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    Structured Review

    Thermo Fisher qrt pcr analysis
    ATRA and Ro41 reciprocally regulate IL5 gene expression. In vitro differentiated 2xTh2 (open symbols) and 3xTh2 (closed symbols) cells were cultured with ATRA, Ro41, or DMSO vehicle for 6hrs. The expression of IL-5, IL-4, and IL-13 were determined by <t>qRT-PCR</t> and fold change (treated/vehicle) plotted. Th2 cell lines stimulated in the presence of IL-2 (50 U/ml) only. Data points represent individual Th2 cell lines. Paired Student’s t test was used to evaluate the data. Each symbol represents a specific cell line.
    Qrt Pcr Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1896 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The retinoic acid receptor-? modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression"

    Article Title: The retinoic acid receptor-? modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression

    Journal: Clinical and Molecular Allergy : CMA

    doi: 10.1186/1476-7961-11-4

    ATRA and Ro41 reciprocally regulate IL5 gene expression. In vitro differentiated 2xTh2 (open symbols) and 3xTh2 (closed symbols) cells were cultured with ATRA, Ro41, or DMSO vehicle for 6hrs. The expression of IL-5, IL-4, and IL-13 were determined by qRT-PCR and fold change (treated/vehicle) plotted. Th2 cell lines stimulated in the presence of IL-2 (50 U/ml) only. Data points represent individual Th2 cell lines. Paired Student’s t test was used to evaluate the data. Each symbol represents a specific cell line.
    Figure Legend Snippet: ATRA and Ro41 reciprocally regulate IL5 gene expression. In vitro differentiated 2xTh2 (open symbols) and 3xTh2 (closed symbols) cells were cultured with ATRA, Ro41, or DMSO vehicle for 6hrs. The expression of IL-5, IL-4, and IL-13 were determined by qRT-PCR and fold change (treated/vehicle) plotted. Th2 cell lines stimulated in the presence of IL-2 (50 U/ml) only. Data points represent individual Th2 cell lines. Paired Student’s t test was used to evaluate the data. Each symbol represents a specific cell line.

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

    2) Product Images from "The aspirin-induced long non-coding RNA OLA1P2 blocks phosphorylated STAT3 homodimer formation"

    Article Title: The aspirin-induced long non-coding RNA OLA1P2 blocks phosphorylated STAT3 homodimer formation

    Journal: Genome Biology

    doi: 10.1186/s13059-016-0892-5

    OLA1P2 blocked the formation of phosphorylated STAT3 homodimers. a A total of 293 T cells were transfected with indicated plasmids for 48 h and then treated with IL-6 for 24 h. Immunoprecipitation and immunoblotting analysis were performed to analyze STAT3-Flag and STAT3-HA protein levels. b COLO205 cells were subjected to RNA pull-down analysis using 5′ biotin-linked RNAs, and the eluted proteins were determined using immunoblotting analysis following polyacrylamide gel electrophoresis under non-denaturing conditions. c Schematic diagram showing the experimental design of purified OLA1P2 blocking the formation of phosphorylated STAT3 homodimers. We used 4 μg purified STAT3 (2 μg STAT3-Flag combined with 2 μg STAT3-HA) incubated with 2 μg purified JAK2. After incubation, purified OLA1P2 was then added to the reaction mixture. d Different amount of purified OLA1P2 blocking the formation of phosphorylated STAT3 homodimers. e QRT-PCR analysis of the amount of lncRNA OLA1P2 in COLO205 cells treated with aspirin and shRNA-OLA1P2. f Schematic diagram showing the modified experimental design of total RNA containing OLA1P2 blocking the formation of phosphorylated STAT3 homodimers. We used 4 μg purified STAT3 (2 μg STAT3-Flag combined with 2 μg STAT3-HA) incubated with 2 μg purified JAK2. After incubation, total RNA extracted from COLO205 cells were then added to the reaction mixture. g The total RNA of OLA1P2 silencing COLO205 cells failed to block the formation of phosphorylated STAT3 homodimers
    Figure Legend Snippet: OLA1P2 blocked the formation of phosphorylated STAT3 homodimers. a A total of 293 T cells were transfected with indicated plasmids for 48 h and then treated with IL-6 for 24 h. Immunoprecipitation and immunoblotting analysis were performed to analyze STAT3-Flag and STAT3-HA protein levels. b COLO205 cells were subjected to RNA pull-down analysis using 5′ biotin-linked RNAs, and the eluted proteins were determined using immunoblotting analysis following polyacrylamide gel electrophoresis under non-denaturing conditions. c Schematic diagram showing the experimental design of purified OLA1P2 blocking the formation of phosphorylated STAT3 homodimers. We used 4 μg purified STAT3 (2 μg STAT3-Flag combined with 2 μg STAT3-HA) incubated with 2 μg purified JAK2. After incubation, purified OLA1P2 was then added to the reaction mixture. d Different amount of purified OLA1P2 blocking the formation of phosphorylated STAT3 homodimers. e QRT-PCR analysis of the amount of lncRNA OLA1P2 in COLO205 cells treated with aspirin and shRNA-OLA1P2. f Schematic diagram showing the modified experimental design of total RNA containing OLA1P2 blocking the formation of phosphorylated STAT3 homodimers. We used 4 μg purified STAT3 (2 μg STAT3-Flag combined with 2 μg STAT3-HA) incubated with 2 μg purified JAK2. After incubation, total RNA extracted from COLO205 cells were then added to the reaction mixture. g The total RNA of OLA1P2 silencing COLO205 cells failed to block the formation of phosphorylated STAT3 homodimers

    Techniques Used: Transfection, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Purification, Blocking Assay, Incubation, Quantitative RT-PCR, shRNA, Modification

    OLA1P2 mediated the aspirin-induced anti-metastatic phenotype and were associated with lower overall survival in CRC. a Stable OLA1P2-silencing COLO205 cells were injected into the immunodeficient mice, which were evaluated to determine the lung colonization capacity in tail vein assays. The bar graph represents 72 h time point measurements of the normalized photon flux for the animals. Representative images are shown; n = 6 for each group. b Number of metastases in the lungs of mice 4 weeks after tail vein injection; mean nodules per lung values are shown (bottom). Representative H E staining of metastatic lung tumor tissues are shown (top). c Increased levels of OLA1P2 in clinical CRC tissues obtained from patients with regular use of aspirin were determined by qRT-PCR. d Immunoblotting analysis determined the protein levels of FOXD3 and phosphorylated STAT3 (Tyr705) in nuclear extract of clinical CRC samples obtained from patients with or without regular use of aspirin. e Linear regression analysis revealed the inverse correlation between FOXD3 and phosphorylated STAT3 (Tyr705) protein levels in nuclear extract of CRC tissues obtained from patients with regular use of aspirin (n = 46). f Lower OLA1P2 levels were correlated with increased pathological grade of CRC. g The curves show the lower overall survival of CRC patients with low OLA1P2 levels compared with CRC patients with high OLA1P2 levels. h A model illustrating the putative roles of aspirin-induced OLA1P2 in controlling the formation of phosphorylated STAT3 homodimers. *: P
    Figure Legend Snippet: OLA1P2 mediated the aspirin-induced anti-metastatic phenotype and were associated with lower overall survival in CRC. a Stable OLA1P2-silencing COLO205 cells were injected into the immunodeficient mice, which were evaluated to determine the lung colonization capacity in tail vein assays. The bar graph represents 72 h time point measurements of the normalized photon flux for the animals. Representative images are shown; n = 6 for each group. b Number of metastases in the lungs of mice 4 weeks after tail vein injection; mean nodules per lung values are shown (bottom). Representative H E staining of metastatic lung tumor tissues are shown (top). c Increased levels of OLA1P2 in clinical CRC tissues obtained from patients with regular use of aspirin were determined by qRT-PCR. d Immunoblotting analysis determined the protein levels of FOXD3 and phosphorylated STAT3 (Tyr705) in nuclear extract of clinical CRC samples obtained from patients with or without regular use of aspirin. e Linear regression analysis revealed the inverse correlation between FOXD3 and phosphorylated STAT3 (Tyr705) protein levels in nuclear extract of CRC tissues obtained from patients with regular use of aspirin (n = 46). f Lower OLA1P2 levels were correlated with increased pathological grade of CRC. g The curves show the lower overall survival of CRC patients with low OLA1P2 levels compared with CRC patients with high OLA1P2 levels. h A model illustrating the putative roles of aspirin-induced OLA1P2 in controlling the formation of phosphorylated STAT3 homodimers. *: P

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

    Aspirin-induced lncRNA OLA1P2 upregulation in cancer cells. a Primary cultured colon cancer cells were treated with aspirin (100 μM) for 48 h and then evaluated to determine their lncRNA profiles using an lncRNA expression microarray. Red indicates high expression; blue indicates low expression. b A volcano plot showing the relationship between the P values and the magnitude of the differences in the expression values of the samples in the different groups. c The expression level of OLA1P2 was validated by qRT-PCR analysis in all eight pairs of CRC cells with or without aspirin treatment. d Primary cultured colon cancer cells were treated with aspirin (100 μM) for 48 h and then evaluated for OLA1P2 expression using northern blotting analysis. e Cancer cell lines were treated with aspirin (100 μM) for 48 h and then evaluated for OLA1P2 expression using qRT-PCR analysis
    Figure Legend Snippet: Aspirin-induced lncRNA OLA1P2 upregulation in cancer cells. a Primary cultured colon cancer cells were treated with aspirin (100 μM) for 48 h and then evaluated to determine their lncRNA profiles using an lncRNA expression microarray. Red indicates high expression; blue indicates low expression. b A volcano plot showing the relationship between the P values and the magnitude of the differences in the expression values of the samples in the different groups. c The expression level of OLA1P2 was validated by qRT-PCR analysis in all eight pairs of CRC cells with or without aspirin treatment. d Primary cultured colon cancer cells were treated with aspirin (100 μM) for 48 h and then evaluated for OLA1P2 expression using northern blotting analysis. e Cancer cell lines were treated with aspirin (100 μM) for 48 h and then evaluated for OLA1P2 expression using qRT-PCR analysis

    Techniques Used: Cell Culture, Expressing, Microarray, Quantitative RT-PCR, Northern Blot

    Aspirin promoted OLA1P2 transcription through FOXD3 upregulation. a The biotin-labeled OLA1P2 promoter was mixed with the nuclear extract separated from DMSO/aspirin-treated primary culture cancer cells. The eluted proteins were then analyzed using mass spectrometry methods. b The protein levels in the nuclei of primary cultured cells treated with aspirin (100 μM) for 48 h were analyzed using immunoblotting methods. c Protein levels in primary culture cancer cells transfected with shRNA vectors for 48 h were analyzed by immunoblot. d OLA1P2 expression was analyzed by qRT-PCR methods in primary culture cancer cells transfected with the shRNA vector for 48 h and then treated with aspirin (100 μM) for 48 h. e QRT-PCR analysis of FOXD3 mRNA expression in CRC cells treated with aspirin (top lanes). Immunoblot analysis of FOXD3 protein levels in CRC cells treated with aspirin (middle lanes). Quantitative methylation-specific PCR analysis of FOXD3 promoter methylation levels in CRC cells treated with aspirin (bottom lanes). M: methylation-specific primer; U: unmethylation-specific primer. f Diagram showing conserved FOXD3 response elements in the OLA1P2 promoter. g ChIP assays using anti-FOXD3 or anti-IgG antibodies were performed to determine the affinity of FOXD3 for the OLA1P2 promoter in primary culture cancer cells. h Primary culture cancer cells were co-transfected with the lenti-FOXD3 vector and a luciferase reporter for 48 h. i Primary culture cancer cells were transfected with a luciferase reporter for 12 h and were then treated with aspirin (100 μM) for 48 h. **: P
    Figure Legend Snippet: Aspirin promoted OLA1P2 transcription through FOXD3 upregulation. a The biotin-labeled OLA1P2 promoter was mixed with the nuclear extract separated from DMSO/aspirin-treated primary culture cancer cells. The eluted proteins were then analyzed using mass spectrometry methods. b The protein levels in the nuclei of primary cultured cells treated with aspirin (100 μM) for 48 h were analyzed using immunoblotting methods. c Protein levels in primary culture cancer cells transfected with shRNA vectors for 48 h were analyzed by immunoblot. d OLA1P2 expression was analyzed by qRT-PCR methods in primary culture cancer cells transfected with the shRNA vector for 48 h and then treated with aspirin (100 μM) for 48 h. e QRT-PCR analysis of FOXD3 mRNA expression in CRC cells treated with aspirin (top lanes). Immunoblot analysis of FOXD3 protein levels in CRC cells treated with aspirin (middle lanes). Quantitative methylation-specific PCR analysis of FOXD3 promoter methylation levels in CRC cells treated with aspirin (bottom lanes). M: methylation-specific primer; U: unmethylation-specific primer. f Diagram showing conserved FOXD3 response elements in the OLA1P2 promoter. g ChIP assays using anti-FOXD3 or anti-IgG antibodies were performed to determine the affinity of FOXD3 for the OLA1P2 promoter in primary culture cancer cells. h Primary culture cancer cells were co-transfected with the lenti-FOXD3 vector and a luciferase reporter for 48 h. i Primary culture cancer cells were transfected with a luciferase reporter for 12 h and were then treated with aspirin (100 μM) for 48 h. **: P

    Techniques Used: Labeling, Mass Spectrometry, Cell Culture, Transfection, shRNA, Expressing, Quantitative RT-PCR, Plasmid Preparation, Methylation, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Luciferase

    OLA1P2 affected the translocation of the phosphorylated STAT3 protein. a GSEA demonstrated enrichment of STAT3 target genes. The top of the panel shows the enrichment scores for genes associated with STAT3 signaling pathway targets. The black lines represent the 17 most highly correlated targets. The bottom of the panel shows the ranking scores (correlations of all genes associated with STAT3 signaling pathway targets with aspirin). b QRT-PCR analysis of OLA1P2 expression in the colon cancer cell line COLO205 transfected with lenti-OLA1P2, shRNA-OLA1P2, or negative controls. c Total STAT3 protein and phosphorylated STAT3 protein levels were analyzed by immunoblot in COLO205 cells transfected with lenti-OLA1P2 or shRNA-OLA1P2. d RNA FISH analysis of OLA1P2 localization in COLO205 cells. e QRT-PCR analysis of OLA1P2 localization. OLA1P2 was mainly present in the cytoplasm. U2 RNA was used as a positive control for nuclear RNA. f Phosphorylated STAT3 protein levels in the cytoplasm and nuclei of COLO205 cells transfected with lenti-OLA1P2 were analyzed by immunoblot analysis. g Phosphorylated STAT3 protein levels in the cytoplasm and nuclei of COLO205 cells transfected with shRNA-OLA1P2 were analyzed by immunoblot analysis. ***: P
    Figure Legend Snippet: OLA1P2 affected the translocation of the phosphorylated STAT3 protein. a GSEA demonstrated enrichment of STAT3 target genes. The top of the panel shows the enrichment scores for genes associated with STAT3 signaling pathway targets. The black lines represent the 17 most highly correlated targets. The bottom of the panel shows the ranking scores (correlations of all genes associated with STAT3 signaling pathway targets with aspirin). b QRT-PCR analysis of OLA1P2 expression in the colon cancer cell line COLO205 transfected with lenti-OLA1P2, shRNA-OLA1P2, or negative controls. c Total STAT3 protein and phosphorylated STAT3 protein levels were analyzed by immunoblot in COLO205 cells transfected with lenti-OLA1P2 or shRNA-OLA1P2. d RNA FISH analysis of OLA1P2 localization in COLO205 cells. e QRT-PCR analysis of OLA1P2 localization. OLA1P2 was mainly present in the cytoplasm. U2 RNA was used as a positive control for nuclear RNA. f Phosphorylated STAT3 protein levels in the cytoplasm and nuclei of COLO205 cells transfected with lenti-OLA1P2 were analyzed by immunoblot analysis. g Phosphorylated STAT3 protein levels in the cytoplasm and nuclei of COLO205 cells transfected with shRNA-OLA1P2 were analyzed by immunoblot analysis. ***: P

    Techniques Used: Translocation Assay, Quantitative RT-PCR, Expressing, Transfection, shRNA, Fluorescence In Situ Hybridization, Positive Control

    3) Product Images from "MicroRNA Regulatory Mechanisms on Citrus sinensis leaves to Magnesium-Deficiency"

    Article Title: MicroRNA Regulatory Mechanisms on Citrus sinensis leaves to Magnesium-Deficiency

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2016.00201

    Relative abundances of selected known miRNAs in Mg-deficient and -sufficient (control) leaves revealed by qRT-PCR . Bars represent mean ± SD ( n = 3). Significant differences were tested between control and Mg-deficient leaves for the same miRNA. Different letters above the bars indicate a significant difference at P
    Figure Legend Snippet: Relative abundances of selected known miRNAs in Mg-deficient and -sufficient (control) leaves revealed by qRT-PCR . Bars represent mean ± SD ( n = 3). Significant differences were tested between control and Mg-deficient leaves for the same miRNA. Different letters above the bars indicate a significant difference at P

