF, and given ciprofloxacin in drinking water for the time period shown. For all analyses, bacterial colonization levels were assessed by real-time qRT-PCR from stool DNA (n=4 animals/group). Results are representative of at least 2 independent trials per experiments. Error bars indicate SEM.* p
    Figure Legend Snippet: B. fragilis requires the ccf genes for stable and resilient colonization of mice a , Groups of SPF Rag-/- mice were gavaged with either WT B. fragilis or B. fragilis ΔCCF. b , SPF Rag-/- mice were given a 1:1 co-inoculum of WT B. fragilis and B. fragilis ΔCCF by single gavage. c , SPF NOD mice were gavaged with either WT B. fragilis or B. fragilis ΔCCF. d , SPF NOD mice were given a 1:1 co-inoculum of WT B. fragilis and B. fragilis ΔCCF by single gavage. e , SPF mice were co-associated with WT B. fragilis and B. fragilis ΔCCF, and infected with Citrobacter rodentium . f , SPF mice were co-associated with WT B. fragilis and B. fragilis ΔCCF, and given ciprofloxacin in drinking water for the time period shown. For all analyses, bacterial colonization levels were assessed by real-time qRT-PCR from stool DNA (n=4 animals/group). Results are representative of at least 2 independent trials per experiments. Error bars indicate SEM.* p

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

    29) Product Images from "Only a minority of broad-range detoxification genes respond to a variety of phytotoxins in generalist Bemisia tabaci species"

    Article Title: Only a minority of broad-range detoxification genes respond to a variety of phytotoxins in generalist Bemisia tabaci species

    Journal: Scientific Reports

    doi: 10.1038/srep17975

    Expression of gut specific genes. Total RNA was separately extracted from the gut (MG) and the rest of the body (WB-MG) of MED females. The relative expression of seven detoxification genes and Unigene9390 (a gene reported to be transcribed in the salivary gland) was tested by qRT-PCR. Genes were considered significantly over- or under-transcribed when the ∆CT values of samples from the gut were different from ∆CT values of samples from the rest of the body at P ≤ 0.05.Values presented are mean 2 −ΔΔct ± SE. Asterisks indicate significant differences (one-way ANOVA model).
    Figure Legend Snippet: Expression of gut specific genes. Total RNA was separately extracted from the gut (MG) and the rest of the body (WB-MG) of MED females. The relative expression of seven detoxification genes and Unigene9390 (a gene reported to be transcribed in the salivary gland) was tested by qRT-PCR. Genes were considered significantly over- or under-transcribed when the ∆CT values of samples from the gut were different from ∆CT values of samples from the rest of the body at P ≤ 0.05.Values presented are mean 2 −ΔΔct ± SE. Asterisks indicate significant differences (one-way ANOVA model).

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

    30) Product Images from "Identification of genes regulating breast cancer dormancy in 3D bone endosteal niche cultures"

    Article Title: Identification of genes regulating breast cancer dormancy in 3D bone endosteal niche cultures

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-18-0956

    Analysis of HBP1 and WNT3 as potential DSRG. ( A ) Gene Set Enrichment Analysis of module-1 DSRG candidates identified 18 of 65 hits as being MYC target genes. ( B ) qRT-PCR showing knockdown of HBP1 in MDA-MB-231 cells. ( C and D ) Knockdown of HBP1 ( C ) or WNT3 ( D ) induces proliferation in 3D-EN vs. 3D (“C”) or 2D cultures. Error bars, SEM of three independent replicates; *, p
    Figure Legend Snippet: Analysis of HBP1 and WNT3 as potential DSRG. ( A ) Gene Set Enrichment Analysis of module-1 DSRG candidates identified 18 of 65 hits as being MYC target genes. ( B ) qRT-PCR showing knockdown of HBP1 in MDA-MB-231 cells. ( C and D ) Knockdown of HBP1 ( C ) or WNT3 ( D ) induces proliferation in 3D-EN vs. 3D (“C”) or 2D cultures. Error bars, SEM of three independent replicates; *, p

    Techniques Used: Quantitative RT-PCR, Multiple Displacement Amplification

    31) Product Images from "Inhibition of dihydrotestosterone synthesis in prostate cancer by combined frontdoor and backdoor pathway blockade"

    Article Title: Inhibition of dihydrotestosterone synthesis in prostate cancer by combined frontdoor and backdoor pathway blockade

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24107

    3α-oxidoreductases were expressed in CaP cell lines and xenografts qRT-PCR results were shown for 3α-oxidoreductase ( A ), SRD5A ( B ), and AR ( C ) mRNA levels for CaP cell lines and CWR22 and rCWR22 xenografts. Data were presented as mean +/- SEM. Gene expression was modeled as a function of cell line and a random replicate effect using a LMM. Mean expression was compared among cell lines using Tukey-Kramer adjusted tests about the least square means.
    Figure Legend Snippet: 3α-oxidoreductases were expressed in CaP cell lines and xenografts qRT-PCR results were shown for 3α-oxidoreductase ( A ), SRD5A ( B ), and AR ( C ) mRNA levels for CaP cell lines and CWR22 and rCWR22 xenografts. Data were presented as mean +/- SEM. Gene expression was modeled as a function of cell line and a random replicate effect using a LMM. Mean expression was compared among cell lines using Tukey-Kramer adjusted tests about the least square means.

    Techniques Used: Quantitative RT-PCR, Expressing

    32) Product Images from "Cytokinesis arrest and multiple centrosomes in B cell chronic lymphocytic leukaemia. Cytokinesis arrest and multiple centrosomes in B cell chronic lymphocytic leukaemia"

    Article Title: Cytokinesis arrest and multiple centrosomes in B cell chronic lymphocytic leukaemia. Cytokinesis arrest and multiple centrosomes in B cell chronic lymphocytic leukaemia

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13579

    Shown are genes involved in centrosome assembly, duplication and regulation as well as genes regulated by TP53. qRT‐PCR analyses on the indicated number of CLL vs healthy control (n = 3) samples demonstrated significant differences in mRNA expression levels for (B) CDK4 (* P ≤ .05), (C) ING4 (* P ≤ .05), (D) ING5 (* P ≤ .05), (E) TP53I3 (** P ≤ .005) and (F) CDKN1A/p21 (** P ≤ .005)
    Figure Legend Snippet: Shown are genes involved in centrosome assembly, duplication and regulation as well as genes regulated by TP53. qRT‐PCR analyses on the indicated number of CLL vs healthy control (n = 3) samples demonstrated significant differences in mRNA expression levels for (B) CDK4 (* P ≤ .05), (C) ING4 (* P ≤ .05), (D) ING5 (* P ≤ .05), (E) TP53I3 (** P ≤ .005) and (F) CDKN1A/p21 (** P ≤ .005)

    Techniques Used: Quantitative RT-PCR, Expressing

    33) Product Images from "Terpene profiling, transcriptome analysis and characterization of cis-β-terpineol synthase from Ocimum"

    Article Title: Terpene profiling, transcriptome analysis and characterization of cis-β-terpineol synthase from Ocimum