    Techniques Used: Quantitative RT-PCR

    4) Product Images from "Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs"

    Article Title: Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8420

    RIG-I binds U1 snRNA accumulated in the cytoplasm to mediate radiation-induced IFN-beta response A. RIG-I binds diverse non-coding RNA molecules, majority of which are snRNAs. Graphic representation indicating the distribution of non-coding and repetitive RNA molecules bound to RIG-I following exposure to IR as compared to total irradiated cellular RNA. Transcripts were mapped to reference genomes using RepeatMasker. See Methods for further details. B. qRT-PCR quantification of U1 RNA from purified RNA bound to ectopically expressing WT and K858A-K861A mutant RIG-I HEK293 cells exposed to IR (6 Gy) or left untreated. Cells were UV crosslinked at 150mJ/cm 2 48 hours post-IR treatment prior to cell lysis. U1 RNA levels were normalized to the geometric average of 3 housekeeping genes (18S rDNA, GAPDH, and β-actin). Fold change was determined relative to un-irradiated controls. C. U1 RNA levels quantified by qRT-PCR from total cellular and RIG-I pulldowns in RIG-I overexpressing HEK293 and HCT116 cells. U1 RNA levels were normalized to the geometric average of 3 housekeeping genes (18S rDNA, GAPDH, and β-actin). Fold change was determined relative to un-irradiated controls. Time course of cytosolic accumulation of U1 RNA measured by qRT-PCR from purified total cellular RNA following cellular fractionation of nuclear/mitochondrial and cytoplasmic fractions of HEK293 D. and HCT116 cells E. exposed to IR (6 Gy) or left untreated. F. The structure of the U1 snRNA illustrating the four stem loop (SL) regions. G. Relative IFN-beta luciferase reporter activity of HEK293 cells following a 24 hour stimulation with synthetic oligonucleotides corresponding to U1 RNA stem loop (SL) regions I to IV or a combination of SL I + II and SL II + III. H. IFN-b levels in culture supernatant from ICR RIG-I +/+ and RIG-I −/− primary MEFs 24 hours post-stimulation with the same set of synthetic U1 oligonucleotides used in G. The amount of U1 synthetic oligonucleotides used in all stimulation experiments was 1μg. P values were determined using unpaired Student's t -test. Error bars are SEM. *** P
    Figure Legend Snippet: RIG-I binds U1 snRNA accumulated in the cytoplasm to mediate radiation-induced IFN-beta response A. RIG-I binds diverse non-coding RNA molecules, majority of which are snRNAs. Graphic representation indicating the distribution of non-coding and repetitive RNA molecules bound to RIG-I following exposure to IR as compared to total irradiated cellular RNA. Transcripts were mapped to reference genomes using RepeatMasker. See Methods for further details. B. qRT-PCR quantification of U1 RNA from purified RNA bound to ectopically expressing WT and K858A-K861A mutant RIG-I HEK293 cells exposed to IR (6 Gy) or left untreated. Cells were UV crosslinked at 150mJ/cm 2 48 hours post-IR treatment prior to cell lysis. U1 RNA levels were normalized to the geometric average of 3 housekeeping genes (18S rDNA, GAPDH, and β-actin). Fold change was determined relative to un-irradiated controls. C. U1 RNA levels quantified by qRT-PCR from total cellular and RIG-I pulldowns in RIG-I overexpressing HEK293 and HCT116 cells. U1 RNA levels were normalized to the geometric average of 3 housekeeping genes (18S rDNA, GAPDH, and β-actin). Fold change was determined relative to un-irradiated controls. Time course of cytosolic accumulation of U1 RNA measured by qRT-PCR from purified total cellular RNA following cellular fractionation of nuclear/mitochondrial and cytoplasmic fractions of HEK293 D. and HCT116 cells E. exposed to IR (6 Gy) or left untreated. F. The structure of the U1 snRNA illustrating the four stem loop (SL) regions. G. Relative IFN-beta luciferase reporter activity of HEK293 cells following a 24 hour stimulation with synthetic oligonucleotides corresponding to U1 RNA stem loop (SL) regions I to IV or a combination of SL I + II and SL II + III. H. IFN-b levels in culture supernatant from ICR RIG-I +/+ and RIG-I −/− primary MEFs 24 hours post-stimulation with the same set of synthetic U1 oligonucleotides used in G. The amount of U1 synthetic oligonucleotides used in all stimulation experiments was 1μg. P values were determined using unpaired Student's t -test. Error bars are SEM. *** P

    Techniques Used: Irradiation, Quantitative RT-PCR, Purification, Expressing, Mutagenesis, Lysis, Cell Fractionation, Luciferase, Activity Assay

    MAVS is necessary for ionizing radiation-induced Type I interferon signaling A. Proposed mechanism of MAVS-dependent activation of Type I IFN signaling in the cellular response to IR. B. Transcriptional profiling of C57BL/6 wild-type (WT) and MAVS −/− primary MEFs demonstrating MAVS-dependent expression of Type I IFN-stimulated genes (ISGs) 48 hours following exposure to IR (6 Gy). Heatmap displays differences in gene expression values between WT and MAVS −/− MEFs; red indicates high expression and blue low expression. Inset shows qRT-PCR validation of Usp18 , Ifit3 , Stat1 , Ddx58 , and Cdkn1a gene expression values in WT and MAVS −/− MEFs after IR treatment. C. Top-ranked cellular pathways (top) and functions (bottom) (Ingenuity Pathway Analysis) activated by IR in WT MEFs. Pie-chart displays the relative abundance of each functional category among all significant functions ( P
    Figure Legend Snippet: MAVS is necessary for ionizing radiation-induced Type I interferon signaling A. Proposed mechanism of MAVS-dependent activation of Type I IFN signaling in the cellular response to IR. B. Transcriptional profiling of C57BL/6 wild-type (WT) and MAVS −/− primary MEFs demonstrating MAVS-dependent expression of Type I IFN-stimulated genes (ISGs) 48 hours following exposure to IR (6 Gy). Heatmap displays differences in gene expression values between WT and MAVS −/− MEFs; red indicates high expression and blue low expression. Inset shows qRT-PCR validation of Usp18 , Ifit3 , Stat1 , Ddx58 , and Cdkn1a gene expression values in WT and MAVS −/− MEFs after IR treatment. C. Top-ranked cellular pathways (top) and functions (bottom) (Ingenuity Pathway Analysis) activated by IR in WT MEFs. Pie-chart displays the relative abundance of each functional category among all significant functions ( P

    Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Functional Assay

    5) Product Images from "Large-scale screening identifies a novel microRNA, miR-15a-3p, which induces apoptosis in human cancer cell lines"

    Article Title: Large-scale screening identifies a novel microRNA, miR-15a-3p, which induces apoptosis in human cancer cell lines

    Journal: RNA Biology

    doi: 10.4161/rna.23339

    Figure 4. qRT-PCR comparison of mRNA levels for bcl-x L gene in HeLa and AsPc-1 cells 24 and 48 h after transfection. ( A ) HeLa cells. ( B ) AsPc-1 cells. 18S mRNA levels were used as a control for 2 -ΔΔCt analysis. Error bars represent
    Figure Legend Snippet: Figure 4. qRT-PCR comparison of mRNA levels for bcl-x L gene in HeLa and AsPc-1 cells 24 and 48 h after transfection. ( A ) HeLa cells. ( B ) AsPc-1 cells. 18S mRNA levels were used as a control for 2 -ΔΔCt analysis. Error bars represent

    Techniques Used: Quantitative RT-PCR, Transfection

    6) Product Images from "The role of microRNA deregulation in the pathogenesis of adrenocortical carcinoma"

    Article Title: The role of microRNA deregulation in the pathogenesis of adrenocortical carcinoma

    Journal: Endocrine-Related Cancer

    doi: 10.1530/ERC-11-0082

    miRNAs associated with survival of carcinoma patients. (A) Clustering analysis of 11 miRNAs identified by SAM survival analysis of microarray data, and subsequent Kaplan–Meier analysis for cases in cluster 1–3. (B) Kaplan–Meier curves for miR-503 , miR-1202 , and miR-1275 based on qRT-PCR results. Differences in survival were calculated using log-rank test.
    Figure Legend Snippet: miRNAs associated with survival of carcinoma patients. (A) Clustering analysis of 11 miRNAs identified by SAM survival analysis of microarray data, and subsequent Kaplan–Meier analysis for cases in cluster 1–3. (B) Kaplan–Meier curves for miR-503 , miR-1202 , and miR-1275 based on qRT-PCR results. Differences in survival were calculated using log-rank test.

    Techniques Used: Microarray, Quantitative RT-PCR

    Relative expression levels of miR-483-3p, miR-483-5p, miR-210, miR-21, miR-195, miR-497 , and miR-1974 in the different sample groups. Box plots show miRNA expression levels determined by qRT-PCR in adrenocortical carcinomas, adenomas, and adrenal cortices. Statistical significances between the groups were determined with two-tailed unpaired t -test and P
    Figure Legend Snippet: Relative expression levels of miR-483-3p, miR-483-5p, miR-210, miR-21, miR-195, miR-497 , and miR-1974 in the different sample groups. Box plots show miRNA expression levels determined by qRT-PCR in adrenocortical carcinomas, adenomas, and adrenal cortices. Statistical significances between the groups were determined with two-tailed unpaired t -test and P

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

    7) Product Images from "miR-424 coordinates multilayered regulation of cell cycle progression to promote esophageal squamous cell carcinoma cell proliferation"

    Article Title: miR-424 coordinates multilayered regulation of cell cycle progression to promote esophageal squamous cell carcinoma cell proliferation

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2018.10.043

    miR-424 promotes ESCC cell proliferation in vitro and in vivo . (a) qRT-PCR analyses showed the relative miR-424 expression levels in KYSE-410 and KYSE-510 ESCC cells and NE1 immortalized esophageal epithelial cells expressing the miR-424 precursor (lenti- miR-424 ) or control vectors. Data are presented as the mean ± SD of three independent experiments. **, p
    Figure Legend Snippet: miR-424 promotes ESCC cell proliferation in vitro and in vivo . (a) qRT-PCR analyses showed the relative miR-424 expression levels in KYSE-410 and KYSE-510 ESCC cells and NE1 immortalized esophageal epithelial cells expressing the miR-424 precursor (lenti- miR-424 ) or control vectors. Data are presented as the mean ± SD of three independent experiments. **, p

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

    Characterization of the miR-424 promoter and transcriptional regulation of miR-424 by E2F1 during G1/S transition. (a) KYSE-410 and KYSE-510 cells were synchronized in G0/G1-phase by serum starvation and released. qRT-PCR analysis shows the expression kinetics of miR-424 , pri- miR-424 , and pre- miR-424 in KYSE-410 and KYSE-510 cells after release from G0/G1-phase for the indicated time points. Data are presented as the mean ± SD of three independent experiments. (b) A schematic illustration shows two binding sites for E2F1 in the putative promoter region of the miR-424 gene. Specific primers surrounding the promoter were designed. A fragment of the promoter region encompassing the wild-type or mutant E2F1 binding sites was cloned into a reporter vector. (c) Expression of E2F1 was silenced using siRNA (left panel). qRT-PCR showed changes in the expression kinetics of miR-424 , pri- miR-424 , and pre- miR-424 after knocking down E2F1 in KYSE-410 cells during G1/S transition (right panel). Data are presented as the mean ± SD of three independent experiments. *, p
    Figure Legend Snippet: Characterization of the miR-424 promoter and transcriptional regulation of miR-424 by E2F1 during G1/S transition. (a) KYSE-410 and KYSE-510 cells were synchronized in G0/G1-phase by serum starvation and released. qRT-PCR analysis shows the expression kinetics of miR-424 , pri- miR-424 , and pre- miR-424 in KYSE-410 and KYSE-510 cells after release from G0/G1-phase for the indicated time points. Data are presented as the mean ± SD of three independent experiments. (b) A schematic illustration shows two binding sites for E2F1 in the putative promoter region of the miR-424 gene. Specific primers surrounding the promoter were designed. A fragment of the promoter region encompassing the wild-type or mutant E2F1 binding sites was cloned into a reporter vector. (c) Expression of E2F1 was silenced using siRNA (left panel). qRT-PCR showed changes in the expression kinetics of miR-424 , pri- miR-424 , and pre- miR-424 after knocking down E2F1 in KYSE-410 cells during G1/S transition (right panel). Data are presented as the mean ± SD of three independent experiments. *, p

    Techniques Used: Quantitative RT-PCR, Expressing, Binding Assay, Mutagenesis, Clone Assay, Plasmid Preparation

    8) Product Images from "A Cyclin Dependent Kinase Regulatory Subunit (CKS) Gene of Pigeonpea Imparts Abiotic Stress Tolerance and Regulates Plant Growth and Development in Arabidopsis"

    Article Title: A Cyclin Dependent Kinase Regulatory Subunit (CKS) Gene of Pigeonpea Imparts Abiotic Stress Tolerance and Regulates Plant Growth and Development in Arabidopsis

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2017.00165

    qRT-PCR analysis for the expression profiles of selected genes in wild type and transgenic Arabidopsis under drought stress . Comparisons of the relative transcript levels of different genes in CcCKS- transgenic (T) and wild-type (WT) plants under normal (A) and 150 mM mannitol (B) stress conditions. Actin was used as a reference gene. The vertical column indicates the relative transcript level. Bar represents mean and error bars represents SE from three independent experiments. Asterisks indicate significant differences in comparison with the WT at P
    Figure Legend Snippet: qRT-PCR analysis for the expression profiles of selected genes in wild type and transgenic Arabidopsis under drought stress . Comparisons of the relative transcript levels of different genes in CcCKS- transgenic (T) and wild-type (WT) plants under normal (A) and 150 mM mannitol (B) stress conditions. Actin was used as a reference gene. The vertical column indicates the relative transcript level. Bar represents mean and error bars represents SE from three independent experiments. Asterisks indicate significant differences in comparison with the WT at P

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

    Analysis of Cajanus cajan cyclin dependent kinase regulatory subunit gene ( CcCKS ) expression in pigeonpea under different treatment conditions by qRT- PCR . Comparison of the relative transcript levels of CcCKS gene under (PEG) 6000 (20% w/v), Mannitol (400 mM), NaCl (1.0 M), and ABA (100 μM) stress for 6 h (A) and 12 h (B) . Actin has been used as reference gene. The vertical column indicates the relative transcript level. Bar represents mean and error bars represents SE from three independent experiments. Asterisks indicate significant differences in comparison with the WT at P
    Figure Legend Snippet: Analysis of Cajanus cajan cyclin dependent kinase regulatory subunit gene ( CcCKS ) expression in pigeonpea under different treatment conditions by qRT- PCR . Comparison of the relative transcript levels of CcCKS gene under (PEG) 6000 (20% w/v), Mannitol (400 mM), NaCl (1.0 M), and ABA (100 μM) stress for 6 h (A) and 12 h (B) . Actin has been used as reference gene. The vertical column indicates the relative transcript level. Bar represents mean and error bars represents SE from three independent experiments. Asterisks indicate significant differences in comparison with the WT at P

    Techniques Used: Expressing, Quantitative RT-PCR

    9) Product Images from "bHLH142 regulates various metabolic pathway-related genes to affect pollen development and anther dehiscence in rice"

    Article Title: bHLH142 regulates various metabolic pathway-related genes to affect pollen development and anther dehiscence in rice

    Journal: Scientific Reports

    doi: 10.1038/srep43397

    bHLH142 overexpressing anthers are defective in septum and stomium degeneration. (a) Transverse sections of pre-anthesis staged WT and transgenic anthers (L42) undergoing dehiscence. Green and red arrows indicate intact septum and stomium in transgenic anthers, respectively. (b) WT and transgenic anthers analyzed under UV light to visualize lignin autofluorescence of endothecium regions. Red and yellow arrows indicate presence and absence secondary thickening of endothecium, respectively. (c) qRT-PCR expression data of anther dehiscence-related protein genes. Error bars indicate standard deviation (SD). The data are presented as the mean ± SD (n = 3). Asterisks indicate significant difference with respect to WT (‘***’ indicates t -test p-value ≤ 0.001, while ‘**’ indicates p-value ≤ 0.01). WT, Wild Type. Scales: A, 50 μm; B, 100 μm.
    Figure Legend Snippet: bHLH142 overexpressing anthers are defective in septum and stomium degeneration. (a) Transverse sections of pre-anthesis staged WT and transgenic anthers (L42) undergoing dehiscence. Green and red arrows indicate intact septum and stomium in transgenic anthers, respectively. (b) WT and transgenic anthers analyzed under UV light to visualize lignin autofluorescence of endothecium regions. Red and yellow arrows indicate presence and absence secondary thickening of endothecium, respectively. (c) qRT-PCR expression data of anther dehiscence-related protein genes. Error bars indicate standard deviation (SD). The data are presented as the mean ± SD (n = 3). Asterisks indicate significant difference with respect to WT (‘***’ indicates t -test p-value ≤ 0.001, while ‘**’ indicates p-value ≤ 0.01). WT, Wild Type. Scales: A, 50 μm; B, 100 μm.