    Journal: Physiology and Molecular Biology of Plants

    doi: 10.1007/s12298-018-0612-6

    Expression analysis of TPS 1, TPS 2 and TPS 3 by a sqRT–PCR using 18S rRNA as reference gene in leaves tissue of five Ocimum species and b qRT–PCR analysis with EF1 as endogenous reference in different tissue of five species of Ocimum . Og
    Figure Legend Snippet: Expression analysis of TPS 1, TPS 2 and TPS 3 by a sqRT–PCR using 18S rRNA as reference gene in leaves tissue of five Ocimum species and b qRT–PCR analysis with EF1 as endogenous reference in different tissue of five species of Ocimum . Og

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

    34) Product Images from "γδ-T cells promote IFN-γ–dependent Plasmodium pathogenesis upon liver-stage infection"

    Article Title: γδ-T cells promote IFN-γ–dependent Plasmodium pathogenesis upon liver-stage infection

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

    doi: 10.1073/pnas.1814440116

    γδ-T cells promote a proinflammatory microenvironment in the liver without affecting P. berghei development. ( A ) Relative expression of parasite 18S P.b. rRNA in liver tissue of WT or TCRδ −/− mice measured by qRT-PCR at 48 h after Spz infection ( n = 9–10). ( B ) Assessment of prepatent period at 48 h, 52 h, 56 h, and 60 h after infection with 5 × 10 4 luciferase P. berghei ANKA parasites using bioluminescence quantification ( n = 9–10 mice per group). Each symbol in A and B represents an individual mouse. ( C ) Spleen and liver representative microphotographs of sections by H E of WT or TCRδ −/− mice at 48 h after Spz infection ( n = 6 mice per group). (Scale bars, 50–300 μm.) ( D – F ) Absolute number of IFN-γ producers among CD4 + and CD8 + T cells, and NK cells from livers of WT mice at 48 h after Spz infection ( n = 6–7 mice per group). ( G – I ) Relative expression levels of il6 , il1b , and ifng in liver tissue of WT or TCRδ −/− mice measured by qRT-PCR at 48 h after Spz infection ( n = 6 mice per group). Data are represented as mean ± SEM. * P
    Figure Legend Snippet: γδ-T cells promote a proinflammatory microenvironment in the liver without affecting P. berghei development. ( A ) Relative expression of parasite 18S P.b. rRNA in liver tissue of WT or TCRδ −/− mice measured by qRT-PCR at 48 h after Spz infection ( n = 9–10). ( B ) Assessment of prepatent period at 48 h, 52 h, 56 h, and 60 h after infection with 5 × 10 4 luciferase P. berghei ANKA parasites using bioluminescence quantification ( n = 9–10 mice per group). Each symbol in A and B represents an individual mouse. ( C ) Spleen and liver representative microphotographs of sections by H E of WT or TCRδ −/− mice at 48 h after Spz infection ( n = 6 mice per group). (Scale bars, 50–300 μm.) ( D – F ) Absolute number of IFN-γ producers among CD4 + and CD8 + T cells, and NK cells from livers of WT mice at 48 h after Spz infection ( n = 6–7 mice per group). ( G – I ) Relative expression levels of il6 , il1b , and ifng in liver tissue of WT or TCRδ −/− mice measured by qRT-PCR at 48 h after Spz infection ( n = 6 mice per group). Data are represented as mean ± SEM. * P

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Infection, Luciferase

    35) Product Images from "Regulation of MYB mediated cisplatin resistance of ovarian cancer cells involves miR-21-wnt signaling axis"

    Article Title: Regulation of MYB mediated cisplatin resistance of ovarian cancer cells involves miR-21-wnt signaling axis

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-63396-8

    miR-21 reverses effect of c-MYB silencing on wnt signaling. (A) Levels of miR-21 were assessed, by qRT-PCR, in two ovarian cancer cell lines (ES2 and OVCAR3). Expression of miR-21 in control cells was set as 1 (black bars) and the altered expression of miR-21 in c-MYB silenced cells is shown as gray bars. Wnt signaling was assessed through phosphorylation of β-catenin, as detected by ELISA, and endpoint measurement of absorbance at 450 nm. ES2 ( B ) and OVCAR3 ( C ) were transfected either with siRNA against c-MYB alone or with pre-miR-21, and then β-catenin was detected by ELISA. *p
    Figure Legend Snippet: miR-21 reverses effect of c-MYB silencing on wnt signaling. (A) Levels of miR-21 were assessed, by qRT-PCR, in two ovarian cancer cell lines (ES2 and OVCAR3). Expression of miR-21 in control cells was set as 1 (black bars) and the altered expression of miR-21 in c-MYB silenced cells is shown as gray bars. Wnt signaling was assessed through phosphorylation of β-catenin, as detected by ELISA, and endpoint measurement of absorbance at 450 nm. ES2 ( B ) and OVCAR3 ( C ) were transfected either with siRNA against c-MYB alone or with pre-miR-21, and then β-catenin was detected by ELISA. *p

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

    c-MYB overexpression alters the levels of several miRNAs. (A–F) Levels of several miRNAs, as indicated on Y-axis, were assessed, by qRT-PCR, in two ovarian cancer cell lines (ES2 and OVCAR3). Expression of miRNAs in control cells was set as 1 (black bars) and the altered expression of same miRNAs in c-MYB overexpressing cells is shown as gray bars. # p
    Figure Legend Snippet: c-MYB overexpression alters the levels of several miRNAs. (A–F) Levels of several miRNAs, as indicated on Y-axis, were assessed, by qRT-PCR, in two ovarian cancer cell lines (ES2 and OVCAR3). Expression of miRNAs in control cells was set as 1 (black bars) and the altered expression of same miRNAs in c-MYB overexpressing cells is shown as gray bars. # p

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

    miR-21 affects EMT. (A–D) Levels of EMT markers, as indicated on Y-axis, were assessed, by qRT-PCR, in two ovarian cancer cell lines (ES2 and OVCAR3). Expression of genes in control cells was set as 1 (black bars) and the altered expression of same genes in presence of anti-miR-21 (A,B) or pre-miR-21 (C,D) transfections is shown as gray bars. # p
    Figure Legend Snippet: miR-21 affects EMT. (A–D) Levels of EMT markers, as indicated on Y-axis, were assessed, by qRT-PCR, in two ovarian cancer cell lines (ES2 and OVCAR3). Expression of genes in control cells was set as 1 (black bars) and the altered expression of same genes in presence of anti-miR-21 (A,B) or pre-miR-21 (C,D) transfections is shown as gray bars. # p

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection

    36) Product Images from "Replication study: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET"

    Article Title: Replication study: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET

    Journal: eLife

    doi: 10.7554/eLife.39944

    Multiplicity of infection (MOI) ratios tested for stable cell line generation. B16-F10 cells infected with various MOI ratios were characterized for shMet, Met , and Met expression. ( A ) Relative expression levels of shMet (normalized to U6) was determined by qRT-PCR for each cell line. For each MOI tested, fold change in shMet expression in shMet cells relative to shScr cells was determined. Expression level of shScr cells was assigned a value of 1, which is indicated by the dashed line. Means reported from one biological repeat. ( B ) Relative expression levels of Met (normalized to Gapdh ) was determined by qRT-PCR for each cell line. For each MOI tested, fold change in Met expression in shMet cells relative to shScr cells was determined. Expression levels of shScr cells was assigned a value of 1, which is indicated by the dashed line. Means reported from one biological repeat. ( C ) Western blots using anti-Met (top panel) and anti-Gapdh (bottom panel) antibodies. Membranes were cut at ~75 kDa so that Met and Gapdh could be probed in parallel. Repeat indicates the number of independently isolated cell lysate preparations from the same batch of infected cells. Additional details for this experiment can be found at https://osf.io/aqm2m/ .
    Figure Legend Snippet: Multiplicity of infection (MOI) ratios tested for stable cell line generation. B16-F10 cells infected with various MOI ratios were characterized for shMet, Met , and Met expression. ( A ) Relative expression levels of shMet (normalized to U6) was determined by qRT-PCR for each cell line. For each MOI tested, fold change in shMet expression in shMet cells relative to shScr cells was determined. Expression level of shScr cells was assigned a value of 1, which is indicated by the dashed line. Means reported from one biological repeat. ( B ) Relative expression levels of Met (normalized to Gapdh ) was determined by qRT-PCR for each cell line. For each MOI tested, fold change in Met expression in shMet cells relative to shScr cells was determined. Expression levels of shScr cells was assigned a value of 1, which is indicated by the dashed line. Means reported from one biological repeat. ( C ) Western blots using anti-Met (top panel) and anti-Gapdh (bottom panel) antibodies. Membranes were cut at ~75 kDa so that Met and Gapdh could be probed in parallel. Repeat indicates the number of independently isolated cell lysate preparations from the same batch of infected cells. Additional details for this experiment can be found at https://osf.io/aqm2m/ .

    Techniques Used: Infection, Stable Transfection, Expressing, Quantitative RT-PCR, Western Blot, Isolation

    37) Product Images from "Sphingosine 1‐phosphate and its carrier apolipoprotein M in human sepsis and in Escherichia coli sepsis in baboons"

    Article Title: Sphingosine 1‐phosphate and its carrier apolipoprotein M in human sepsis and in Escherichia coli sepsis in baboons

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12831

    Liver and kidney mRNA levels of apolipoproteins are decreased in sepsis. mRNA from liver (A, n = 23) and kidney (B, n = 15) tissue was extracted from baboons challenged with 10 9 cfu/kg of E. coli ( LD 50 , open circles) or 1–2 × 10 10 cfu/kg ( LD 100 , closed circles) dose of E. coli or saline solution alone (triangles). Baboons receiving the saline infusion were sacrificed directly after the infusion ( t = 0 h). Tissue samples from the liver in septic baboons were taken after 2 ( n = 3), 6 ( n = 1), 8 ( n = 5), 12 ( n = 1), 24 ( n = 6), 27 ( n = 1) and 34 ( n = 1) hours and from the kidney after 2 ( n = 3), 6 ( n = 1), 8 ( n = 4) and 24 ( n = 4) hours. mRNA levels were measured by qRT ‐ PCR , normalized against GAPDH and are given relative to one reference baboon. Statistical analysis was performed with Kruskal–Wallis test, * P
    Figure Legend Snippet: Liver and kidney mRNA levels of apolipoproteins are decreased in sepsis. mRNA from liver (A, n = 23) and kidney (B, n = 15) tissue was extracted from baboons challenged with 10 9 cfu/kg of E. coli ( LD 50 , open circles) or 1–2 × 10 10 cfu/kg ( LD 100 , closed circles) dose of E. coli or saline solution alone (triangles). Baboons receiving the saline infusion were sacrificed directly after the infusion ( t = 0 h). Tissue samples from the liver in septic baboons were taken after 2 ( n = 3), 6 ( n = 1), 8 ( n = 5), 12 ( n = 1), 24 ( n = 6), 27 ( n = 1) and 34 ( n = 1) hours and from the kidney after 2 ( n = 3), 6 ( n = 1), 8 ( n = 4) and 24 ( n = 4) hours. mRNA levels were measured by qRT ‐ PCR , normalized against GAPDH and are given relative to one reference baboon. Statistical analysis was performed with Kruskal–Wallis test, * P

    Techniques Used: Quantitative RT-PCR

    38) Product Images from "Deep sequencing of mitochondrial genomes reveals increased mutation load in Friedreich's ataxia"

    Article Title: Deep sequencing of mitochondrial genomes reveals increased mutation load in Friedreich's ataxia

    Journal: Annals of Clinical and Translational Neurology

    doi: 10.1002/acn3.322

    Increased mtDNA damage in FRDA fibroblasts. (A) PCR analysis of GAA repeat length in FRDA and control fibroblasts; M1 = HyperLadder Plus 1 kbp ladder, C1–C5 = controls, F1–F5 = FRDA. (B) qRT‐PCR analysis of FXN mRNA expression in fibroblast lines used in this study; C1–C5 shown in black, F1–F5 shown in gray. FXN expression was normalized to GAPDH mRNA level. (C) Left panel: Representative agarose gel electrophoresis and amplicons for mtDNA damage qPCR products; M1 = HyperLadder ™ 1 kb Plus ladder (catalog # BIO‐33068, Bioline, Taunton, MA); M2 = HyperLadder ™ 100 bp Plus ladder (catalog # BIO‐33071, Bioline); long = long PCR product, ~8.8 kbp; short = short PCR product, 222 bp. Long amplicon shown in gray, short amplicon shown in black. Right panel: qPCR analysis of mtDNA copy number in control (C) and FRDA (F) fibroblasts. Results shown are from two independent experiments with five biological replicates for each group. (D) mtDNA damage qPCR analyses of short and long fragments were performed for control and FRDA fibroblasts; results from at least three independent experiments are shown. Controls (C1–C5) are depicted in black and FRDA (F1–F5) are depicted in gray. Cumulative analysis of the data for entire C and F cohort is shown; **** indicates P
    Figure Legend Snippet: Increased mtDNA damage in FRDA fibroblasts. (A) PCR analysis of GAA repeat length in FRDA and control fibroblasts; M1 = HyperLadder Plus 1 kbp ladder, C1–C5 = controls, F1–F5 = FRDA. (B) qRT‐PCR analysis of FXN mRNA expression in fibroblast lines used in this study; C1–C5 shown in black, F1–F5 shown in gray. FXN expression was normalized to GAPDH mRNA level. (C) Left panel: Representative agarose gel electrophoresis and amplicons for mtDNA damage qPCR products; M1 = HyperLadder ™ 1 kb Plus ladder (catalog # BIO‐33068, Bioline, Taunton, MA); M2 = HyperLadder ™ 100 bp Plus ladder (catalog # BIO‐33071, Bioline); long = long PCR product, ~8.8 kbp; short = short PCR product, 222 bp. Long amplicon shown in gray, short amplicon shown in black. Right panel: qPCR analysis of mtDNA copy number in control (C) and FRDA (F) fibroblasts. Results shown are from two independent experiments with five biological replicates for each group. (D) mtDNA damage qPCR analyses of short and long fragments were performed for control and FRDA fibroblasts; results from at least three independent experiments are shown. Controls (C1–C5) are depicted in black and FRDA (F1–F5) are depicted in gray. Cumulative analysis of the data for entire C and F cohort is shown; **** indicates P

    Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Amplification

    Downregulation of NTHL1 expression in FRDA fibroblasts. (A) NTHL1 expression was determined by RNA‐seq 30 (control cohort‐circles, FRDA cohort‐squares) or by qRT‐PCR (triangles; in the case where no RNA‐seq analysis was available). Samples C1–C5 and F1–F5 used in mtDNA damage and frequency analyses are indicated in gray; **** P = 0.0002 and was calculated for RNA‐seq samples only. (B) The Pearson correlation coefficient was determined for FXN mRNA and NTHL1 mRNA expression in control and FRDA fibroblasts. Designations of symbols as described in A. Pearson's correlation coefficient ( r ) and statistical significance ( P ) values are indicated on the graph.
    Figure Legend Snippet: Downregulation of NTHL1 expression in FRDA fibroblasts. (A) NTHL1 expression was determined by RNA‐seq 30 (control cohort‐circles, FRDA cohort‐squares) or by qRT‐PCR (triangles; in the case where no RNA‐seq analysis was available). Samples C1–C5 and F1–F5 used in mtDNA damage and frequency analyses are indicated in gray; **** P = 0.0002 and was calculated for RNA‐seq samples only. (B) The Pearson correlation coefficient was determined for FXN mRNA and NTHL1 mRNA expression in control and FRDA fibroblasts. Designations of symbols as described in A. Pearson's correlation coefficient ( r ) and statistical significance ( P ) values are indicated on the graph.

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

    39) Product Images from "Improving protein content and quality by over-expressing artificially synthetic fusion proteins with high lysine and threonine constituent in rice plants"

    Article Title: Improving protein content and quality by over-expressing artificially synthetic fusion proteins with high lysine and threonine constituent in rice plants

    Journal: Scientific Reports

    doi: 10.1038/srep34427

    Molecular characterization of 35S::TKTKK2 transgenic plants. (a,b) show the expression patterns of the TKTKK2 gene in 60 transgenic plants by qRT-PCR. The mRNA relative amount (Y axis) was calculated as shown in Materials and methods. The eEF-1a gene was used as an internal control to normalize the data as shown in Fig. 2b . ( c ) Copy number detection of T-DNA insertion in transgenic plants by Southern blot hybridization. DNA samples from top 12 transgenic plants in expression were restricted by Eco RV and then transferred into nylon membrane for hybridization using the HPT probe. The red star “*” in (b,c) indicated the line with high level of expression signal and with single copy of T-DNA insertion, which was selected for further analysis.
    Figure Legend Snippet: Molecular characterization of 35S::TKTKK2 transgenic plants. (a,b) show the expression patterns of the TKTKK2 gene in 60 transgenic plants by qRT-PCR. The mRNA relative amount (Y axis) was calculated as shown in Materials and methods. The eEF-1a gene was used as an internal control to normalize the data as shown in Fig. 2b . ( c ) Copy number detection of T-DNA insertion in transgenic plants by Southern blot hybridization. DNA samples from top 12 transgenic plants in expression were restricted by Eco RV and then transferred into nylon membrane for hybridization using the HPT probe. The red star “*” in (b,c) indicated the line with high level of expression signal and with single copy of T-DNA insertion, which was selected for further analysis.

    Techniques Used: Transgenic Assay, Expressing, Quantitative RT-PCR, Southern Blot, Hybridization

    Molecular characterization of 35S::TKTKK1 transgenic plants. (a) Copy number detection of T-DNA insertion in transgenic plants by Southern blot hybridization. DNA samples from a total of 18 transgenic plants were restricted by Eco RV and then transferred onto nylon membrane for hybridization using the HPT probe. The red triangle indicated the lines with single copy of T-DNA insertion. (b) Bar diagrams showing expression patterns of the TKTKK1 gene in the 18 independent transgenic plants by qRT-PCR. The mRNA relative amount (Y axis) was calculated according to the description in Materials and methods. The amplification of an eEF-1a gene was used as an internal control to normalize the data. The red arrows in (a) indicated the lines with single copy of T-DNA insertion by Southern blotting analysis. The red stars “*” in (a , b) indicated the lines with single copy of T-DNA insertion and with relatively higher expression level. These three independent lines were selected for further investigation.
    Figure Legend Snippet: Molecular characterization of 35S::TKTKK1 transgenic plants. (a) Copy number detection of T-DNA insertion in transgenic plants by Southern blot hybridization. DNA samples from a total of 18 transgenic plants were restricted by Eco RV and then transferred onto nylon membrane for hybridization using the HPT probe. The red triangle indicated the lines with single copy of T-DNA insertion. (b) Bar diagrams showing expression patterns of the TKTKK1 gene in the 18 independent transgenic plants by qRT-PCR. The mRNA relative amount (Y axis) was calculated according to the description in Materials and methods. The amplification of an eEF-1a gene was used as an internal control to normalize the data. The red arrows in (a) indicated the lines with single copy of T-DNA insertion by Southern blotting analysis. The red stars “*” in (a , b) indicated the lines with single copy of T-DNA insertion and with relatively higher expression level. These three independent lines were selected for further investigation.

    Techniques Used: Transgenic Assay, Southern Blot, Hybridization, Expressing, Quantitative RT-PCR, Amplification

    Molecular characterization of 35S::TKTKK3 transgenic plants and western blot hybridization. (a,b) show the bar diagrams of expression patterns of both genes TKTKK1 and TKTKK2 in 55 35S::TKTKK3 transgenic plants by qRT-PCR, respectively. (c) Southern blot hybridization for detecting copy numbers of T-DNA insertion. DNA samples (line numbers were labelled on the top of the panel) were digested by the restriction enzyme Eco RV and were then transferred into nylon member for hybridization with the HPT probe. The red stars “*” indicated the lines with single copy number of T-DNA insertion. (d,e) Western blot hybridization using crude proteins from E. coli (d) and T2 rice grains (e) , respectively. The “M” in (d,e) indicates the protein marker used for the hybridization. The numbers in (d) indicate the crude proteins extracted from E. coli carrying the plasmid pGEX-6P-1 (1 and 2) and pGEX-6P-1::TKTKK1 (3 and 4). The white arrows indicated the GST expression in the E. coli carrying the plasmid pGEX-6P-1 . The green arrows indicated the GST fusion protein expression in the E. coli carrying pGEX-6P-1::TKTKK1 . The numbers in (e) indicate the different transgenic lines used for crude proteins extraction from rice seeds. Three lines (1, line 9, 2, line 14 and 3, line 21) were from 35S::TKTKK1 . Line 46 (indicated by 4) and Line 5 (indicated by 5) were from 35S::TKTKK2 and 35S::TKTKK3 , respectively. The number 6 indicates WT and 7 indicates negative control. The green arrows indicated the stable expression of TKTKK1 protein in lines 9, 14 and 21 by Western blot hybridization.
    Figure Legend Snippet: Molecular characterization of 35S::TKTKK3 transgenic plants and western blot hybridization. (a,b) show the bar diagrams of expression patterns of both genes TKTKK1 and TKTKK2 in 55 35S::TKTKK3 transgenic plants by qRT-PCR, respectively. (c) Southern blot hybridization for detecting copy numbers of T-DNA insertion. DNA samples (line numbers were labelled on the top of the panel) were digested by the restriction enzyme Eco RV and were then transferred into nylon member for hybridization with the HPT probe. The red stars “*” indicated the lines with single copy number of T-DNA insertion. (d,e) Western blot hybridization using crude proteins from E. coli (d) and T2 rice grains (e) , respectively. The “M” in (d,e) indicates the protein marker used for the hybridization. The numbers in (d) indicate the crude proteins extracted from E. coli carrying the plasmid pGEX-6P-1 (1 and 2) and pGEX-6P-1::TKTKK1 (3 and 4). The white arrows indicated the GST expression in the E. coli carrying the plasmid pGEX-6P-1 . The green arrows indicated the GST fusion protein expression in the E. coli carrying pGEX-6P-1::TKTKK1 . The numbers in (e) indicate the different transgenic lines used for crude proteins extraction from rice seeds. Three lines (1, line 9, 2, line 14 and 3, line 21) were from 35S::TKTKK1 . Line 46 (indicated by 4) and Line 5 (indicated by 5) were from 35S::TKTKK2 and 35S::TKTKK3 , respectively. The number 6 indicates WT and 7 indicates negative control. The green arrows indicated the stable expression of TKTKK1 protein in lines 9, 14 and 21 by Western blot hybridization.