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

    Overexpression of bHLH142 causes anther indehiscence in rice. ( a) bHLH142 OE plant (L42) showing growth compared to WT. (b) Post-anthesis panicles of WT and bHLH142 OE transgenics (L40 and L42) showing dehisced anthers in WT and indehisced anther in transgenic plants. (c) SEM images of WT and transgenic anthers showing that apical and basal part of WT anther is ruptured while that of transgenics is intact. (d) qRT-PCR expression analysis showing high accumulation of bHLH142 transcripts in transgenic anthers at tetrad and MP stages. Error bars indicate standard deviation (SD). The data are presented as the mean ± SD (n = 3). Asterisks indicate significant difference with respect to WT (‘*’ indicates t -test p-value ≤ 0.05 while ‘***’ indicates p-value ≤ 0.001). (e) Immunoblot showing higher accumulation of bHLH142 polypeptide in transgenic plants in MP anther. WT, Wild Type.
    Figure Legend Snippet: Overexpression of bHLH142 causes anther indehiscence in rice. ( a) bHLH142 OE plant (L42) showing growth compared to WT. (b) Post-anthesis panicles of WT and bHLH142 OE transgenics (L40 and L42) showing dehisced anthers in WT and indehisced anther in transgenic plants. (c) SEM images of WT and transgenic anthers showing that apical and basal part of WT anther is ruptured while that of transgenics is intact. (d) qRT-PCR expression analysis showing high accumulation of bHLH142 transcripts in transgenic anthers at tetrad and MP stages. Error bars indicate standard deviation (SD). The data are presented as the mean ± SD (n = 3). Asterisks indicate significant difference with respect to WT (‘*’ indicates t -test p-value ≤ 0.05 while ‘***’ indicates p-value ≤ 0.001). (e) Immunoblot showing higher accumulation of bHLH142 polypeptide in transgenic plants in MP anther. WT, Wild Type.

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

    bHLH142 OE anthers show changed in expression of various metabolic pathway-related genes. (a) Venn diagram showing common and unique genes differentially regulated in tetrad and MP anther. (b) Histogram showing number of genes upregulated and downregulated in tetrad and MP stage of transgenic anthers compared to WT. Heat map showing log 2 fold changed in expression of cell wall modification (c), ROS signaling (d) and cell death (e) -related genes. (f) Validation of changed in expression of different metabolism-related genes through qRT-PCR. The data are presented as the mean ± SD (n = 3). Asterisks indicate significant difference with respect to WT (‘***’ indicates t -test p-value ≤ 0.001, ‘**’ p-value ≤ 0.01 and ‘*’ p-value ≤ 0.05).
    Figure Legend Snippet: bHLH142 OE anthers show changed in expression of various metabolic pathway-related genes. (a) Venn diagram showing common and unique genes differentially regulated in tetrad and MP anther. (b) Histogram showing number of genes upregulated and downregulated in tetrad and MP stage of transgenic anthers compared to WT. Heat map showing log 2 fold changed in expression of cell wall modification (c), ROS signaling (d) and cell death (e) -related genes. (f) Validation of changed in expression of different metabolism-related genes through qRT-PCR. The data are presented as the mean ± SD (n = 3). Asterisks indicate significant difference with respect to WT (‘***’ indicates t -test p-value ≤ 0.001, ‘**’ p-value ≤ 0.01 and ‘*’ p-value ≤ 0.05).

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

    Temporal expression pattern of transcript and protein for bHLH142 are different. (a) qRT-PCR expression analysis of bHLH142 transcript in different stages of rice anthers. Error bars indicate standard deviation (SD). The data are presented as the mean ± SD (n = 3). Asterisks indicate significant difference with respect to Tetrad (‘***’ indicates t -test, p-value ≤ 0.001). (b) Immunoblot showing accumulation of bHLH142 polypeptide in different stages of rice spikelets. (c) In situ immunolocalization showing spatio-temporal presence of bHLH142 polypeptide during different stages of rice anthers. Right most panels show enlarged view of the selected region (shown as rectangle) of MP stage of the anther. Control panel shows samples without bHLH142 primary antibody treatment. M, Microspore; sp, Septum; st, Stomium; T, Tapetum; V, Vascular tissue. Scales: 100 μm.
    Figure Legend Snippet: Temporal expression pattern of transcript and protein for bHLH142 are different. (a) qRT-PCR expression analysis of bHLH142 transcript in different stages of rice anthers. Error bars indicate standard deviation (SD). The data are presented as the mean ± SD (n = 3). Asterisks indicate significant difference with respect to Tetrad (‘***’ indicates t -test, p-value ≤ 0.001). (b) Immunoblot showing accumulation of bHLH142 polypeptide in different stages of rice spikelets. (c) In situ immunolocalization showing spatio-temporal presence of bHLH142 polypeptide during different stages of rice anthers. Right most panels show enlarged view of the selected region (shown as rectangle) of MP stage of the anther. Control panel shows samples without bHLH142 primary antibody treatment. M, Microspore; sp, Septum; st, Stomium; T, Tapetum; V, Vascular tissue. Scales: 100 μm.

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

    10) Product Images from "Collagen I promotes hepatocellular carcinoma cell proliferation by regulating integrin β1/FAK signaling pathway in nonalcoholic fatty liver"

    Article Title: Collagen I promotes hepatocellular carcinoma cell proliferation by regulating integrin β1/FAK signaling pathway in nonalcoholic fatty liver

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21525

    Collagen I contributes to the NAFLD-related HCC ( A ) qRT-PCR analysis showed that Collagen I levels were 2.8 fold higher in HCC samples than those in adjacent non-tumor liver tissues. ( B ) Immunofluorescence staining confirmed the higher expression of Collagen I (green) in HCC tissues compared to normal liver tissues. ( C ) Immunofluorescence staining revealed a higher expression of Collagen I (green) in human fatty liver compared to human normal liver. ( D ) In the mouse models of NAFLD/NASH, Collagen I was found to be upregulated. ( E , F ) HCC cells cultured on different ECM or different concentration of Collagen I to determine the effect of Collagen I on HCC cell proliferation. The vitality of cells at each time point was detected by CCK-8 assay. (E) SMMC-7721 and HepG2 cells cultured on Collagen I proliferated significantly faster than those on either Collagen IV or fibronectin. (F) The effect of Collagen I on cell proliferation appeared to be dose-dependent. Scale bar: 50 μm (B); 100 μm (C, D), * P
    Figure Legend Snippet: Collagen I contributes to the NAFLD-related HCC ( A ) qRT-PCR analysis showed that Collagen I levels were 2.8 fold higher in HCC samples than those in adjacent non-tumor liver tissues. ( B ) Immunofluorescence staining confirmed the higher expression of Collagen I (green) in HCC tissues compared to normal liver tissues. ( C ) Immunofluorescence staining revealed a higher expression of Collagen I (green) in human fatty liver compared to human normal liver. ( D ) In the mouse models of NAFLD/NASH, Collagen I was found to be upregulated. ( E , F ) HCC cells cultured on different ECM or different concentration of Collagen I to determine the effect of Collagen I on HCC cell proliferation. The vitality of cells at each time point was detected by CCK-8 assay. (E) SMMC-7721 and HepG2 cells cultured on Collagen I proliferated significantly faster than those on either Collagen IV or fibronectin. (F) The effect of Collagen I on cell proliferation appeared to be dose-dependent. Scale bar: 50 μm (B); 100 μm (C, D), * P

    Techniques Used: Quantitative RT-PCR, Immunofluorescence, Staining, Expressing, Cell Culture, Concentration Assay, CCK-8 Assay

    The expression of integrin β1 in NAFLD-related HCC ( A – C ) The expression of integrin β1 in HCC. (A) qRT-PCR analysis showed that integrin β1 levels were 1.1 fold higher in HCC samples than those in adjacent non-tumor liver tissues. (B) Correlation analysis indicated that the expression level of integrin β1 were positively correlated with the level of Collagen I, r = 0.43, P = 0.004. (C) Immunohistochemical staining revealed a high expression of integrin β1 in HCC tissues. ( D ) HepG2 cells were seeded into the NLM and FLM and cultured for 15 days. Western blot indicated that cells cultured in FLM had a higher expression of integrin β1 than those in NLM. ( E , F ) HCC cells cultured on different ECM or different concentration of Collagen I. (E) For SMMC-7721 and HepG2, integrin β1 was higher expressed in Collagen I compared to Collagen IV and fibronectin. (F) Increasing Collagen I concentration could up regulated the expression of integrin β1 in both cell lines. ( G , H ) The expression of integrin β1 in mouse orthotopic tumor model. (G) Western blot indicated that tumors grown in HF- or MCD-fed mice expressed higher level of integrin β1. (H) Immunohistochemical staining of integrin β1 confirmed the same results. ( I – J ) Integrin β1 was knocked down in HCC cells by shRNA technology through lentiviral transduction. (I) Western blot confirmed the stable knock-down of integrin β1 in both cell lines. (J) HepG2 and SMMC-7721 cells cultured on Collagen I grew significantly slowly after knocking down integrin β1. Scale bar: 100 μm (C), 50 μm (H), * P
    Figure Legend Snippet: The expression of integrin β1 in NAFLD-related HCC ( A – C ) The expression of integrin β1 in HCC. (A) qRT-PCR analysis showed that integrin β1 levels were 1.1 fold higher in HCC samples than those in adjacent non-tumor liver tissues. (B) Correlation analysis indicated that the expression level of integrin β1 were positively correlated with the level of Collagen I, r = 0.43, P = 0.004. (C) Immunohistochemical staining revealed a high expression of integrin β1 in HCC tissues. ( D ) HepG2 cells were seeded into the NLM and FLM and cultured for 15 days. Western blot indicated that cells cultured in FLM had a higher expression of integrin β1 than those in NLM. ( E , F ) HCC cells cultured on different ECM or different concentration of Collagen I. (E) For SMMC-7721 and HepG2, integrin β1 was higher expressed in Collagen I compared to Collagen IV and fibronectin. (F) Increasing Collagen I concentration could up regulated the expression of integrin β1 in both cell lines. ( G , H ) The expression of integrin β1 in mouse orthotopic tumor model. (G) Western blot indicated that tumors grown in HF- or MCD-fed mice expressed higher level of integrin β1. (H) Immunohistochemical staining of integrin β1 confirmed the same results. ( I – J ) Integrin β1 was knocked down in HCC cells by shRNA technology through lentiviral transduction. (I) Western blot confirmed the stable knock-down of integrin β1 in both cell lines. (J) HepG2 and SMMC-7721 cells cultured on Collagen I grew significantly slowly after knocking down integrin β1. Scale bar: 100 μm (C), 50 μm (H), * P

    Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Cell Culture, Western Blot, Concentration Assay, Mouse Assay, shRNA, Transduction

    11) Product Images from "Myeloid-derived suppressor cell and macrophage exert distinct angiogenic and immunosuppressive effects in breast cancer"

    Article Title: Myeloid-derived suppressor cell and macrophage exert distinct angiogenic and immunosuppressive effects in breast cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17013

    TiMDSC expresses lower levels of anti-inflammatory factors compared to those of TAM in MCaP0008 breast cancers Gene expression profiles of tiMDSC and TAM were analyzed by qRT-PCR. The experiment procedure was the same as described in Figure 2 . Data were shown as mean values ± SEM ( n = 6–8 mice per group). Experiments were repeated four times. * denotes P
    Figure Legend Snippet: TiMDSC expresses lower levels of anti-inflammatory factors compared to those of TAM in MCaP0008 breast cancers Gene expression profiles of tiMDSC and TAM were analyzed by qRT-PCR. The experiment procedure was the same as described in Figure 2 . Data were shown as mean values ± SEM ( n = 6–8 mice per group). Experiments were repeated four times. * denotes P

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

    12) Product Images from "De novo transcriptome sequencing and comparative analysis to discover genes related to floral development in Cymbidium faberi Rolfe"

    Article Title: De novo transcriptome sequencing and comparative analysis to discover genes related to floral development in Cymbidium faberi Rolfe

    Journal: SpringerPlus

    doi: 10.1186/s40064-016-3089-1

    qRT-PCR analysis of ten selected genes in different organs of C . faberi. a – j indicated the relative expression level of DEF , PI , AG , AP , TCP4 , BRC2 , CO , SOC1 , CCA1 , LFY gene, respectively. In these pictures, F flower buds; V vegetative buds; Asterisk indicated significantly higher expression
    Figure Legend Snippet: qRT-PCR analysis of ten selected genes in different organs of C . faberi. a – j indicated the relative expression level of DEF , PI , AG , AP , TCP4 , BRC2 , CO , SOC1 , CCA1 , LFY gene, respectively. In these pictures, F flower buds; V vegetative buds; Asterisk indicated significantly higher expression

    Techniques Used: Quantitative RT-PCR, Expressing

    13) Product Images from "Overexpression of the WOX gene STENOFOLIA improves biomass yield and sugar release in transgenic grasses and display altered cytokinin homeostasis"

    Article Title: Overexpression of the WOX gene STENOFOLIA improves biomass yield and sugar release in transgenic grasses and display altered cytokinin homeostasis

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006649

    STF overexpression in switchgrass improves biomass yield and release of solubilized sugars. (A) Morphology of switchgrass plants overexpressing different levels of STF at flowering. One representative from each group was shown. Bars = 10 cm for plants, 1cm for leaves. (B) Transcript abundance of STF in transgenic plants revealed by qRT-PCR. UBI :: GUS-1 switchgrass plant was used as the control. Bars represent means ± SE of three technical replicates. (C) Comparison of postharvest dry weights of total above-ground biomass of three control ( UBI :: GUS plants) and three classes of STF overexpressors shown in (B) at maturity. Bars represent means ± SE (n = 3 independent plants for Control, Group Ⅰ, Ⅱ, and 2 plants for group Ⅲ), the asterisks indicate significant differences (*p
    Figure Legend Snippet: STF overexpression in switchgrass improves biomass yield and release of solubilized sugars. (A) Morphology of switchgrass plants overexpressing different levels of STF at flowering. One representative from each group was shown. Bars = 10 cm for plants, 1cm for leaves. (B) Transcript abundance of STF in transgenic plants revealed by qRT-PCR. UBI :: GUS-1 switchgrass plant was used as the control. Bars represent means ± SE of three technical replicates. (C) Comparison of postharvest dry weights of total above-ground biomass of three control ( UBI :: GUS plants) and three classes of STF overexpressors shown in (B) at maturity. Bars represent means ± SE (n = 3 independent plants for Control, Group Ⅰ, Ⅱ, and 2 plants for group Ⅲ), the asterisks indicate significant differences (*p

    Techniques Used: Over Expression, Transgenic Assay, Quantitative RT-PCR

    STF directly binds and represses the expression of several CKX genes in vitro and in vivo . (A) Schematic presentation of putative STF binding sites in CKX promoters. (B) DNA binding assay corresponding to the putative binding sites of STF. DNA fragments bound to His-TF-STF(HD) fusion protein or His-TF control were quantified by qRT-PCR after elution. Bars represent means ± SE of three technical replicates and two biological replicates. The asterisks indicate significant differences (** p
    Figure Legend Snippet: STF directly binds and represses the expression of several CKX genes in vitro and in vivo . (A) Schematic presentation of putative STF binding sites in CKX promoters. (B) DNA binding assay corresponding to the putative binding sites of STF. DNA fragments bound to His-TF-STF(HD) fusion protein or His-TF control were quantified by qRT-PCR after elution. Bars represent means ± SE of three technical replicates and two biological replicates. The asterisks indicate significant differences (** p

    Techniques Used: Expressing, In Vitro, In Vivo, Binding Assay, DNA Binding Assay, Quantitative RT-PCR

    14) Product Images from "AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells"

    Article Title: AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-020-1565-6

    Effect of AICAR and NAM treatment on differentiation potential of MSCs after long-term in vitro culture. MSCs at P8 were cultured in osteogenic or adipogenic induction media in the presence of AICAR (1 mM), NAM (5 mM), or AICAR+NAM (1 mM and 5 mM, respectively) or in the absence of AICAR and NAM (control group). a Cells were stained with Alizarin Red S, and the accumulation of calcium deposits was visualized using light microscopy (scale bar = 100 μm) and quantified by spectrophotometry ( n = 3 independent experiments). Each bar indicates mean ± SD. b Expression of markers of osteogenesis—Runx-2, osteopontin, and ALP—was determined by qRT-PCR analysis ( n = 3 independent experiments). Each bar indicates mean ± SD. c For the evaluation of adipogenesis, cells were stained with Oil Red O, and lipid accumulation was visualized using light microscopy (scale bar = 100 μm) and quantified by spectrophotometry ( n = 3 independent experiments). Each bar indicates mean ± SD. d mRNA expression of markers of adipogenesis—LPL and PPAR-γ—was determined by qRT-PCR analysis ( n = 3 independent experiments). Each bar indicates mean ± SD (* p
    Figure Legend Snippet: Effect of AICAR and NAM treatment on differentiation potential of MSCs after long-term in vitro culture. MSCs at P8 were cultured in osteogenic or adipogenic induction media in the presence of AICAR (1 mM), NAM (5 mM), or AICAR+NAM (1 mM and 5 mM, respectively) or in the absence of AICAR and NAM (control group). a Cells were stained with Alizarin Red S, and the accumulation of calcium deposits was visualized using light microscopy (scale bar = 100 μm) and quantified by spectrophotometry ( n = 3 independent experiments). Each bar indicates mean ± SD. b Expression of markers of osteogenesis—Runx-2, osteopontin, and ALP—was determined by qRT-PCR analysis ( n = 3 independent experiments). Each bar indicates mean ± SD. c For the evaluation of adipogenesis, cells were stained with Oil Red O, and lipid accumulation was visualized using light microscopy (scale bar = 100 μm) and quantified by spectrophotometry ( n = 3 independent experiments). Each bar indicates mean ± SD. d mRNA expression of markers of adipogenesis—LPL and PPAR-γ—was determined by qRT-PCR analysis ( n = 3 independent experiments). Each bar indicates mean ± SD (* p