    Techniques Used: Transgenic Assay, Western Blot, Hybridization, Expressing, Quantitative RT-PCR, Southern Blot, Marker, Plasmid Preparation, Negative Control

    Expression analysis of synthetic genes and measurement of amino acid and crude protein contents in transgenic rice seeds. ( a ) The qRT-PCR analysis of synthetic genes in transgenic seeds from 3 constructs. ( b – e ) shows the content (percentage) of lysine ( b ), threonine ( c ), total amino acids ( d ) and crude protein ( e ) in 5 independent transgenic plants. Matured seeds were dried at 37 °C for 3 days and were then subjected to amino acid and protein measurement. Asterisks “*” and “**” indicate significant differences at P
    Figure Legend Snippet: Expression analysis of synthetic genes and measurement of amino acid and crude protein contents in transgenic rice seeds. ( a ) The qRT-PCR analysis of synthetic genes in transgenic seeds from 3 constructs. ( b – e ) shows the content (percentage) of lysine ( b ), threonine ( c ), total amino acids ( d ) and crude protein ( e ) in 5 independent transgenic plants. Matured seeds were dried at 37 °C for 3 days and were then subjected to amino acid and protein measurement. Asterisks “*” and “**” indicate significant differences at P

    Techniques Used: Expressing, Transgenic Assay, Quantitative RT-PCR, Construct

    40) Product Images from "Inhibition of dihydrotestosterone synthesis in prostate cancer by combined frontdoor and backdoor pathway blockade"

    Article Title: Inhibition of dihydrotestosterone synthesis in prostate cancer by combined frontdoor and backdoor pathway blockade

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24107

    3α-oxidoreductases were expressed in CaP cell lines and xenografts qRT-PCR results were shown for 3α-oxidoreductase ( A ), SRD5A ( B ), and AR ( C ) mRNA levels for CaP cell lines and CWR22 and rCWR22 xenografts. Data were presented as mean +/- SEM. Gene expression was modeled as a function of cell line and a random replicate effect using a LMM. Mean expression was compared among cell lines using Tukey-Kramer adjusted tests about the least square means.
    Figure Legend Snippet: 3α-oxidoreductases were expressed in CaP cell lines and xenografts qRT-PCR results were shown for 3α-oxidoreductase ( A ), SRD5A ( B ), and AR ( C ) mRNA levels for CaP cell lines and CWR22 and rCWR22 xenografts. Data were presented as mean +/- SEM. Gene expression was modeled as a function of cell line and a random replicate effect using a LMM. Mean expression was compared among cell lines using Tukey-Kramer adjusted tests about the least square means.

    Techniques Used: Quantitative RT-PCR, Expressing

    Related Articles

    SYBR Green Assay:

    Article Title: A Dominant-Negative Isoform of IKAROS Expands Primitive Normal Human Hematopoietic Cells
    Article Snippet: .. Primers for qRT-PCR were designed to amplify across at least one intron and produce a product of 80–100 bp using Primer3 software ( http://frodo.wi.mit.edu/ ). qRT-PCR reactions were performed using Fast SYBR green (Life Technologies) and a 7500 Fast Real-Time PCR analyzer (Applied Biosystems). ..

    Article Title: Tuba1a is uniquely important for axon guidance through midline commissural structures
    Article Snippet: .. RNA concentration and purity were determined using a spectrophotometer, then cDNA was synthesized using the RT2 First Strand Kit (Qiagen, Hilden, Germany; 330401). qRT-PCR reactions were prepared with SYBR Green RT-PCR Master mix (Thermo; S9194) and run with a CFX Connect Real-Time System (Bio-Rad). .. All qPCR data presented in this manuscript was normalized to expression of GFP, which was present on the same plasmid as TUBA1A -His6 constructs.

    Synthesized:

    Article Title: Tuba1a is uniquely important for axon guidance through midline commissural structures
    Article Snippet: .. RNA concentration and purity were determined using a spectrophotometer, then cDNA was synthesized using the RT2 First Strand Kit (Qiagen, Hilden, Germany; 330401). qRT-PCR reactions were prepared with SYBR Green RT-PCR Master mix (Thermo; S9194) and run with a CFX Connect Real-Time System (Bio-Rad). .. All qPCR data presented in this manuscript was normalized to expression of GFP, which was present on the same plasmid as TUBA1A -His6 constructs.

    Article Title: Protein Kinase C ? Regulates the Phenotype of Murine CD4+ Th17 Cells
    Article Snippet: .. Quantitative Real-Time PCR (qRT-PCR) RNA was isolated using RNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's instructions. cDNA was synthesized using Omniscript Kit (Qiagen) and oligo-dT primers (Promega, Fitschburg, Wisconsin, USA) and qRT-PCR reactions were conducted using TaqMan technology (reagents were purchased from Life Technologies; Applied Biosystem, Foster City, California, USA). .. The reactions were run on 7500 FAST and ABI PRIM 7000 instruments (Life Technologies).

    Isolation:

    Article Title: Protein Kinase C ? Regulates the Phenotype of Murine CD4+ Th17 Cells
    Article Snippet: .. Quantitative Real-Time PCR (qRT-PCR) RNA was isolated using RNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's instructions. cDNA was synthesized using Omniscript Kit (Qiagen) and oligo-dT primers (Promega, Fitschburg, Wisconsin, USA) and qRT-PCR reactions were conducted using TaqMan technology (reagents were purchased from Life Technologies; Applied Biosystem, Foster City, California, USA). .. The reactions were run on 7500 FAST and ABI PRIM 7000 instruments (Life Technologies).

    Methylation:

    Article Title: Epigenetic silencing of miR-145-5p contributes to brain metastasis
    Article Snippet: .. Immunoprecipitation of methylated DNA was prepared as Weber et al, 2005; the antibody against 5-methyl-cytosine used for immunoprecipitation was from Abcam ab1884, San Diego, CA. qRT-PCR reactions were carried out in duplicate on specific genomic regions using TaqMan Master Mix (Applied Biosystem). .. The resulting signals were normalized for primer efficiency by carrying out qRT-PCR for each primer pair using Input DNA.

    Quantitative RT-PCR:

    Article Title: Epigenetic silencing of miR-145-5p contributes to brain metastasis
    Article Snippet: .. Immunoprecipitation of methylated DNA was prepared as Weber et al, 2005; the antibody against 5-methyl-cytosine used for immunoprecipitation was from Abcam ab1884, San Diego, CA. qRT-PCR reactions were carried out in duplicate on specific genomic regions using TaqMan Master Mix (Applied Biosystem). .. The resulting signals were normalized for primer efficiency by carrying out qRT-PCR for each primer pair using Input DNA.

    Article Title: A Dominant-Negative Isoform of IKAROS Expands Primitive Normal Human Hematopoietic Cells
    Article Snippet: .. Primers for qRT-PCR were designed to amplify across at least one intron and produce a product of 80–100 bp using Primer3 software ( http://frodo.wi.mit.edu/ ). qRT-PCR reactions were performed using Fast SYBR green (Life Technologies) and a 7500 Fast Real-Time PCR analyzer (Applied Biosystems). ..

    Article Title: Transcriptome Analysis Reveals New Insights into the Bacterial Wilt Resistance Mechanism Mediated by Silicon in Tomato
    Article Snippet: .. All qRT-PCR reactions were performed in a 20-μL volume composed of 4 μL of cDNA, 0.6 μL of each primer (10 µM µL−1 ), 4.8 μL of sterile water, and 10 μL of qPCR master mix in the ABI Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). .. The amplification cycling program was as follows: 90 s at 95 °C, followed by 40 cycles of 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 20 s. All primers for qRT-PCR are listed in .

    Article Title: Tuba1a is uniquely important for axon guidance through midline commissural structures
    Article Snippet: .. RNA concentration and purity were determined using a spectrophotometer, then cDNA was synthesized using the RT2 First Strand Kit (Qiagen, Hilden, Germany; 330401). qRT-PCR reactions were prepared with SYBR Green RT-PCR Master mix (Thermo; S9194) and run with a CFX Connect Real-Time System (Bio-Rad). .. All qPCR data presented in this manuscript was normalized to expression of GFP, which was present on the same plasmid as TUBA1A -His6 constructs.

    Article Title: miR-103 Regulates Oxidative Stress by Targeting the BCL2/Adenovirus E1B 19 kDa Interacting Protein 3 in HUVECs
    Article Snippet: .. All qRT-PCR reactions were performed with the Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA) in triplicate. ..

    Article Title: Long Non-Coding RNA (LncRNA) Urothelial Carcinoma Associated 1 (UCA1) Increases Multi-Drug Resistance of Gastric Cancer via Downregulating miR-27b
    Article Snippet: .. All qRT-PCR reactions were performed using an ABI Prism 7500 (Applied Biosystems). ..

    Article Title: Protein Kinase C ? Regulates the Phenotype of Murine CD4+ Th17 Cells
    Article Snippet: .. Quantitative Real-Time PCR (qRT-PCR) RNA was isolated using RNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's instructions. cDNA was synthesized using Omniscript Kit (Qiagen) and oligo-dT primers (Promega, Fitschburg, Wisconsin, USA) and qRT-PCR reactions were conducted using TaqMan technology (reagents were purchased from Life Technologies; Applied Biosystem, Foster City, California, USA). .. The reactions were run on 7500 FAST and ABI PRIM 7000 instruments (Life Technologies).

    Article Title: Broad anti-coronaviral activity of FDA approved drugs against SARS-CoV-2 in vitro and SARS-CoV in vivo
    Article Snippet: .. The qRT-PCR reactions were performed with a QuantStudio 5 (Applied Biosystems). .. To normalize loading, 18S RNA was used as a control, assessed with TaqMan Gene Expression Assays (Applied Biosystems) and TaqMan Fast Advanced Master Mix.

    Immunoprecipitation:

    Article Title: Epigenetic silencing of miR-145-5p contributes to brain metastasis
    Article Snippet: .. Immunoprecipitation of methylated DNA was prepared as Weber et al, 2005; the antibody against 5-methyl-cytosine used for immunoprecipitation was from Abcam ab1884, San Diego, CA. qRT-PCR reactions were carried out in duplicate on specific genomic regions using TaqMan Master Mix (Applied Biosystem). .. The resulting signals were normalized for primer efficiency by carrying out qRT-PCR for each primer pair using Input DNA.

    Real-time Polymerase Chain Reaction:

    Article Title: A Dominant-Negative Isoform of IKAROS Expands Primitive Normal Human Hematopoietic Cells
    Article Snippet: .. Primers for qRT-PCR were designed to amplify across at least one intron and produce a product of 80–100 bp using Primer3 software ( http://frodo.wi.mit.edu/ ). qRT-PCR reactions were performed using Fast SYBR green (Life Technologies) and a 7500 Fast Real-Time PCR analyzer (Applied Biosystems). ..

    Article Title: Transcriptome Analysis Reveals New Insights into the Bacterial Wilt Resistance Mechanism Mediated by Silicon in Tomato
    Article Snippet: .. All qRT-PCR reactions were performed in a 20-μL volume composed of 4 μL of cDNA, 0.6 μL of each primer (10 µM µL−1 ), 4.8 μL of sterile water, and 10 μL of qPCR master mix in the ABI Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). .. The amplification cycling program was as follows: 90 s at 95 °C, followed by 40 cycles of 95 °C for 5 s, 60 °C for 15 s, and 72 °C for 20 s. All primers for qRT-PCR are listed in .

    Article Title: miR-103 Regulates Oxidative Stress by Targeting the BCL2/Adenovirus E1B 19 kDa Interacting Protein 3 in HUVECs
    Article Snippet: .. All qRT-PCR reactions were performed with the Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA) in triplicate. ..

    Article Title: Protein Kinase C ? Regulates the Phenotype of Murine CD4+ Th17 Cells
    Article Snippet: .. Quantitative Real-Time PCR (qRT-PCR) RNA was isolated using RNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's instructions. cDNA was synthesized using Omniscript Kit (Qiagen) and oligo-dT primers (Promega, Fitschburg, Wisconsin, USA) and qRT-PCR reactions were conducted using TaqMan technology (reagents were purchased from Life Technologies; Applied Biosystem, Foster City, California, USA). .. The reactions were run on 7500 FAST and ABI PRIM 7000 instruments (Life Technologies).

    Concentration Assay:

    Article Title: Tuba1a is uniquely important for axon guidance through midline commissural structures
    Article Snippet: .. RNA concentration and purity were determined using a spectrophotometer, then cDNA was synthesized using the RT2 First Strand Kit (Qiagen, Hilden, Germany; 330401). qRT-PCR reactions were prepared with SYBR Green RT-PCR Master mix (Thermo; S9194) and run with a CFX Connect Real-Time System (Bio-Rad). .. All qPCR data presented in this manuscript was normalized to expression of GFP, which was present on the same plasmid as TUBA1A -His6 constructs.

    Spectrophotometry:

    Article Title: Tuba1a is uniquely important for axon guidance through midline commissural structures
    Article Snippet: .. RNA concentration and purity were determined using a spectrophotometer, then cDNA was synthesized using the RT2 First Strand Kit (Qiagen, Hilden, Germany; 330401). qRT-PCR reactions were prepared with SYBR Green RT-PCR Master mix (Thermo; S9194) and run with a CFX Connect Real-Time System (Bio-Rad). .. All qPCR data presented in this manuscript was normalized to expression of GFP, which was present on the same plasmid as TUBA1A -His6 constructs.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Tuba1a is uniquely important for axon guidance through midline commissural structures
    Article Snippet: .. RNA concentration and purity were determined using a spectrophotometer, then cDNA was synthesized using the RT2 First Strand Kit (Qiagen, Hilden, Germany; 330401). qRT-PCR reactions were prepared with SYBR Green RT-PCR Master mix (Thermo; S9194) and run with a CFX Connect Real-Time System (Bio-Rad). .. All qPCR data presented in this manuscript was normalized to expression of GFP, which was present on the same plasmid as TUBA1A -His6 constructs.