    Techniques Used: In Vitro, Cell Culture, Staining, Light Microscopy, Spectrophotometry, Expressing, Quantitative RT-PCR

    15) Product Images from "Endogenous retroviruses and neighboring genes are coordinately repressed by LSD1/KDM1A"

    Article Title: Endogenous retroviruses and neighboring genes are coordinately repressed by LSD1/KDM1A

    Journal: Genes & Development

    doi: 10.1101/gad.2008511

    KDM1A represses MERVL retroviruses in ES cells and preimplantation development. ( A–C ) mRNA-seq was performed on polyA-enriched mRNAs from Kdm1a +/+ and GT/GT ES cells ( A ), or Kdm1a FL/FL Cre-ERT ES cells treated with vehicle or 4OHT and passaged two ( B ) or six ( C ) additional times. Each plot displays the expression value (in RPKM) of repetitive sequences in wild-type ( x -axis) and mutant ( Y -axis) ES cells. Data points in red indicate a greater than fourfold difference in the mutants, while those in blue indicate a greater than twofold difference. Colored arrows label the following: MERVL (purple), L1- (orange), and SINE-B2 (green). ( D ) qRT–PCR analysis was performed on Kdm1a +/+, GT/GT, and GT/GT rescued clones with MERVL Pol-specific primers and plotted relative to Gapdh . ( E , F ) Kdm1a FL/FL ES cells stably expressing a neo resistance control plasmid (Con; lanes 1 , 2 ), Flag-hKDM1A (WT; lanes 3 , 4 ), or catalytically inactive Flag-hKDM1A (MUT; lanes 5 , 6 ) were transfected with either GFP alone or Cre-GFP as indicated to remove the endogenous mouse Kdm1a alleles. GFP + cells were collected 24 h later and plated for an additional 48 h before being subjected to immunoblot analysis with the indicated antibody ( E ) or qRT–PCR with MERVL primers and plotted relative to Gapdh ( F ). Error bars represent SD. ( G ) Whole-cell extracts from ES cells of the indicated genotype were subjected to immunoblots with the indicated antibodies. ( H ) Immunofluorescence microscopy using anti-MERVL Gag antibodies (red) was performed on Kdm1a +/KO or KO/KO blastocysts and overlaid with DAPI (blue).
    Figure Legend Snippet: KDM1A represses MERVL retroviruses in ES cells and preimplantation development. ( A–C ) mRNA-seq was performed on polyA-enriched mRNAs from Kdm1a +/+ and GT/GT ES cells ( A ), or Kdm1a FL/FL Cre-ERT ES cells treated with vehicle or 4OHT and passaged two ( B ) or six ( C ) additional times. Each plot displays the expression value (in RPKM) of repetitive sequences in wild-type ( x -axis) and mutant ( Y -axis) ES cells. Data points in red indicate a greater than fourfold difference in the mutants, while those in blue indicate a greater than twofold difference. Colored arrows label the following: MERVL (purple), L1- (orange), and SINE-B2 (green). ( D ) qRT–PCR analysis was performed on Kdm1a +/+, GT/GT, and GT/GT rescued clones with MERVL Pol-specific primers and plotted relative to Gapdh . ( E , F ) Kdm1a FL/FL ES cells stably expressing a neo resistance control plasmid (Con; lanes 1 , 2 ), Flag-hKDM1A (WT; lanes 3 , 4 ), or catalytically inactive Flag-hKDM1A (MUT; lanes 5 , 6 ) were transfected with either GFP alone or Cre-GFP as indicated to remove the endogenous mouse Kdm1a alleles. GFP + cells were collected 24 h later and plated for an additional 48 h before being subjected to immunoblot analysis with the indicated antibody ( E ) or qRT–PCR with MERVL primers and plotted relative to Gapdh ( F ). Error bars represent SD. ( G ) Whole-cell extracts from ES cells of the indicated genotype were subjected to immunoblots with the indicated antibodies. ( H ) Immunofluorescence microscopy using anti-MERVL Gag antibodies (red) was performed on Kdm1a +/KO or KO/KO blastocysts and overlaid with DAPI (blue).

    Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR, Clone Assay, Stable Transfection, Plasmid Preparation, Transfection, Western Blot, Immunofluorescence, Microscopy

    Kdm1a mutant ES cells have increased potency to generate extraembryonic lineages. ( A , B ) Kdm1a FL/FL Cre-ERT ES cells were treated with 4OHT and maintained for 48 h before growing in bacterial-grade dishes in the absence of LIF to induce differentiation. ( A ) After 48 h, embryoid bodies were imaged by phase-contrast microscopy. After 8 d of differentiation, embryoid bodies were immunostained with HNF4 and SOX2 antibodies and overlaid with DAPI. Optical confocal sections were then taken through the entire embryoid body. ( C ) qRT–PCR analysis with the indicated primers was performed on tumors derived from Kmd1a Fl/FL CreERT ES cells treated with vehicle (WT) or 4OHT (Mut). Error bars represent SD. (Ecto) Ectoderm; (Meso) mesoderm; (ExEn) extraembryonic endoderm; (PE) parietal endoderm; (VE) visceral endoderm. ( D ) Tumors derived from Kdm1a wild-type (WT) and mutant (Mut) ES cells were immunostained with the VE marker HNF4 and the neural marker TUJ1 and counterstained with DAPI. ( E ) Cryosections from Kdm1a wild-type (WT) and KO/KO (Mut) embryos at embryonic day 6.5 were immunostained with anti-collagen IV antibodies and counterstained with DAPI. ( F ) Model of KDM1A function in the coordinated repression of MERVL and extraembryonic cell fate potential. In ES cells derived from the ICM, MERVL retroviruses and a subset of ZGA genes that are LTR-linked are repressed by KDM1A, and cell fate is restricted to embryonic lineages. In Kdm1a mutants, MERVL and LTR-linked ZGA genes are improperly activated, resulting in an expanded fate potential to generate extraembryonic tissues.
    Figure Legend Snippet: Kdm1a mutant ES cells have increased potency to generate extraembryonic lineages. ( A , B ) Kdm1a FL/FL Cre-ERT ES cells were treated with 4OHT and maintained for 48 h before growing in bacterial-grade dishes in the absence of LIF to induce differentiation. ( A ) After 48 h, embryoid bodies were imaged by phase-contrast microscopy. After 8 d of differentiation, embryoid bodies were immunostained with HNF4 and SOX2 antibodies and overlaid with DAPI. Optical confocal sections were then taken through the entire embryoid body. ( C ) qRT–PCR analysis with the indicated primers was performed on tumors derived from Kmd1a Fl/FL CreERT ES cells treated with vehicle (WT) or 4OHT (Mut). Error bars represent SD. (Ecto) Ectoderm; (Meso) mesoderm; (ExEn) extraembryonic endoderm; (PE) parietal endoderm; (VE) visceral endoderm. ( D ) Tumors derived from Kdm1a wild-type (WT) and mutant (Mut) ES cells were immunostained with the VE marker HNF4 and the neural marker TUJ1 and counterstained with DAPI. ( E ) Cryosections from Kdm1a wild-type (WT) and KO/KO (Mut) embryos at embryonic day 6.5 were immunostained with anti-collagen IV antibodies and counterstained with DAPI. ( F ) Model of KDM1A function in the coordinated repression of MERVL and extraembryonic cell fate potential. In ES cells derived from the ICM, MERVL retroviruses and a subset of ZGA genes that are LTR-linked are repressed by KDM1A, and cell fate is restricted to embryonic lineages. In Kdm1a mutants, MERVL and LTR-linked ZGA genes are improperly activated, resulting in an expanded fate potential to generate extraembryonic tissues.

    Techniques Used: Mutagenesis, Microscopy, Quantitative RT-PCR, Derivative Assay, Marker

    KDM1A, HDACs, and KAP1 cooperate to repress endogenous retroviral sequences in ES cells. ( A , B ) KDM1A complexes were affinity-purified from ES cells and subjected to LDS-PAGE and silver staining ( A ) or immunoblotting ( B ) with the indicated antibodies. ( C ) Wild-type ES cells were immunostained with KDM1A and KAP1 antibodies and subjected to confocal microscopy. ( D ) Bisulfite sequencing was performed on Kdm1a +/+ or GT/GT ES cells with primers amplifying the MERVL retrovirus. ( E–G ) Kdm1a +/+ or GT/GT ES cells were treated with TSA or 5Aza for 24 h. qRT–PCR was performed using primers specific for MERVL Pol ( E ), Tcstv3 ( F ), or Eif1a ( Gm2022 ) ( G ) and normalized to Gapdh . The fold change was then plotted relative to untreated Kdm1a +/+ ES cells, with error bars representing SD. ( H ) Kdm1a +/+, +/GT, or GT/GT ES cells were treated with TSA for 24 h, and cells were subjected to immunoblotting with the indicated antibodies. ( I ) Kdm1a FL/FL Cre-ERT ES cells were treated with vehicle or 4OHT for 48 h, followed by treatment with TSA for 24 h. Immunoblotting was then performed with the indicated antibodies.
    Figure Legend Snippet: KDM1A, HDACs, and KAP1 cooperate to repress endogenous retroviral sequences in ES cells. ( A , B ) KDM1A complexes were affinity-purified from ES cells and subjected to LDS-PAGE and silver staining ( A ) or immunoblotting ( B ) with the indicated antibodies. ( C ) Wild-type ES cells were immunostained with KDM1A and KAP1 antibodies and subjected to confocal microscopy. ( D ) Bisulfite sequencing was performed on Kdm1a +/+ or GT/GT ES cells with primers amplifying the MERVL retrovirus. ( E–G ) Kdm1a +/+ or GT/GT ES cells were treated with TSA or 5Aza for 24 h. qRT–PCR was performed using primers specific for MERVL Pol ( E ), Tcstv3 ( F ), or Eif1a ( Gm2022 ) ( G ) and normalized to Gapdh . The fold change was then plotted relative to untreated Kdm1a +/+ ES cells, with error bars representing SD. ( H ) Kdm1a +/+, +/GT, or GT/GT ES cells were treated with TSA for 24 h, and cells were subjected to immunoblotting with the indicated antibodies. ( I ) Kdm1a FL/FL Cre-ERT ES cells were treated with vehicle or 4OHT for 48 h, followed by treatment with TSA for 24 h. Immunoblotting was then performed with the indicated antibodies.

    Techniques Used: Affinity Purification, Polyacrylamide Gel Electrophoresis, Silver Staining, Confocal Microscopy, Methylation Sequencing, Quantitative RT-PCR

    16) Product Images from "Identification of a Protein Network Interacting with TdRF1, a Wheat RING Ubiquitin Ligase with a Protective Role against Cellular Dehydration 1Identification of a Protein Network Interacting with TdRF1, a Wheat RING Ubiquitin Ligase with a Protective Role against Cellular Dehydration 1 [C]Identification of a Protein Network Interacting with TdRF1, a Wheat RING Ubiquitin Ligase with a Protective Role against Cellular Dehydration 1 [C] [W]"

    Article Title: Identification of a Protein Network Interacting with TdRF1, a Wheat RING Ubiquitin Ligase with a Protective Role against Cellular Dehydration 1Identification of a Protein Network Interacting with TdRF1, a Wheat RING Ubiquitin Ligase with a Protective Role against Cellular Dehydration 1 [C]Identification of a Protein Network Interacting with TdRF1, a Wheat RING Ubiquitin Ligase with a Protective Role against Cellular Dehydration 1 [C] [W]

    Journal: Plant Physiology

    doi: 10.1104/pp.111.183988

    Gene expression analysis of TdRF1 , WBLH1 , TdWNK5 , and WVIP2 in durum wheat plants exposed to cold (A), dehydration (B), or ABA (C) treatment. Leaves of treated or control untreated plants were used for RNA extraction and qRT-PCR analysis. The log(2) of
    Figure Legend Snippet: Gene expression analysis of TdRF1 , WBLH1 , TdWNK5 , and WVIP2 in durum wheat plants exposed to cold (A), dehydration (B), or ABA (C) treatment. Leaves of treated or control untreated plants were used for RNA extraction and qRT-PCR analysis. The log(2) of

    Techniques Used: Expressing, RNA Extraction, Quantitative RT-PCR

    17) Product Images from "Functional Analysis of an Arabidopsis thaliana Abiotic Stress-inducible Facilitated Diffusion Transporter for Monosaccharides *"

    Article Title: Functional Analysis of an Arabidopsis thaliana Abiotic Stress-inducible Facilitated Diffusion Transporter for Monosaccharides *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.054288

    Kinetics analysis of glucose uptake activity of ESL1(LLL/AAA). A , relative expression of ESL1 in transgenic BY-2 cells determined by qRT-PCR. The highest expression level was set to 1.0. To normalize ESL1 expression, 18 S rRNA was amplified as an internal
    Figure Legend Snippet: Kinetics analysis of glucose uptake activity of ESL1(LLL/AAA). A , relative expression of ESL1 in transgenic BY-2 cells determined by qRT-PCR. The highest expression level was set to 1.0. To normalize ESL1 expression, 18 S rRNA was amplified as an internal

    Techniques Used: Activity Assay, Expressing, Transgenic Assay, Quantitative RT-PCR, Amplification

    Expression analysis of ERD6 and ESL1 under abiotic stress conditions and ABA treatment. Total RNA from whole plants, leaves, and roots were used for Northern blot ( A ) and qRT-PCR analyses ( B ), respectively. The value of control (leaf) was set to 1.0.
    Figure Legend Snippet: Expression analysis of ERD6 and ESL1 under abiotic stress conditions and ABA treatment. Total RNA from whole plants, leaves, and roots were used for Northern blot ( A ) and qRT-PCR analyses ( B ), respectively. The value of control (leaf) was set to 1.0.