    Software:

    Article Title: A Dominant-Negative Isoform of IKAROS Expands Primitive Normal Human Hematopoietic Cells
    Article Snippet: .. Primers for qRT-PCR were designed to amplify across at least one intron and produce a product of 80–100 bp using Primer3 software ( http://frodo.wi.mit.edu/ ). qRT-PCR reactions were performed using Fast SYBR green (Life Technologies) and a 7500 Fast Real-Time PCR analyzer (Applied Biosystems). ..

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    Thermo Fisher quan titative reverse transcriptase polymerase chain reaction qrt pcr
    miR-138 up regulation leads to hTERT suppression in NB4 cells. A: total RNAs from NB4 cells that were transduced with GFP hsa-miR-138-expressing lentiviruses were extracted at 96 h after removal of the lentivirus-containing medium. hTERT mRNA expression was measured by <t>qRT-PCR.</t> Values were normalized to GAPDH . (n=3; *P
    Quan Titative Reverse Transcriptase Polymerase Chain Reaction Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MARF1 binds the CCR4-NOT deadenylase complex and cyclin A mRNA. a Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1) and those from w 1118 negative control were tested. b Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-tagged MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1 full-length or fragments) and those from w 1118 negative control were tested. Black triangles indicate the detected 3xHA-tagged MARF1 proteins. c Co-immunoprecipitation using ovary lysates from w 1118 and anti-Not1 antibody or negative control IgG-bound protein G beads followed by Western blotting. d Fold enrichment of mRNAs relative to a control gapdh mRNA that were co-immunoprecipitated with MARF1 by anti-HA antibody-conjugated beads and were eluted from the beads normalized by w 1118 negative control, determined by <t>RNA-immunoprecipitation</t> followed by <t>qRT-PCR.</t> Mean ± SD ( n = 3). P -value
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    Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) <t>qRT-PCR</t> indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p
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    Thermo Fisher quantitative real time pcr qrt pcr
    LINC01296 knockdown suppressed the migration of bladder cancer cells. Notes: ( A , B ) Transwell assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( C , D ) Wound healing assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( E ) <t>qRT-</t> <t>PCR</t> analyses were used to measure mRNA levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. ( F ) Western blot assays were used to measure protein levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P
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    miR-138 up regulation leads to hTERT suppression in NB4 cells. A: total RNAs from NB4 cells that were transduced with GFP hsa-miR-138-expressing lentiviruses were extracted at 96 h after removal of the lentivirus-containing medium. hTERT mRNA expression was measured by qRT-PCR. Values were normalized to GAPDH . (n=3; *P

    Journal: International Journal of Molecular and Cellular Medicine

    Article Title: Overexpression of MiR-138 Inhibits Cell Growth and Induces Caspase-mediated Apoptosis in Acute Promyelocytic Leukemia Cell Line

    doi: 10.22088/IJMCM.BUMS.7.1.24

    Figure Lengend Snippet: miR-138 up regulation leads to hTERT suppression in NB4 cells. A: total RNAs from NB4 cells that were transduced with GFP hsa-miR-138-expressing lentiviruses were extracted at 96 h after removal of the lentivirus-containing medium. hTERT mRNA expression was measured by qRT-PCR. Values were normalized to GAPDH . (n=3; *P

    Article Snippet: The prepared cDNA was subjected to quan-titative reverse-transcriptase polymerase chain reaction (qRT-PCR), using Maxima SYBR Green Master mix (Thermo Scientific, Waltham, Massa-chusetts, USA) in the Rotor Gene 6000 Real Time PCR inst rument (Corbett Research, Hilden, Germany).

    Techniques: Transduction, Expressing, Quantitative RT-PCR

    MARF1 binds the CCR4-NOT deadenylase complex and cyclin A mRNA. a Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1) and those from w 1118 negative control were tested. b Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-tagged MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1 full-length or fragments) and those from w 1118 negative control were tested. Black triangles indicate the detected 3xHA-tagged MARF1 proteins. c Co-immunoprecipitation using ovary lysates from w 1118 and anti-Not1 antibody or negative control IgG-bound protein G beads followed by Western blotting. d Fold enrichment of mRNAs relative to a control gapdh mRNA that were co-immunoprecipitated with MARF1 by anti-HA antibody-conjugated beads and were eluted from the beads normalized by w 1118 negative control, determined by RNA-immunoprecipitation followed by qRT-PCR. Mean ± SD ( n = 3). P -value

    Journal: Nature Communications

    Article Title: LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes

    doi: 10.1038/s41467-018-06404-w

    Figure Lengend Snippet: MARF1 binds the CCR4-NOT deadenylase complex and cyclin A mRNA. a Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1) and those from w 1118 negative control were tested. b Co-immunoprecipitation using anti-HA antibody-conjugated beads followed by Western blotting. Ovary lysates expressing 3xHA-tagged MARF1 (MAT67Tub-Gal4 → UASP-3xHA-MARF1 full-length or fragments) and those from w 1118 negative control were tested. Black triangles indicate the detected 3xHA-tagged MARF1 proteins. c Co-immunoprecipitation using ovary lysates from w 1118 and anti-Not1 antibody or negative control IgG-bound protein G beads followed by Western blotting. d Fold enrichment of mRNAs relative to a control gapdh mRNA that were co-immunoprecipitated with MARF1 by anti-HA antibody-conjugated beads and were eluted from the beads normalized by w 1118 negative control, determined by RNA-immunoprecipitation followed by qRT-PCR. Mean ± SD ( n = 3). P -value

    Article Snippet: qRT-PCR RNA from oocytes was prepared using miRVana (Thermo Fisher Scientific).

    Techniques: Immunoprecipitation, Western Blot, Expressing, Negative Control, Quantitative RT-PCR

    Tethered MARF1 shortens reporter mRNA poly-A tail and reduces reporter protein level. a GFP-5x BoxB reporter structure, harboring a ubiquitous tubulin promoter, GFP-coding sequence, and a 3′ UTR containing five BoxB hairpins. LambdaN-HA-fused control peptide, MARF1, GW182, and Piwi under a UASP promoter were expressed in germline cells using maternal-alpha-tubulin-Gal4 driver. b Confocal images of GFP signal in stage 14 oocytes. Scale bar is 100 μm. c Western blots using stage 14 oocyte lysates. Black triangles indicate the transgenic lambda-HA-fused proteins. d Quantification of band intensities in c . e ePAT assay measuring GFP reporter mRNA poly-A tail length in stage 14 oocytes. The amplified DNA sizes were analyzed on an agarose gel. f ePAT assay measuring poly-A tail lengths of GFP reporter mRNA and a negative control cdk1 mRNA in stage 14 oocytes. Amplified DNA sizes were analyzed by capillary fragment analysis. g Relative abundance of GFP-5xBoxB mRNA normalized by gapdh mRNA determined by qRT-PCR. Mean ± SD ( n = 6 for the control and n = 4 for all the others)