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

    18) Product Images from "PINK1 and Parkin Control Localized Translation of Respiratory Chain Component mRNAs on Mitochondria Outer Membrane"

    Article Title: PINK1 and Parkin Control Localized Translation of Respiratory Chain Component mRNAs on Mitochondria Outer Membrane

    Journal: Cell metabolism

    doi: 10.1016/j.cmet.2014.12.007

    Parkin Cooperates with PINK1 to Promote Glo/hnRNP-F Ubiquitination and nRCC mRNA Translation (A) Association of endogenous (left) and exogenous (right) Parkin with Pum-1 in co-IP assays. (B, C) m 7 -GTP sepharose binding showing RNA-dependent association of myc-Parkin (B) and endogenous Parkin (C) with TIC. (D) m 7 -GTP sepharose binding showing impaired association of Parkin (V56E, C212Y) with the TIC. (E) Association of endogenous Parkin with exogenous PABP in co-IP assays. (F) RT-PCR and WB showing the effects of parkin mutation on the MOM-targeting and translation of nRCC mRNAs (n=3 experiments). (G) qRT-PCR showing the effect of parkin mutation on RCC mRNA levels in the total and subcellular fractions (n=3 experiments). (H) RT-PCR and WB showing effects of Parkin or PINK1 OE on the defective MOM-targeting and translation of nRCC mRNAs caused by Pum-m OE (n=3 experiments). (I) RNA-IP showing the effect of parkin mutation on C-I 30 mRNA binding by Glo. (J) Effect of PINK1 and parkin mutations on Glo ubiquitination in fly muscle. Glo IP from control or mutant tissues was probed with anti-Ub. Red arrow: Glo; Green arrow: Ub-Glo. (K) Effects of WT and mutant forms of hParkin on hnRNP-F ubiquitination. Hela cells transfected with the indicated constructs were subjected to hnRNP-F IP, followed by WB. *: Ub-hnRNP-F. Left two lanes of the top panel are from a different gel and used to show that the band below Ub-hnRNP-F is non-specific. Error bar: SEM; *, p
    Figure Legend Snippet: Parkin Cooperates with PINK1 to Promote Glo/hnRNP-F Ubiquitination and nRCC mRNA Translation (A) Association of endogenous (left) and exogenous (right) Parkin with Pum-1 in co-IP assays. (B, C) m 7 -GTP sepharose binding showing RNA-dependent association of myc-Parkin (B) and endogenous Parkin (C) with TIC. (D) m 7 -GTP sepharose binding showing impaired association of Parkin (V56E, C212Y) with the TIC. (E) Association of endogenous Parkin with exogenous PABP in co-IP assays. (F) RT-PCR and WB showing the effects of parkin mutation on the MOM-targeting and translation of nRCC mRNAs (n=3 experiments). (G) qRT-PCR showing the effect of parkin mutation on RCC mRNA levels in the total and subcellular fractions (n=3 experiments). (H) RT-PCR and WB showing effects of Parkin or PINK1 OE on the defective MOM-targeting and translation of nRCC mRNAs caused by Pum-m OE (n=3 experiments). (I) RNA-IP showing the effect of parkin mutation on C-I 30 mRNA binding by Glo. (J) Effect of PINK1 and parkin mutations on Glo ubiquitination in fly muscle. Glo IP from control or mutant tissues was probed with anti-Ub. Red arrow: Glo; Green arrow: Ub-Glo. (K) Effects of WT and mutant forms of hParkin on hnRNP-F ubiquitination. Hela cells transfected with the indicated constructs were subjected to hnRNP-F IP, followed by WB. *: Ub-hnRNP-F. Left two lanes of the top panel are from a different gel and used to show that the band below Ub-hnRNP-F is non-specific. Error bar: SEM; *, p

    Techniques Used: Co-Immunoprecipitation Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Mutagenesis, Quantitative RT-PCR, Transfection, Construct

    PINK1 Regulates mRNA Localization and Protein Expression of Select nRCC Genes in Mammalian Cells (A) Semi-quantitative RT-PCR of mito-bound nRCC mRNAs in HEK293 cells with hPINK1 . Bar graph shows normalized mito-bound nRCC mRNA levels (n=3 experiments). (B) qRT-PCR of RCC mRNAs in total and subcellular fractions of HEK293 cells transfected with PINK1-WT or PINK1-G309D (n=3 experiments). (C) WB of RCC proteins in HEK293 cells transfected with PINK1-WT or PINK1-G309D. (D) Mitochondrial morphology in fibroblasts or iDNs from control subject (WT) and PINK1(G309D) patient. (E) Quantification of mito size in the periphery of fibroblasts and iDNs (left), and signal intensity ratios of mito-GFP versus Tuj-1 in iDN neurites (right). For each sample at least 4 cells were analyzed (* p
    Figure Legend Snippet: PINK1 Regulates mRNA Localization and Protein Expression of Select nRCC Genes in Mammalian Cells (A) Semi-quantitative RT-PCR of mito-bound nRCC mRNAs in HEK293 cells with hPINK1 . Bar graph shows normalized mito-bound nRCC mRNA levels (n=3 experiments). (B) qRT-PCR of RCC mRNAs in total and subcellular fractions of HEK293 cells transfected with PINK1-WT or PINK1-G309D (n=3 experiments). (C) WB of RCC proteins in HEK293 cells transfected with PINK1-WT or PINK1-G309D. (D) Mitochondrial morphology in fibroblasts or iDNs from control subject (WT) and PINK1(G309D) patient. (E) Quantification of mito size in the periphery of fibroblasts and iDNs (left), and signal intensity ratios of mito-GFP versus Tuj-1 in iDN neurites (right). For each sample at least 4 cells were analyzed (* p

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

    Involvement of Pum and TOM/TIM Complex in the MOM Localization and Translation of Select nRCC mRNAs (A) WB showing the presence of Pum in the mito fraction of Mhc-Gal4 > Pum-WT muscle. C-I 30 serves as mito marker and Actin as loading control. (B) Effects of PINK1 mutation on the MOM recruitment of Pum. (C) RT-PCR and WB showing the effect of Pum RNAi on the MOM localization and protein expression of nRCC mRNAs (n=3 experiments). (D) qRT-PCR showing the effect of Pum RNAi on RCC mRNA levels in the total and subcellular fractions (n=3–4 experiments). (E) RT-PCR and WB showing the effects of Pum, Tom40, or Tim8 RNAi on the defective MOM localization and translation of nRCC mRNAs in PINK1 RNAi flies (n=3 experiments). (F, G, H) Effects of Pum, TOM40, or TIM8 RNAi on the wing posture, jumping ability, ATP level, and DN loss (F), and on the mito aggregation defect caused by PINK1 mutation in flight muscle (G) or DNs (H) (n=3 experiments, 20–25 flies per genotype per experiment; For DN counting, 7–8 animals were assayed). Scale bar (G): 25μm. (I, J) Effects of Pum-1 RNAi on C-I 30 protein expression (I) and ATP level (J) in control and PINK1(G309D) iDNs (n=3 experiments). Error bar: SEM; *, p
    Figure Legend Snippet: Involvement of Pum and TOM/TIM Complex in the MOM Localization and Translation of Select nRCC mRNAs (A) WB showing the presence of Pum in the mito fraction of Mhc-Gal4 > Pum-WT muscle. C-I 30 serves as mito marker and Actin as loading control. (B) Effects of PINK1 mutation on the MOM recruitment of Pum. (C) RT-PCR and WB showing the effect of Pum RNAi on the MOM localization and protein expression of nRCC mRNAs (n=3 experiments). (D) qRT-PCR showing the effect of Pum RNAi on RCC mRNA levels in the total and subcellular fractions (n=3–4 experiments). (E) RT-PCR and WB showing the effects of Pum, Tom40, or Tim8 RNAi on the defective MOM localization and translation of nRCC mRNAs in PINK1 RNAi flies (n=3 experiments). (F, G, H) Effects of Pum, TOM40, or TIM8 RNAi on the wing posture, jumping ability, ATP level, and DN loss (F), and on the mito aggregation defect caused by PINK1 mutation in flight muscle (G) or DNs (H) (n=3 experiments, 20–25 flies per genotype per experiment; For DN counting, 7–8 animals were assayed). Scale bar (G): 25μm. (I, J) Effects of Pum-1 RNAi on C-I 30 protein expression (I) and ATP level (J) in control and PINK1(G309D) iDNs (n=3 experiments). Error bar: SEM; *, p

    Techniques Used: Western Blot, Marker, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

    Effects of Pum Overexpression and a TOM20-PINK1 Complex on the Localization and Translation of nRCC mRNAs (A) Effects of Pum OE on wing posture, jumping ability, and ATP levels. Weak (Pum-w) and moderate (Pum-m) Tg lines were used (n=3 experiments, 20–25 flies per genotype per experiment). (B) RT-PCR and WB showing the effect of Pum-m OE on the MOM localization and translation of nRCC mRNAs (n=3 experiments). (C) qRT-PCR showing the effect of Pum-m OE on RCC mRNA levels in the total and subcellular fractions (n=3 experiments). (D) Effects of Pum-w and Pum-m OE on mito morphology and survival of DNs (n=7–8 flies). Scale bar: 5μm. (E) RNA-dependent interaction of Tom20 with PINK1 or Pum as detected by co-IP using fly muscle extracts. (F) Tom20 interacting proteins as detected by co-IP. Extracts made from CCCP-treated HEK293 cells were mock treated or treated with RNase A before IP. *: Pum-1 signal. (G) RT-PCR and WB showing the effect of Tom20 RNAi on the MOM localization and translation of nRCC mRNAs in fly muscle (n=3 experiments). (H) qRT-PCR showing the effect of Tom20 RNAi on RCC mRNA levels in the total and subcellular fractions (n=3–4 experiments). (I) RT-PCR and WB showing the effect of Tom20 RNAi on the defective localization and translation of nRCC mRNAs in PINK1 mutant (n=3 experiments). Error bar: SEM; *, p
    Figure Legend Snippet: Effects of Pum Overexpression and a TOM20-PINK1 Complex on the Localization and Translation of nRCC mRNAs (A) Effects of Pum OE on wing posture, jumping ability, and ATP levels. Weak (Pum-w) and moderate (Pum-m) Tg lines were used (n=3 experiments, 20–25 flies per genotype per experiment). (B) RT-PCR and WB showing the effect of Pum-m OE on the MOM localization and translation of nRCC mRNAs (n=3 experiments). (C) qRT-PCR showing the effect of Pum-m OE on RCC mRNA levels in the total and subcellular fractions (n=3 experiments). (D) Effects of Pum-w and Pum-m OE on mito morphology and survival of DNs (n=7–8 flies). Scale bar: 5μm. (E) RNA-dependent interaction of Tom20 with PINK1 or Pum as detected by co-IP using fly muscle extracts. (F) Tom20 interacting proteins as detected by co-IP. Extracts made from CCCP-treated HEK293 cells were mock treated or treated with RNase A before IP. *: Pum-1 signal. (G) RT-PCR and WB showing the effect of Tom20 RNAi on the MOM localization and translation of nRCC mRNAs in fly muscle (n=3 experiments). (H) qRT-PCR showing the effect of Tom20 RNAi on RCC mRNA levels in the total and subcellular fractions (n=3–4 experiments). (I) RT-PCR and WB showing the effect of Tom20 RNAi on the defective localization and translation of nRCC mRNAs in PINK1 mutant (n=3 experiments). Error bar: SEM; *, p

    Techniques Used: Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Mutagenesis

    PINK1 Regulates mRNA Localization and Protein Expression of Select nRCC Genes in Drosophila (A) RT-PCR and western blot (WB) analyses of RCC mRNAs and proteins in PINK1 thoracic muscle. mRNAs or proteins prepared from total, cyto, or mito fractions were used. Mito-bound nRCC mRNA levels were normalized with Porin protein level, total and cyto mRNA levels with rps49 , and protein levels with actin (n=3 experiments). Since the expression of the mtRCC gene C-IV s1 is not altered by PINK1, it is used later to normalize RNA and protein levels of nRCC genes. (B) RT-PCR and WB of RCC mRNAs and proteins in PINK1 head tissues. Experiments were done as in A (n=3 experiments). (C) qRT-PCR of RCC mRNAs in total and subcellular fractions from PINK1 muscle tissues (n=3–4 experiments). (D, E) RNA FISH of nRCC mRNAs and control marf mRNA in wild type muscle. Mito and nuclei were labelled with mito-GFP and TOPRO3, respectively. Arrows: RNA particles colocalizing (white) or not colocalizing (red) with mito. Scale bar: 25μm. Bar graph in E shows quantification of RNA signals colocalizing with mito (150 signals from 3 individual animals). Error bar: SEM; *, p
    Figure Legend Snippet: PINK1 Regulates mRNA Localization and Protein Expression of Select nRCC Genes in Drosophila (A) RT-PCR and western blot (WB) analyses of RCC mRNAs and proteins in PINK1 thoracic muscle. mRNAs or proteins prepared from total, cyto, or mito fractions were used. Mito-bound nRCC mRNA levels were normalized with Porin protein level, total and cyto mRNA levels with rps49 , and protein levels with actin (n=3 experiments). Since the expression of the mtRCC gene C-IV s1 is not altered by PINK1, it is used later to normalize RNA and protein levels of nRCC genes. (B) RT-PCR and WB of RCC mRNAs and proteins in PINK1 head tissues. Experiments were done as in A (n=3 experiments). (C) qRT-PCR of RCC mRNAs in total and subcellular fractions from PINK1 muscle tissues (n=3–4 experiments). (D, E) RNA FISH of nRCC mRNAs and control marf mRNA in wild type muscle. Mito and nuclei were labelled with mito-GFP and TOPRO3, respectively. Arrows: RNA particles colocalizing (white) or not colocalizing (red) with mito. Scale bar: 25μm. Bar graph in E shows quantification of RNA signals colocalizing with mito (150 signals from 3 individual animals). Error bar: SEM; *, p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Fluorescence In Situ Hybridization

    19) Product Images from "Presence and Persistence of Salmonella enterica Serotype Typhimurium in the Phyllosphere and Rhizosphere of Spray-Irrigated Parsley"

    Article Title: Presence and Persistence of Salmonella enterica Serotype Typhimurium in the Phyllosphere and Rhizosphere of Spray-Irrigated Parsley

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00087-12

    Implementation of qRT-PCR with or without enrichment for detection of Salmonella in plants irrigated with low levels of bacteria.
    Figure Legend Snippet: Implementation of qRT-PCR with or without enrichment for detection of Salmonella in plants irrigated with low levels of bacteria.

    Techniques Used: Quantitative RT-PCR

    20) Product Images from "miRNA-34c regulates Notch signaling during bone development"

    Article Title: miRNA-34c regulates Notch signaling during bone development

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/dds129

    miR-34c inhibits osteoblast differentiation and increases osteoclastogenesis. ( A ) Induction of miR-34c was quantified by qRT–PCR after osteoblast differentiation from both WT and miR-34c Tg BMSC ( n = 5 per group). * P
    Figure Legend Snippet: miR-34c inhibits osteoblast differentiation and increases osteoclastogenesis. ( A ) Induction of miR-34c was quantified by qRT–PCR after osteoblast differentiation from both WT and miR-34c Tg BMSC ( n = 5 per group). * P

    Techniques Used: Quantitative RT-PCR

    21) Product Images from "Targeting polyIC to EGFR over-expressing cells using a dsRNA binding protein domain tethered to EGF"

    Article Title: Targeting polyIC to EGFR over-expressing cells using a dsRNA binding protein domain tethered to EGF

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0162321

    PolyIC/dsRBEC induces the expression and secretion of pro-inflammatory cytokines in MDA-MB-468 cells. A) qRT-PCR analysis of IFN-β, CCL5, IP10 and TNFα mRNA expression following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 2 and 4 hours. Data were normalized to GAPDH and are expressed as fold change relative to vehicle-treated samples. A representative experiment out of 3 experiments is shown. Error bars represent RQ max. B) Protein levels of IFN-β, CCL5, IP10 and TNFα were measured by ELISA following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 24 hours. Values are averages of triplicate biological samples from one representative experiment. (***,P
    Figure Legend Snippet: PolyIC/dsRBEC induces the expression and secretion of pro-inflammatory cytokines in MDA-MB-468 cells. A) qRT-PCR analysis of IFN-β, CCL5, IP10 and TNFα mRNA expression following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 2 and 4 hours. Data were normalized to GAPDH and are expressed as fold change relative to vehicle-treated samples. A representative experiment out of 3 experiments is shown. Error bars represent RQ max. B) Protein levels of IFN-β, CCL5, IP10 and TNFα were measured by ELISA following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 24 hours. Values are averages of triplicate biological samples from one representative experiment. (***,P

    Techniques Used: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    22) Product Images from "Possible contribution of pannexin-1 to ATP release in human upper airway epithelia"

    Article Title: Possible contribution of pannexin-1 to ATP release in human upper airway epithelia

    Journal: Physiological Reports

    doi: 10.1002/phy2.227

    qRT‐PCR
    Figure Legend Snippet: qRT‐PCR

    Techniques Used: Quantitative RT-PCR

    23) 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

    24) Product Images from "Detection of Zika virus in mouse mammary gland and breast milk"

    Article Title: Detection of Zika virus in mouse mammary gland and breast milk

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0007080

    ZIKV replication in mammary glands of AG129 mice. Postpartum 8-week-old AG129 dams were retro-orbitally inoculated with 1 x 10 2 FFU of ZIKV FSS13025 or 10% FBS-PBS as Mock within 24 hours of parturition. (A-D) Viral titers in mammary gland, brain, spleen and serum were determined via qRT-PCR at 5, 7, 9 and 11 days post infection (dpi). (E) Levels of infectious ZIKV in mammary gland were determined by FFA at 5, 7, 9 and 11 dpi. Negative controls were evaluated at 5 days after mock-infection (A-E). n = 3 mice per time point in each panel. Data represent two independent experiments.
    Figure Legend Snippet: ZIKV replication in mammary glands of AG129 mice. Postpartum 8-week-old AG129 dams were retro-orbitally inoculated with 1 x 10 2 FFU of ZIKV FSS13025 or 10% FBS-PBS as Mock within 24 hours of parturition. (A-D) Viral titers in mammary gland, brain, spleen and serum were determined via qRT-PCR at 5, 7, 9 and 11 days post infection (dpi). (E) Levels of infectious ZIKV in mammary gland were determined by FFA at 5, 7, 9 and 11 dpi. Negative controls were evaluated at 5 days after mock-infection (A-E). n = 3 mice per time point in each panel. Data represent two independent experiments.

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

    Transmission of ZIKV RNA in mouse breast milk. Postpartum AG129 dams were retro-orbitally inoculated with 1 x 10 2 FFU of ZIKV FSS13025 or 10% FBS-PBS as Mock within 24 hours of parturition. Pups were sacrificed on d1, d3, d5 and d7 after birth. (A and B) Levels of ZIKV RNA in the head and the rest of the body were measured by qRT-PCR (n = 6 mice, each day). (C and D) ZIKV RNA levels in stomach milk clots (SMC) and stomach tissues were quantified via qRT-PCR (n = 3 mice, each day). (E and F) Presence of infectious ZIKV in SMC and stomach was assessed using FFA (n = 3 mice, each day). Data represent two independent experiments.
    Figure Legend Snippet: Transmission of ZIKV RNA in mouse breast milk. Postpartum AG129 dams were retro-orbitally inoculated with 1 x 10 2 FFU of ZIKV FSS13025 or 10% FBS-PBS as Mock within 24 hours of parturition. Pups were sacrificed on d1, d3, d5 and d7 after birth. (A and B) Levels of ZIKV RNA in the head and the rest of the body were measured by qRT-PCR (n = 6 mice, each day). (C and D) ZIKV RNA levels in stomach milk clots (SMC) and stomach tissues were quantified via qRT-PCR (n = 3 mice, each day). (E and F) Presence of infectious ZIKV in SMC and stomach was assessed using FFA (n = 3 mice, each day). Data represent two independent experiments.

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

    25) Product Images from "miRNA-132-3p inhibits osteoblast differentiation by targeting Ep300 in simulated microgravity"

    Article Title: miRNA-132-3p inhibits osteoblast differentiation by targeting Ep300 in simulated microgravity

    Journal: Scientific Reports

    doi: 10.1038/srep18655

    miR-132-3p inhibits osteoblast differentiation in vitro . To study the effects of miR-132-3p on osteoblast differentiation, prOB cells were transfected with either a miR-132-3p mimic (miR-mimic, 60 nM), an inhibitor (anti-miR, 80 nM) or its homologous miRNA negative control (miR-N.C.). ( a ) Representative fluorescent images of prOB cells transfected with a miRNA nucleoside analogue for 24 h. The upper panel shows fluorescence in a dark field, and the lower panel shows the same cells in a bright field (original magnification 200×). ( b,c ) Osteoblast differentiation was confirmed by qRT-PCR analysis of osteoblast marker genes (Runx2, Osx and ALP normalized to GAPDH) and activity analysis of the ALP protein at 48 h. (n = 3). ( d,e ) Western blot analyses of Runx2 and Osx protein expression were performed and quantified using ImageJ software. (n = 4). For each group, values are mean ± SD, * P
    Figure Legend Snippet: miR-132-3p inhibits osteoblast differentiation in vitro . To study the effects of miR-132-3p on osteoblast differentiation, prOB cells were transfected with either a miR-132-3p mimic (miR-mimic, 60 nM), an inhibitor (anti-miR, 80 nM) or its homologous miRNA negative control (miR-N.C.). ( a ) Representative fluorescent images of prOB cells transfected with a miRNA nucleoside analogue for 24 h. The upper panel shows fluorescence in a dark field, and the lower panel shows the same cells in a bright field (original magnification 200×). ( b,c ) Osteoblast differentiation was confirmed by qRT-PCR analysis of osteoblast marker genes (Runx2, Osx and ALP normalized to GAPDH) and activity analysis of the ALP protein at 48 h. (n = 3). ( d,e ) Western blot analyses of Runx2 and Osx protein expression were performed and quantified using ImageJ software. (n = 4). For each group, values are mean ± SD, * P

    Techniques Used: In Vitro, Transfection, Negative Control, Fluorescence, Quantitative RT-PCR, Marker, ALP Assay, Activity Assay, Western Blot, Expressing, Software

    Up-regulation of miR-132-3p both in the bone tissue of HU rat femurs and prOB cells cultured in simulated microgravity. ( a ) Alterations of miRNA expression in the femur bone tissue of CON and HU rats examined by Agilent miRNA arrays. Green represents down-regulated miRNAs, and red represents up-regulated miRNAs, with the color scale in the upper right corner indicating the relative expression levels. ( b ) qRT-PCR analysis of miR-139-3p, -339-3p, -132-3p, -487b, -2985 and -34b levels partly selected from the array data (normalized to internal reference U6). ( c ) Expression levels of miR-139-3p, -339-3p, -132-3p, -487b, -2985 and -34b in prOB cells after exposure to clinorotation (Clino) for 48 h examined by qRT-PCR. The relative ratio is shown with that of cells under static control conditions. ( d ) Time course of miR-132-3p expression in prOB cells after exposure to clinorotation. Fold-increase is provided in comparison to the static control group. For each group, values are mean ± SD, n = 3, * P
    Figure Legend Snippet: Up-regulation of miR-132-3p both in the bone tissue of HU rat femurs and prOB cells cultured in simulated microgravity. ( a ) Alterations of miRNA expression in the femur bone tissue of CON and HU rats examined by Agilent miRNA arrays. Green represents down-regulated miRNAs, and red represents up-regulated miRNAs, with the color scale in the upper right corner indicating the relative expression levels. ( b ) qRT-PCR analysis of miR-139-3p, -339-3p, -132-3p, -487b, -2985 and -34b levels partly selected from the array data (normalized to internal reference U6). ( c ) Expression levels of miR-139-3p, -339-3p, -132-3p, -487b, -2985 and -34b in prOB cells after exposure to clinorotation (Clino) for 48 h examined by qRT-PCR. The relative ratio is shown with that of cells under static control conditions. ( d ) Time course of miR-132-3p expression in prOB cells after exposure to clinorotation. Fold-increase is provided in comparison to the static control group. For each group, values are mean ± SD, n = 3, * P

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

    miR-132-3p directly inhibits Ep300 protein expression in prOB cells exposed to a simulated microgravity environment. ( a ) Schematic representation of luciferase constructs used for reporter assays. The two miR-132-3p target sites within the 3′ UTR of Ep300 are depicted as black boxes. Sequences below indicate putative miR-132-3p target sites on the wild type 3′ UTR, the mutated derivative, and the pairing regions of miR-132-3p. ( b ) The effect of the miR-132-3p mimic, the inhibitor or their negative controls on the luciferase activity of the WT Ep300 3′ UTR or the MUT Ep300 3′ UTR reporter in 293T cells. (n = 3). ( c,d ) Ep300 mRNA (n = 3) and protein expression (n = 4) in prOB cells were examined after transfection with miR-N.C. and miR-mimic for 48 h by qRT-PCR and western blot, respectively. ( e,f ) Ep300 mRNA and protein expression was detected by qRT-PCR and western blot, respectively, after exposure to clinorotation for 48 h. (n = 3). For each group, values are mean ± SD, * P
    Figure Legend Snippet: miR-132-3p directly inhibits Ep300 protein expression in prOB cells exposed to a simulated microgravity environment. ( a ) Schematic representation of luciferase constructs used for reporter assays. The two miR-132-3p target sites within the 3′ UTR of Ep300 are depicted as black boxes. Sequences below indicate putative miR-132-3p target sites on the wild type 3′ UTR, the mutated derivative, and the pairing regions of miR-132-3p. ( b ) The effect of the miR-132-3p mimic, the inhibitor or their negative controls on the luciferase activity of the WT Ep300 3′ UTR or the MUT Ep300 3′ UTR reporter in 293T cells. (n = 3). ( c,d ) Ep300 mRNA (n = 3) and protein expression (n = 4) in prOB cells were examined after transfection with miR-N.C. and miR-mimic for 48 h by qRT-PCR and western blot, respectively. ( e,f ) Ep300 mRNA and protein expression was detected by qRT-PCR and western blot, respectively, after exposure to clinorotation for 48 h. (n = 3). For each group, values are mean ± SD, * P

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

    Down-regulation of endogenous miR-132-3p expression partly attenuates inhibition of osteoblast differentiation by clinorotation in vitro. prOB cells were transfected with miR-N.C. and anti-miR for 12 h and then exposed to clinorotation for 48 h. The relative parameter was detected. ( a ) miR-132-3p expression in prOB cells was analyzed by q RT-PCR. (n = 3). ( b ) Osteoblast differentiation was confirmed by qRT-PCR analysis of osteoblast marker genes (Runx2 and Osx normalized to GAPDH). (n = 3). ( c ) Western blot analysis of Runx2 and Osx protein expression was performed and quantified using ImageJ software. (n = 4). ( d ) ALP gene expression and protein activity were measured. (n = 3). For each group, values are mean ± SD, * P
    Figure Legend Snippet: Down-regulation of endogenous miR-132-3p expression partly attenuates inhibition of osteoblast differentiation by clinorotation in vitro. prOB cells were transfected with miR-N.C. and anti-miR for 12 h and then exposed to clinorotation for 48 h. The relative parameter was detected. ( a ) miR-132-3p expression in prOB cells was analyzed by q RT-PCR. (n = 3). ( b ) Osteoblast differentiation was confirmed by qRT-PCR analysis of osteoblast marker genes (Runx2 and Osx normalized to GAPDH). (n = 3). ( c ) Western blot analysis of Runx2 and Osx protein expression was performed and quantified using ImageJ software. (n = 4). ( d ) ALP gene expression and protein activity were measured. (n = 3). For each group, values are mean ± SD, * P

    Techniques Used: Expressing, Inhibition, In Vitro, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Western Blot, Software, ALP Assay, Activity Assay

    Inhibition of Ep300 expression by miR-132-3p decreases the stability and acetylation levels of Runx2. ( a ) qRT-PCR analysis of Ep300 gene expression in prOB cells was performed after transfection with siRNA-Ep300. (n = 3). ( b ) Western blot examination of Runx2 protein expression in prOB cells in which Ep300 expression was inhibited by siRNA. (n = 4). ( c ) Levels of acetylated Runx2 were detected by immunoprecipitation using an anti-acetyl-lysine antibody (Ac-lys), followed by a western blot with an anti-Runx2 antibody. (n = 3). For each group, values are mean ± SD, * P
    Figure Legend Snippet: Inhibition of Ep300 expression by miR-132-3p decreases the stability and acetylation levels of Runx2. ( a ) qRT-PCR analysis of Ep300 gene expression in prOB cells was performed after transfection with siRNA-Ep300. (n = 3). ( b ) Western blot examination of Runx2 protein expression in prOB cells in which Ep300 expression was inhibited by siRNA. (n = 4). ( c ) Levels of acetylated Runx2 were detected by immunoprecipitation using an anti-acetyl-lysine antibody (Ac-lys), followed by a western blot with an anti-Runx2 antibody. (n = 3). For each group, values are mean ± SD, * P

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Immunoprecipitation

    26) Product Images from "Vinexin family (SORBS) proteins regulate mechanotransduction in mesenchymal stem cells"

    Article Title: Vinexin family (SORBS) proteins regulate mechanotransduction in mesenchymal stem cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29700-3

    CAP suppressed, but vinexin promoted, adipocyte differentiation. Vinexin- and CAP-depleted ( a – d ) cells, as well as vinexin α re-expressing cells ( e , g , h ) and CAP re-expressing cells ( f , i , j ) were seeded on plastic dishes at a density of 1.2 × 10 5 cells/35 mm dish and induced to differentiate into adipocyte. RNAs were extracted 6 days after MDI treatment, and the mRNAs expression of PPARγ2 and aP2 was quantified by qRT-PCR ( a , e , f ). The expression levels relative to those in control cells are shown. ( b ) Cell were lysed and equal amounts of cell lysates were analyzed by western blotting using the anti-aP2 and anti-vinculin (loading control) antibodies. Blots were cropped from full-size images (see Supplemental Information). ( c , d , g – j ) Cells were stained with Oil Red O on day 6 after MDI treatment and representative images are shown ( c , g , i ). ( d , h , j ) Nine images were obtained from three independent experiments, and the percentage of area stained were quantified using ImageJ. ( k , l ) Control and CAP depleted cells at the density of 1.0 × 10 5 cells/ 6 well plates were infected with lentiviruses expressing GFP-T12 vinculin. Two days after the infection, cells were induced to differentiate into adipocytes. Differentiated adipocytes were stained with Oil Red O on day 6. (l) Twelve images were obtained from two independent experiments, and the percentage of the stained area was quantified using ImageJ. Scale bars: 200 μm. The values represent the mean ± s.e.m. Statistical significance was determined by one-way ANOVA with Tukey’s test or Student’s t-test ( e , f , h , j ). *p
    Figure Legend Snippet: CAP suppressed, but vinexin promoted, adipocyte differentiation. Vinexin- and CAP-depleted ( a – d ) cells, as well as vinexin α re-expressing cells ( e , g , h ) and CAP re-expressing cells ( f , i , j ) were seeded on plastic dishes at a density of 1.2 × 10 5 cells/35 mm dish and induced to differentiate into adipocyte. RNAs were extracted 6 days after MDI treatment, and the mRNAs expression of PPARγ2 and aP2 was quantified by qRT-PCR ( a , e , f ). The expression levels relative to those in control cells are shown. ( b ) Cell were lysed and equal amounts of cell lysates were analyzed by western blotting using the anti-aP2 and anti-vinculin (loading control) antibodies. Blots were cropped from full-size images (see Supplemental Information). ( c , d , g – j ) Cells were stained with Oil Red O on day 6 after MDI treatment and representative images are shown ( c , g , i ). ( d , h , j ) Nine images were obtained from three independent experiments, and the percentage of area stained were quantified using ImageJ. ( k , l ) Control and CAP depleted cells at the density of 1.0 × 10 5 cells/ 6 well plates were infected with lentiviruses expressing GFP-T12 vinculin. Two days after the infection, cells were induced to differentiate into adipocytes. Differentiated adipocytes were stained with Oil Red O on day 6. (l) Twelve images were obtained from two independent experiments, and the percentage of the stained area was quantified using ImageJ. Scale bars: 200 μm. The values represent the mean ± s.e.m. Statistical significance was determined by one-way ANOVA with Tukey’s test or Student’s t-test ( e , f , h , j ). *p

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

    CAP promoted osteoblast differentiation and vinexin inhibited calcification. Vinexin- or CAP-depleted ( a , b , c , j , k ) cells and vinexin α re-expressing cells ( d , f , h ) or CAP re-expressing cells seeded on plastic dishes were induced to differentiate into osteoblasts by incubating them with αMEM medium. ( a , d , e ) On day 6, RNAs were extracted and the expression of the ALP mRNA was quantified by qRT-PCR. The relative expression compared to control cells is shown from triplicate experiment. ( b , c , f – i ) On day 4, ALP activity was visualized and six images from each condition were obtained from two independent experiments. Representative images are shown. Scale bars: 400 μm. ( c , h , i ) Total intensity was quantified using ImageJ. ( j , k ) On day 6, cells were stained with Alizarin Red S and six images were obtained from two independent experiments. Representative images are shown. Scale bars: 200 μm. ( k ) The relative stained area was quantified using ImageJ. Values represent the mean ± s.e.m. Statistical significance was determined by one-way ANOVA with Tukey’s test ( a , c , k ) and Student’s t-test ( d , e , h , i ). ***p
    Figure Legend Snippet: CAP promoted osteoblast differentiation and vinexin inhibited calcification. Vinexin- or CAP-depleted ( a , b , c , j , k ) cells and vinexin α re-expressing cells ( d , f , h ) or CAP re-expressing cells seeded on plastic dishes were induced to differentiate into osteoblasts by incubating them with αMEM medium. ( a , d , e ) On day 6, RNAs were extracted and the expression of the ALP mRNA was quantified by qRT-PCR. The relative expression compared to control cells is shown from triplicate experiment. ( b , c , f – i ) On day 4, ALP activity was visualized and six images from each condition were obtained from two independent experiments. Representative images are shown. Scale bars: 400 μm. ( c , h , i ) Total intensity was quantified using ImageJ. ( j , k ) On day 6, cells were stained with Alizarin Red S and six images were obtained from two independent experiments. Representative images are shown. Scale bars: 200 μm. ( k ) The relative stained area was quantified using ImageJ. Values represent the mean ± s.e.m. Statistical significance was determined by one-way ANOVA with Tukey’s test ( a , c , k ) and Student’s t-test ( d , e , h , i ). ***p

    Techniques Used: Expressing, ALP Assay, Quantitative RT-PCR, Activity Assay, Staining

    Vinexin and CAP regulated differentiation in an ECM stiffness-dependent manner. ( a , b ) Vinexin- or CAP-depleted cells were seeded onto PAA gels coated with collagen and induced to differentiate into adipocytes. Cells were stained with Oil Red O on day 6 after MDI treatment and representative images are shown. Scale bars: 200 μm. ( b ) Eight images were obtained from two independent experiments, and the percentage of the stained area was quantified using ImageJ. ( c , d ) CAP-depleted cells were transfected with negative control (siNC) or TAZ-targeted siRNA. ( c ) TAZ expression was analyzed with western blotting. Blots were cropped from full-size images (see Supplemental Information). ( d ) On day 6 after siRNA transfection, the expression of aP2 and ALP mRNA was quantified by qRT-PCR. The expression relative to control cells transfected with negative control siRNA from triplicate experiments is shown. Values represent the mean ± s.e.m. Each experiment was repeated twice. Statistical significance was determined by one-way ANOVA with Tukey’s test. **p
    Figure Legend Snippet: Vinexin and CAP regulated differentiation in an ECM stiffness-dependent manner. ( a , b ) Vinexin- or CAP-depleted cells were seeded onto PAA gels coated with collagen and induced to differentiate into adipocytes. Cells were stained with Oil Red O on day 6 after MDI treatment and representative images are shown. Scale bars: 200 μm. ( b ) Eight images were obtained from two independent experiments, and the percentage of the stained area was quantified using ImageJ. ( c , d ) CAP-depleted cells were transfected with negative control (siNC) or TAZ-targeted siRNA. ( c ) TAZ expression was analyzed with western blotting. Blots were cropped from full-size images (see Supplemental Information). ( d ) On day 6 after siRNA transfection, the expression of aP2 and ALP mRNA was quantified by qRT-PCR. The expression relative to control cells transfected with negative control siRNA from triplicate experiments is shown. Values represent the mean ± s.e.m. Each experiment was repeated twice. Statistical significance was determined by one-way ANOVA with Tukey’s test. **p