    Journal: Nature Communications

    Article Title: LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes

    doi: 10.1038/s41467-018-06404-w

    Figure Lengend Snippet: Tethered MARF1 shortens reporter mRNA poly-A tail and reduces reporter protein level. a GFP-5x BoxB reporter structure, harboring a ubiquitous tubulin promoter, GFP-coding sequence, and a 3′ UTR containing five BoxB hairpins. LambdaN-HA-fused control peptide, MARF1, GW182, and Piwi under a UASP promoter were expressed in germline cells using maternal-alpha-tubulin-Gal4 driver. b Confocal images of GFP signal in stage 14 oocytes. Scale bar is 100 μm. c Western blots using stage 14 oocyte lysates. Black triangles indicate the transgenic lambda-HA-fused proteins. d Quantification of band intensities in c . e ePAT assay measuring GFP reporter mRNA poly-A tail length in stage 14 oocytes. The amplified DNA sizes were analyzed on an agarose gel. f ePAT assay measuring poly-A tail lengths of GFP reporter mRNA and a negative control cdk1 mRNA in stage 14 oocytes. Amplified DNA sizes were analyzed by capillary fragment analysis. g Relative abundance of GFP-5xBoxB mRNA normalized by gapdh mRNA determined by qRT-PCR. Mean ± SD ( n = 6 for the control and n = 4 for all the others)

    Article Snippet: qRT-PCR RNA from oocytes was prepared using miRVana (Thermo Fisher Scientific).

    Techniques: Sequencing, Western Blot, Transgenic Assay, Amplification, Agarose Gel Electrophoresis, Negative Control, Quantitative RT-PCR

    Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) qRT-PCR indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p

    Journal: OncoTargets and therapy

    Article Title: Nuclear factor-κB p65 regulates glutaminase 1 expression in human hepatocellular carcinoma

    doi: 10.2147/OTT.S167408

    Figure Lengend Snippet: Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) qRT-PCR indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p

    Article Snippet: Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) Upon relative treatment, cells were lysed, and total RNA was isolated using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR

    LINC01296 knockdown suppressed the migration of bladder cancer cells. Notes: ( A , B ) Transwell assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( C , D ) Wound healing assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( E ) qRT- PCR analyses were used to measure mRNA levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. ( F ) Western blot assays were used to measure protein levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P

    Journal: OncoTargets and therapy

    Article Title: Long noncoding RNA LINC01296 promotes cancer-cell proliferation and metastasis in urothelial carcinoma of the bladder

    doi: 10.2147/OTT.S192809

    Figure Lengend Snippet: LINC01296 knockdown suppressed the migration of bladder cancer cells. Notes: ( A , B ) Transwell assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( C , D ) Wound healing assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( E ) qRT- PCR analyses were used to measure mRNA levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. ( F ) Western blot assays were used to measure protein levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 (Thermo Fisher Scientific) instrument using a KAPA SYBR Green FAST qPCR Kit (KAPA, Wilmington, MA, US) according to the manufacturer’s instructions.

    Techniques: Migration, Transfection, Quantitative RT-PCR, Western Blot

    LINC01296 knockdown inhibited the proliferation of bladder cancer cells. Notes: ( A ) Relative LINC01296 expressions in human bladder cancer cell lines (RT4, T24, and 5637). ( B , C ) MTT assays were performed to measure the proliferation viability of T24 and 5637 cells after transfection. ( D , E ) qRT-PCR results of LINC01296 expressions following the treatment of T24 and 5637 cells with anti-LINC01296 siRNA. ( F , G ) Colony formation assays were used to measure the number of clone formation of bladder cancer cells after transfection. ( H , I ) Flow cytometry assays were performed to analyze the cell cycle progression of T24 and 5637 cells after they were transfected with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P

    Journal: OncoTargets and therapy

    Article Title: Long noncoding RNA LINC01296 promotes cancer-cell proliferation and metastasis in urothelial carcinoma of the bladder

    doi: 10.2147/OTT.S192809

    Figure Lengend Snippet: LINC01296 knockdown inhibited the proliferation of bladder cancer cells. Notes: ( A ) Relative LINC01296 expressions in human bladder cancer cell lines (RT4, T24, and 5637). ( B , C ) MTT assays were performed to measure the proliferation viability of T24 and 5637 cells after transfection. ( D , E ) qRT-PCR results of LINC01296 expressions following the treatment of T24 and 5637 cells with anti-LINC01296 siRNA. ( F , G ) Colony formation assays were used to measure the number of clone formation of bladder cancer cells after transfection. ( H , I ) Flow cytometry assays were performed to analyze the cell cycle progression of T24 and 5637 cells after they were transfected with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 (Thermo Fisher Scientific) instrument using a KAPA SYBR Green FAST qPCR Kit (KAPA, Wilmington, MA, US) according to the manufacturer’s instructions.

    Techniques: MTT Assay, Transfection, Quantitative RT-PCR, Flow Cytometry, Cytometry

    LINC01296 was upregulated in bladder cancer and the expression level of LINC01296 was correlated with clinicopathological features of bladder cancer patients. Notes: ( A ) Comparison of relative expressions of LINC01296 in cancer and normal tissues using the GEPIA database. ( B ) Comparison of relative expressions of LINC01296 in bladder cancer and adjacent normal tissues using the GEPIA database. ( C ) A microarray containing 37,000 lncRNA and 34,000 mRNA probes was used to screen differentially expressed lncRNAs in four pairs of bladder cancer patients. ( D ) Comparison of relative expressions of LINC01296 in 54 bladder cancer tissues and 24 normal bladder tissues using qRT-PCR. ( E ) Two groups were set up according to the mean expression of LINC01296 in tumor tissues. ( F ) The LINC01296 expression was significantly higher in patients with a higher tumor stage, lymph node metastasis and a higher pathologic grade. * P

    Journal: OncoTargets and therapy

    Article Title: Long noncoding RNA LINC01296 promotes cancer-cell proliferation and metastasis in urothelial carcinoma of the bladder

    doi: 10.2147/OTT.S192809

    Figure Lengend Snippet: LINC01296 was upregulated in bladder cancer and the expression level of LINC01296 was correlated with clinicopathological features of bladder cancer patients. Notes: ( A ) Comparison of relative expressions of LINC01296 in cancer and normal tissues using the GEPIA database. ( B ) Comparison of relative expressions of LINC01296 in bladder cancer and adjacent normal tissues using the GEPIA database. ( C ) A microarray containing 37,000 lncRNA and 34,000 mRNA probes was used to screen differentially expressed lncRNAs in four pairs of bladder cancer patients. ( D ) Comparison of relative expressions of LINC01296 in 54 bladder cancer tissues and 24 normal bladder tissues using qRT-PCR. ( E ) Two groups were set up according to the mean expression of LINC01296 in tumor tissues. ( F ) The LINC01296 expression was significantly higher in patients with a higher tumor stage, lymph node metastasis and a higher pathologic grade. * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 (Thermo Fisher Scientific) instrument using a KAPA SYBR Green FAST qPCR Kit (KAPA, Wilmington, MA, US) according to the manufacturer’s instructions.

    Techniques: Expressing, Microarray, Quantitative RT-PCR