    Techniques Used: Staining, Transfection, Negative Control, Expressing, Western Blot, ALP Assay, Quantitative RT-PCR

    27) Product Images from "Autophagy is a gatekeeper of hepatic differentiation and carcinogenesis by controlling the degradation of Yap"

    Article Title: Autophagy is a gatekeeper of hepatic differentiation and carcinogenesis by controlling the degradation of Yap

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07338-z

    Yap-dependent proliferation in livers with impaired autophagy is potentially druggable. a Controls ( Atg7 F/F ) and Atg7 KO ( Alb-CRE:Atg7 F/F ) mice were treated for 21 days with either Yap/Tead inhibitor verteporfin or vehicle. N = 4 per group. VP, verteporfin; Veh, vehicle. b Immunostaining for Ki67 in liver sections from control and Atg7 KO mice treated with either verteporfin or vehicle. Scale bar 100 µm. Large inserts are enlargements of small inserts. c Quantitation of Ki67 + nuclei per 100x field per mouse treated with either verteporfin or vehicle. Ten 100x fields per mouse were analyzed. d qRT-PCR analysis of whole liver RNA from control and Atg7 KO mice, treated with verteporfin or vehicle. Cyr61 mRNA expression was normalized to Gapdh expression and then normalized to control animals. Data from two independent experiments. e [ 3 H]-thymidine incorporation assay in scram- and shAtg7-AML12 cells after incubation with verteporfin or vehicle. Data from three independent experiments, measurements in triplicates. **** P = 0.0003. f Tead4-Luciferase assay of scram- and shAtg7-infected AML12 cells after incubation with verteporfin or vehicle. Data from two independent experiments Data represent mean ± SD. P -values analyzed by two-way ANOVA and Tukey’s HSD. * P
    Figure Legend Snippet: Yap-dependent proliferation in livers with impaired autophagy is potentially druggable. a Controls ( Atg7 F/F ) and Atg7 KO ( Alb-CRE:Atg7 F/F ) mice were treated for 21 days with either Yap/Tead inhibitor verteporfin or vehicle. N = 4 per group. VP, verteporfin; Veh, vehicle. b Immunostaining for Ki67 in liver sections from control and Atg7 KO mice treated with either verteporfin or vehicle. Scale bar 100 µm. Large inserts are enlargements of small inserts. c Quantitation of Ki67 + nuclei per 100x field per mouse treated with either verteporfin or vehicle. Ten 100x fields per mouse were analyzed. d qRT-PCR analysis of whole liver RNA from control and Atg7 KO mice, treated with verteporfin or vehicle. Cyr61 mRNA expression was normalized to Gapdh expression and then normalized to control animals. Data from two independent experiments. e [ 3 H]-thymidine incorporation assay in scram- and shAtg7-AML12 cells after incubation with verteporfin or vehicle. Data from three independent experiments, measurements in triplicates. **** P = 0.0003. f Tead4-Luciferase assay of scram- and shAtg7-infected AML12 cells after incubation with verteporfin or vehicle. Data from two independent experiments Data represent mean ± SD. P -values analyzed by two-way ANOVA and Tukey’s HSD. * P

    Techniques Used: Mouse Assay, Immunostaining, Quantitation Assay, Quantitative RT-PCR, Expressing, Thymidine Incorporation Assay, Incubation, Luciferase, Infection

    Intact Nrf2 signaling in Atg7/Yap DKO mice; Atg7/Nrf2 DKO mice do not have evidence of increased Yap activation. a Analysis of control ( Atg7 F/F and Atg7 F/F ,Yap F/F ), Atg7 KO ( ERT2-Alb-CRE:Atg7 F/F ), Atg7/Yap DKO ( ERT2-Alb-CRE:Atg7 F/F , Yap F/F ) 4 weeks post TAM injection, Atg7/Nrf2 DKO ( Alb-CRE:Atg7 F/F , Nrf2 −/− ) and Nrf2 KO (Nrf2 − / − ) at 9 weeks of age. b Immunoblot analysis of whole liver lysate from control, Atg7 KO, Atg7/Yap DKO, Atg7/Nrf2 DKO, and Nrf2 KO mice. Immunoblotting for Atg7, LC3, Yap, Nrf2, p62/Sqstm1, β-Tubulin. c qRT-PCR analysis of whole liver RNA for Nqo , Srxn1, Cyr61 , Areg . Data normalized to β-actin expression and fold control. Data from 3 ( Cyr61 , Nqo , Areg ) and 2 ( Srxn1 ) independent experiments, respectively. N = 3–4 animals per group. Mean ± SD. **** P
    Figure Legend Snippet: Intact Nrf2 signaling in Atg7/Yap DKO mice; Atg7/Nrf2 DKO mice do not have evidence of increased Yap activation. a Analysis of control ( Atg7 F/F and Atg7 F/F ,Yap F/F ), Atg7 KO ( ERT2-Alb-CRE:Atg7 F/F ), Atg7/Yap DKO ( ERT2-Alb-CRE:Atg7 F/F , Yap F/F ) 4 weeks post TAM injection, Atg7/Nrf2 DKO ( Alb-CRE:Atg7 F/F , Nrf2 −/− ) and Nrf2 KO (Nrf2 − / − ) at 9 weeks of age. b Immunoblot analysis of whole liver lysate from control, Atg7 KO, Atg7/Yap DKO, Atg7/Nrf2 DKO, and Nrf2 KO mice. Immunoblotting for Atg7, LC3, Yap, Nrf2, p62/Sqstm1, β-Tubulin. c qRT-PCR analysis of whole liver RNA for Nqo , Srxn1, Cyr61 , Areg . Data normalized to β-actin expression and fold control. Data from 3 ( Cyr61 , Nqo , Areg ) and 2 ( Srxn1 ) independent experiments, respectively. N = 3–4 animals per group. Mean ± SD. **** P

    Techniques Used: Mouse Assay, Activation Assay, Injection, Quantitative RT-PCR, Expressing

    28) Product Images from "Delta-Opioid Receptor Analgesia Is Independent of Microglial Activation in a Rat Model of Neuropathic Pain"

    Article Title: Delta-Opioid Receptor Analgesia Is Independent of Microglial Activation in a Rat Model of Neuropathic Pain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0104420

    MOR , DOR , KOR mRNAs in spinal cord and DRGs in vehicle- and minocycline-treated CCI-exposed rats. Minocycline (MC; 30 mg/kg; i.p.) was administered intraperitoneally pre-emptively 16 h and 1 h before CCI, and then repeatedly twice daily for 7 days. On the seventh day, spinal cords (L4–L6) and DRG were collected for the qRT-PCR analysis of MOR (A, B), KOR (C, D) and DOR (E, F) gene expression. The data are presented as the means ± SEM and represent the normalised averages derived from the threshold qRT-PCR cycles from four to eight samples for each group. Intergroup differences were analysed using ANOVAs followed by Bonferroni's multiple comparison tests. * P
    Figure Legend Snippet: MOR , DOR , KOR mRNAs in spinal cord and DRGs in vehicle- and minocycline-treated CCI-exposed rats. Minocycline (MC; 30 mg/kg; i.p.) was administered intraperitoneally pre-emptively 16 h and 1 h before CCI, and then repeatedly twice daily for 7 days. On the seventh day, spinal cords (L4–L6) and DRG were collected for the qRT-PCR analysis of MOR (A, B), KOR (C, D) and DOR (E, F) gene expression. The data are presented as the means ± SEM and represent the normalised averages derived from the threshold qRT-PCR cycles from four to eight samples for each group. Intergroup differences were analysed using ANOVAs followed by Bonferroni's multiple comparison tests. * P

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

    29) Product Images from "Characterization of Somatic Embryogenesis Receptor-Like Kinase 4 as a Negative Regulator of Leaf Senescence in Arabidopsis"

    Article Title: Characterization of Somatic Embryogenesis Receptor-Like Kinase 4 as a Negative Regulator of Leaf Senescence in Arabidopsis

    Journal: Cells

    doi: 10.3390/cells8010050

    Expression of SERK4 in the T-DNA insertion line: ( A ) the exon/intron structure of the SERK4 gene and location of the T-DNA insertion; ( B ) the expression level of SERK4 revealed by qRT-PCR in LS leaves of wildtype and the T-DNA insertion line, the ratios of SERK4 gene expression level were calculated relative to the wildtype leaf. CDS, coding sequence; WT, wildtype. The expression data are means ± SD of three biological repeats. *** p
    Figure Legend Snippet: Expression of SERK4 in the T-DNA insertion line: ( A ) the exon/intron structure of the SERK4 gene and location of the T-DNA insertion; ( B ) the expression level of SERK4 revealed by qRT-PCR in LS leaves of wildtype and the T-DNA insertion line, the ratios of SERK4 gene expression level were calculated relative to the wildtype leaf. CDS, coding sequence; WT, wildtype. The expression data are means ± SD of three biological repeats. *** p

    Techniques Used: Expressing, Quantitative RT-PCR, Sequencing

    30) Product Images from "A cotton miRNA is involved in regulation of plant response to salt stress"

    Article Title: A cotton miRNA is involved in regulation of plant response to salt stress

    Journal: Scientific Reports

    doi: 10.1038/srep19736

    Identification of miRNVL5 target from Arabidopsis through degradome sequencing. ( A ) The red line with a dot indicates the significant signature. The arrow indicates the signature produced by miRNA-directed cleavage. ( B ) Target for miRNVL5 identified from degradome of 35S::miRNVL5 Arabidopsis exposed to minus salt (miR5 − S) and plus salt (miR5 + S). Three week-old Arabidopsis seedlings were treated with 0 (control) and 200 mM NaCl for 1 h and the treated seedlings were used for degradome sequencing and qRT-PCR.
    Figure Legend Snippet: Identification of miRNVL5 target from Arabidopsis through degradome sequencing. ( A ) The red line with a dot indicates the significant signature. The arrow indicates the signature produced by miRNA-directed cleavage. ( B ) Target for miRNVL5 identified from degradome of 35S::miRNVL5 Arabidopsis exposed to minus salt (miR5 − S) and plus salt (miR5 + S). Three week-old Arabidopsis seedlings were treated with 0 (control) and 200 mM NaCl for 1 h and the treated seedlings were used for degradome sequencing and qRT-PCR.

    Techniques Used: Sequencing, Produced, Quantitative RT-PCR

    Sodium (Na + )/potassium (K + ) accumulation and expression of SOS1 , NHX1 , AVP1 and P5CS1 in transgenic and wild-type (WT) plants exposed to NaCl. ( A,C ) Na + in shoots and roots. ( B,D ) K + in shoots and roots of three week-old WT, 35S::miRNVLU5 and 35S::mGhCHR transgenic plants exposed to 200 mM NaCl exposure for 3 d. ( E,F ) K + /Na + ratio in WT and the transgenic plants under −NaCl ( E , F ) and +NaCl ( F ) Condition. ( G – J ) qRT-PCR analysis of the indicated gene expression. Three week-old seedlings were exposed to 200 mM NaCl for 1 h. Vertical bars represent SD of the mean with three replicates. Asterisks indicate that mean values are significantly different between the transgenic plants and WT ( p
    Figure Legend Snippet: Sodium (Na + )/potassium (K + ) accumulation and expression of SOS1 , NHX1 , AVP1 and P5CS1 in transgenic and wild-type (WT) plants exposed to NaCl. ( A,C ) Na + in shoots and roots. ( B,D ) K + in shoots and roots of three week-old WT, 35S::miRNVLU5 and 35S::mGhCHR transgenic plants exposed to 200 mM NaCl exposure for 3 d. ( E,F ) K + /Na + ratio in WT and the transgenic plants under −NaCl ( E , F ) and +NaCl ( F ) Condition. ( G – J ) qRT-PCR analysis of the indicated gene expression. Three week-old seedlings were exposed to 200 mM NaCl for 1 h. Vertical bars represent SD of the mean with three replicates. Asterisks indicate that mean values are significantly different between the transgenic plants and WT ( p

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

    31) Product Images from "Puerarin Attenuates Anoxia/Reoxygenation Injury Through Enhancing Bcl-2 Associated Athanogene 3 Expression, a Modulator of Apoptosis and Autophagy"

    Article Title: Puerarin Attenuates Anoxia/Reoxygenation Injury Through Enhancing Bcl-2 Associated Athanogene 3 Expression, a Modulator of Apoptosis and Autophagy

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

    doi: 10.12659/MSM.897379

    Puerarin enhances BAG3 expression in rat primary cardiomyocytes after A/RI. ( A, B ) QRT-PCR analysis ( A ) and Western blot analysis ( B ) of BAG3 expression in rat primary cardiomyocytes without A/RI and in the cells with or without pre-treatment of puerarin (50/100/200 μM) after A/RI. * Comparison with NC; # comparison with A/RI. * and # p
    Figure Legend Snippet: Puerarin enhances BAG3 expression in rat primary cardiomyocytes after A/RI. ( A, B ) QRT-PCR analysis ( A ) and Western blot analysis ( B ) of BAG3 expression in rat primary cardiomyocytes without A/RI and in the cells with or without pre-treatment of puerarin (50/100/200 μM) after A/RI. * Comparison with NC; # comparison with A/RI. * and # p

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

    32) Product Images from "Characterization of Somatic Embryogenesis Receptor-Like Kinase 4 as a Negative Regulator of Leaf Senescence in Arabidopsis"

    Article Title: Characterization of Somatic Embryogenesis Receptor-Like Kinase 4 as a Negative Regulator of Leaf Senescence in Arabidopsis

    Journal: Cells

    doi: 10.3390/cells8010050

    Expression of SERK4 in the T-DNA insertion line: ( A ) the exon/intron structure of the SERK4 gene and location of the T-DNA insertion; ( B ) the expression level of SERK4 revealed by qRT-PCR in LS leaves of wildtype and the T-DNA insertion line, the ratios of SERK4 gene expression level were calculated relative to the wildtype leaf. CDS, coding sequence; WT, wildtype. The expression data are means ± SD of three biological repeats. *** p
    Figure Legend Snippet: Expression of SERK4 in the T-DNA insertion line: ( A ) the exon/intron structure of the SERK4 gene and location of the T-DNA insertion; ( B ) the expression level of SERK4 revealed by qRT-PCR in LS leaves of wildtype and the T-DNA insertion line, the ratios of SERK4 gene expression level were calculated relative to the wildtype leaf. CDS, coding sequence; WT, wildtype. The expression data are means ± SD of three biological repeats. *** p

    Techniques Used: Expressing, Quantitative RT-PCR, Sequencing

    33) Product Images from "Differential Antioxidant Responses and Perturbed Porphyrin Biosynthesis after Exposure to Oxyfluorfen and Methyl Viologen in Oryza sativa"

    Article Title: Differential Antioxidant Responses and Perturbed Porphyrin Biosynthesis after Exposure to Oxyfluorfen and Methyl Viologen in Oryza sativa

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms160716529

    Expression of genes encoding the porphyrin pathway enzymes in rice plants with the foliar application of MV. ( A ) Common branch; ( B ) Mg-porphyrin branch; and ( C ) Fe-porphyrin branch. The plants were subjected to the same treatments as in Figure 2 . Treatment notations are the same as in Figure 2 . Total RNAs were purified from plants and reverse transcribed. The resultant cDNAs were used as templates for qRT-PCR using Actin as an internal control. The control 1 was used for normalization, with the expression level of the sample set to 1. Error bars represent SE, and representative data from three independent experiments are presented.
    Figure Legend Snippet: Expression of genes encoding the porphyrin pathway enzymes in rice plants with the foliar application of MV. ( A ) Common branch; ( B ) Mg-porphyrin branch; and ( C ) Fe-porphyrin branch. The plants were subjected to the same treatments as in Figure 2 . Treatment notations are the same as in Figure 2 . Total RNAs were purified from plants and reverse transcribed. The resultant cDNAs were used as templates for qRT-PCR using Actin as an internal control. The control 1 was used for normalization, with the expression level of the sample set to 1. Error bars represent SE, and representative data from three independent experiments are presented.

    Techniques Used: Expressing, Purification, Quantitative RT-PCR

    Expression of genes encoding the ROS-scavenging enzymes in rice plants with the foliar application of OF or MV treatment. Total RNAs were purified from plants and reverse transcribed. The resultant cDNAs were used as templates for qRT-PCR using Actin as an internal control. The control was used for normalization, with the expression level of the sample set to 1. Error bars represent SE, and representative data from three independent experiments are presented. The plants were subjected to the same treatments as in Figure 2 . Treatment notations are the same as in Figure 2 .
    Figure Legend Snippet: Expression of genes encoding the ROS-scavenging enzymes in rice plants with the foliar application of OF or MV treatment. Total RNAs were purified from plants and reverse transcribed. The resultant cDNAs were used as templates for qRT-PCR using Actin as an internal control. The control was used for normalization, with the expression level of the sample set to 1. Error bars represent SE, and representative data from three independent experiments are presented. The plants were subjected to the same treatments as in Figure 2 . Treatment notations are the same as in Figure 2 .

    Techniques Used: Expressing, Purification, Quantitative RT-PCR

    34) Product Images from "The Mevalonate Pathway of Staphylococcus aureus ▿ ▿ †"

    Article Title: The Mevalonate Pathway of Staphylococcus aureus ▿ ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01357-08

    qRT-PCR analysis.
    Figure Legend Snippet: qRT-PCR analysis.

    Techniques Used: Quantitative RT-PCR

    35) Product Images from "Study on expression of lncRNA RGMB-AS1 and repulsive guidance molecule b in non-small cell lung cancer"

    Article Title: Study on expression of lncRNA RGMB-AS1 and repulsive guidance molecule b in non-small cell lung cancer

    Journal: Diagnostic Pathology

    doi: 10.1186/s13000-015-0297-x

    siRNA of lncRNA RGMB-AS1 promoted the expression of RGMB in A549 and SPC-A-1 cells. a Cells were transfected with siRNA of lncRNA RGMB-AS1 and NC. RGMB mRNA level was detected by qRT-PCR assay. RGMB mRNA expression was upregulated in A549 and SPC-A-1 cells after transfection with siRNA of lncRNA RGMB-AS1 and NC respectively (* P
    Figure Legend Snippet: siRNA of lncRNA RGMB-AS1 promoted the expression of RGMB in A549 and SPC-A-1 cells. a Cells were transfected with siRNA of lncRNA RGMB-AS1 and NC. RGMB mRNA level was detected by qRT-PCR assay. RGMB mRNA expression was upregulated in A549 and SPC-A-1 cells after transfection with siRNA of lncRNA RGMB-AS1 and NC respectively (* P

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    36) Product Images from "Ntann12 annexin expression is induced by auxin in tobacco roots"

    Article Title: Ntann12 annexin expression is induced by auxin in tobacco roots

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/err112

    Ntann12 expression in 4-week-old plants. (A) pNtann12-GUS expression in whole tobacco plant; bar=1 mm. (B) Close-up of (A) showing pNtann12-GUS expression in the root tip; bar=0.5 mm. (C) GUS staining in a longitudinal section of the root; bar=100 μm. (D) Relative expression of Ntann12 in roots and in aerial parts of plants, as determined by qRT-PCR analysis. (E) Western blot analysis of total protein extracts (10 μg) from roots and leaves using anti-Ntann12 antibodies. EZ, elongation zone; L, leaf; M, molecular marker; MZ, maturation zone; RT, root tip; R, root.
    Figure Legend Snippet: Ntann12 expression in 4-week-old plants. (A) pNtann12-GUS expression in whole tobacco plant; bar=1 mm. (B) Close-up of (A) showing pNtann12-GUS expression in the root tip; bar=0.5 mm. (C) GUS staining in a longitudinal section of the root; bar=100 μm. (D) Relative expression of Ntann12 in roots and in aerial parts of plants, as determined by qRT-PCR analysis. (E) Western blot analysis of total protein extracts (10 μg) from roots and leaves using anti-Ntann12 antibodies. EZ, elongation zone; L, leaf; M, molecular marker; MZ, maturation zone; RT, root tip; R, root.

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

    37) Product Images from "HOXA4, down-regulated in lung cancer, inhibits the growth, motility and invasion of lung cancer cells"

    Article Title: HOXA4, down-regulated in lung cancer, inhibits the growth, motility and invasion of lung cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0497-x

    HOXA4 suppressed the Wnt signaling pathway in lung cancer cells. a , b Western blot analysis of GSK3β, β-catenin, Cyclin D1, c-Myc and Survivin in NCI-H1975 and NCI-H446 cells with HOXA4 overexpression ( a ) and NCI-H1299 cells with HOXA4 silencing ( b ). GAPDH was used as a loading control. c , d qRT-PCR analysis of GSK3β mRNA levels in NCI-H1975 and NCI-H446 cells with HOXA4 overexpression ( c ) and NCI-H1299 cells with HOXA4 silencing ( d ). e A luciferase reporter assay was performed to evaluate GSK3β promoter activity in NCI-H1975 and NCI-H446 cells with HOXA4 overexpression or silencing. f ChIP-qPCR for the GSK3β promoter in NCI-H1975 and NCI-H446 cells with HOXA4 overexpression or silencing. *** P
    Figure Legend Snippet: HOXA4 suppressed the Wnt signaling pathway in lung cancer cells. a , b Western blot analysis of GSK3β, β-catenin, Cyclin D1, c-Myc and Survivin in NCI-H1975 and NCI-H446 cells with HOXA4 overexpression ( a ) and NCI-H1299 cells with HOXA4 silencing ( b ). GAPDH was used as a loading control. c , d qRT-PCR analysis of GSK3β mRNA levels in NCI-H1975 and NCI-H446 cells with HOXA4 overexpression ( c ) and NCI-H1299 cells with HOXA4 silencing ( d ). e A luciferase reporter assay was performed to evaluate GSK3β promoter activity in NCI-H1975 and NCI-H446 cells with HOXA4 overexpression or silencing. f ChIP-qPCR for the GSK3β promoter in NCI-H1975 and NCI-H446 cells with HOXA4 overexpression or silencing. *** P

    Techniques Used: Western Blot, Over Expression, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    38) Product Images from "Upregulation of colonic and hepatic tumor overexpressed gene is significantly associated with the unfavorable prognosis marker of human hepatocellular carcinoma"

    Article Title: Upregulation of colonic and hepatic tumor overexpressed gene is significantly associated with the unfavorable prognosis marker of human hepatocellular carcinoma

    Journal: American Journal of Cancer Research

    doi:

    ch-TOG expression is determined in HCC by qRT-PCR and immunohistochemistry. A. The relative expression of ch-TOG mRNA in 207 paired HCC tissues and matched adjacent noncancerous liver tissues (ANLT) and 12 normal liver tissues was evaluated by qRT-PCR.
    Figure Legend Snippet: ch-TOG expression is determined in HCC by qRT-PCR and immunohistochemistry. A. The relative expression of ch-TOG mRNA in 207 paired HCC tissues and matched adjacent noncancerous liver tissues (ANLT) and 12 normal liver tissues was evaluated by qRT-PCR.

    Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry

    39) Product Images from "MicroRNA-135b Regulates Leucine Zipper Tumor Suppressor 1 in Cutaneous Squamous Cell Carcinoma"

    Article Title: MicroRNA-135b Regulates Leucine Zipper Tumor Suppressor 1 in Cutaneous Squamous Cell Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0125412

    Functional assays of miR-135b inhibition and overexpression. A) Baseline mRNA miR-135b expression in 3 different OTR-derived cSCC cell lines was established by qRT-PCR. B) Effect of miR-135b inhibition on LZTS1 mRNA expression in cSCC lines by qRT-PCR within 48 hours post-transfection. C) Effect of miR-135b overexpression on LZTS1 mRNA expression in cSCC lines by qRT-PCR within 48 hours post-transfection. * indicate p values less than 0.005.
    Figure Legend Snippet: Functional assays of miR-135b inhibition and overexpression. A) Baseline mRNA miR-135b expression in 3 different OTR-derived cSCC cell lines was established by qRT-PCR. B) Effect of miR-135b inhibition on LZTS1 mRNA expression in cSCC lines by qRT-PCR within 48 hours post-transfection. C) Effect of miR-135b overexpression on LZTS1 mRNA expression in cSCC lines by qRT-PCR within 48 hours post-transfection. * indicate p values less than 0.005.

    Techniques Used: Functional Assay, Inhibition, Over Expression, Expressing, Derivative Assay, Quantitative RT-PCR, Transfection

    40) Product Images from "Novel pili-like surface structures of Halobacterium salinarum strain R1 are crucial for surface adhesion"

    Article Title: Novel pili-like surface structures of Halobacterium salinarum strain R1 are crucial for surface adhesion

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2014.00755

    Comparative qRT-PCR analyses of planktonic and surface attached cells. (A) Investigation of two representative housekeeping genes encoding RNA polymerase subunit B' ( rpoB1 ) and the translation elongation factor 2 ( aef2 ). Relative expression was normalized to external standard bgaH RNA. (B) Relative transcriptional quantification of the assembly ATPase encoding genes of the archaellum ( flaI ) and the type IV pilus biogenesis complexes pil-1 ( pilB1 ) and pil-2 ( pilB2 ) as well as the constitutively expressed ferredoxin gene ( fdx) . The bars represent the fold change of gene expression shown in base 2 logarithmic scale in adherent cells compared to the planktonic state, which is defined by the baseline.
    Figure Legend Snippet: Comparative qRT-PCR analyses of planktonic and surface attached cells. (A) Investigation of two representative housekeeping genes encoding RNA polymerase subunit B' ( rpoB1 ) and the translation elongation factor 2 ( aef2 ). Relative expression was normalized to external standard bgaH RNA. (B) Relative transcriptional quantification of the assembly ATPase encoding genes of the archaellum ( flaI ) and the type IV pilus biogenesis complexes pil-1 ( pilB1 ) and pil-2 ( pilB2 ) as well as the constitutively expressed ferredoxin gene ( fdx) . The bars represent the fold change of gene expression shown in base 2 logarithmic scale in adherent cells compared to the planktonic state, which is defined by the baseline.

    Techniques Used: Quantitative RT-PCR, Expressing

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    Transfection:

    Article Title: Large-scale screening identifies a novel microRNA, miR-15a-3p, which induces apoptosis in human cancer cell lines
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    Amplification:

    Article Title: Large-scale screening identifies a novel microRNA, miR-15a-3p, which induces apoptosis in human cancer cell lines
    Article Snippet: .. qRT-PCR analysis Total RNA was isolated from HeLa and AsPc-1 cells 24 and 48 h after transfection using mir Vana™ miRNA isolation kit. qRT-PCR analysis of the bcl-xL , mybl2 and fgfr2 genes was performed using TaqMan® mRNA assay (Life technologies Assay IDs: Hs00236329_m1, Hs00942543_m1, Hs01552926_m1) in Prism 7900H Sequence Detector with 40 amplification cycles and normalized to 18S levels (Life Technologies, Assay ID: Hs99999901_s1) in each sample. .. Western blot analysis of Bcl-xL in HeLa and AsPc-1 cells Cells were collected 72 h after transfection and washed with PBS.

    Synthesized:

    Article Title: Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs
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    Isolation:

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    Real-time Polymerase Chain Reaction:

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    Blocking Assay:

    Article Title: The retinoic acid receptor-? modulators ATRA and Ro415253 reciprocally regulate human IL-5+ Th2 cell proliferation and cytokine expression
    Article Snippet: .. qRT-PCR RNA was extracted from cell pellets using the RNeasy mini kit (Qiagen, Gaithersburg, MD), treated with DNase (Qiagen), and cDNA was prepared from 100–300 ng RNA using Qscript cDNA Supermix (Quanta Biosciences, Gaithersburg, MD). qRT-PCR analysis was performed using the delta Ct method of comparison on the Step-One-Plus Real-Time PCR System Thermal Cycling Block (Applied BioSystems, Carlsbad, CA). .. All FAM-MGB-labeled TaqMan probe and primer sets for IL-4 (Hs00166237_m1), IL-5 (Hs01548712_g1), IL-13 (Hs00174379_m1), and GAPDH (Hs_03929097_g1) were purchased from Applied Biosystems.

    Expressing:

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    Sequencing:

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    Thermo Fisher quantitative real time rt pcr qrt pcr analysis
    Validation of hub genes. Notes: ( A ) Survival analysis indicated that ITLN1 was a positive prognosis factor in epithelial ovarian cancer, while patients with a higher expression of ITLN1 had significantly longer overall survival compared to those with higher expression ( P =2.593e–02). ( B ) ITLN1 validation using <t>qRT-PCR</t> analysis. ( C, D ) Validation of ITLN1 expression from the GEO databases. Two datasets showed lower expression of ITLN1 in epithelial ovarian cancer tissues compared with normal ovarian tissues ( P
    Quantitative Real Time Rt Pcr Qrt Pcr Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qrt pcr analysis qrt pcr analysis
    RNAseq analysis identified the ABA transporter AIT1.1 as upregulated by PRO in guard cells. A. Clustered heatmap of PRO regulated genes ( proΔ17 vs. M82, three samples each) generated from RNAseq and shows 81 PRO upregulated and 81 downregulated genes. Genes were grouped based on their pattern of expression. Coloring of the genes is according to the color bar on the upper right side (Log2 fold change). The complete list of PRO-regulated genes are provided in Supplemental dataset 1. B. <t>qRT-PCR</t> analysis of AIT1.1 and AIT1.2 expression in M82, pro and 35S : pro Δ 17 ( proΔ17 ) isolated guard cell. Values are means of three biological replicates ± SE. Lowercase letters represent significant differences between lines (Tukey–Kramer HSD, P
    Qrt Pcr Analysis Qrt Pcr Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1901 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qrt pcr single stranded cdna
    Effects of HWB treatment on the expression of flavonoid biosynthesis-related genes and the occurrence of red lenticel discoloration on mango fruit cv. Shelly. (A) <t>qRT-PCR</t> profile of differentially expressed genes Ugft3, PAL, CFIL and CHSï , which are related to the flavonoid biosynthesis process, naringenin-chalcone synthase activity, and the phenylpropanoid biosynthesis pathway. (B) level of lenticel discoloration of HWB-treated and control fruits, and (C) lenticel discoloration symptoms on mango fruits, cv. Shelly following HWB treatment. qRT-PCR values were normalized to the values obtained in samples from untreated mango fruits at 0 h. Expression data are means of two replicates. Lenticel discoloration was evaluated following 2 weeks of storage at 12°C [ 9 ]. Average values followed by different letters differ significantly at P
    Qrt Pcr Single Stranded Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Validation of hub genes. Notes: ( A ) Survival analysis indicated that ITLN1 was a positive prognosis factor in epithelial ovarian cancer, while patients with a higher expression of ITLN1 had significantly longer overall survival compared to those with higher expression ( P =2.593e–02). ( B ) ITLN1 validation using qRT-PCR analysis. ( C, D ) Validation of ITLN1 expression from the GEO databases. Two datasets showed lower expression of ITLN1 in epithelial ovarian cancer tissues compared with normal ovarian tissues ( P

    Journal: Cancer Management and Research

    Article Title: ITLNI identified by comprehensive bioinformatic analysis as a hub candidate biological target in human epithelial ovarian cancer

    doi: 10.2147/CMAR.S189784

    Figure Lengend Snippet: Validation of hub genes. Notes: ( A ) Survival analysis indicated that ITLN1 was a positive prognosis factor in epithelial ovarian cancer, while patients with a higher expression of ITLN1 had significantly longer overall survival compared to those with higher expression ( P =2.593e–02). ( B ) ITLN1 validation using qRT-PCR analysis. ( C, D ) Validation of ITLN1 expression from the GEO databases. Two datasets showed lower expression of ITLN1 in epithelial ovarian cancer tissues compared with normal ovarian tissues ( P

    Article Snippet: Quantitative real-time RT-PCR (qRT-PCR) analysis We extracted total RNA from tissue samples using TRizol reagent (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Quantitative RT-PCR

    RNAseq analysis identified the ABA transporter AIT1.1 as upregulated by PRO in guard cells. A. Clustered heatmap of PRO regulated genes ( proΔ17 vs. M82, three samples each) generated from RNAseq and shows 81 PRO upregulated and 81 downregulated genes. Genes were grouped based on their pattern of expression. Coloring of the genes is according to the color bar on the upper right side (Log2 fold change). The complete list of PRO-regulated genes are provided in Supplemental dataset 1. B. qRT-PCR analysis of AIT1.1 and AIT1.2 expression in M82, pro and 35S : pro Δ 17 ( proΔ17 ) isolated guard cell. Values are means of three biological replicates ± SE. Lowercase letters represent significant differences between lines (Tukey–Kramer HSD, P

    Journal: bioRxiv

    Article Title: The tomato DELLA protein PROCERA promotes ABA responses in guard cells by upregulating ABA transporter

    doi: 10.1101/2020.04.22.056010

    Figure Lengend Snippet: RNAseq analysis identified the ABA transporter AIT1.1 as upregulated by PRO in guard cells. A. Clustered heatmap of PRO regulated genes ( proΔ17 vs. M82, three samples each) generated from RNAseq and shows 81 PRO upregulated and 81 downregulated genes. Genes were grouped based on their pattern of expression. Coloring of the genes is according to the color bar on the upper right side (Log2 fold change). The complete list of PRO-regulated genes are provided in Supplemental dataset 1. B. qRT-PCR analysis of AIT1.1 and AIT1.2 expression in M82, pro and 35S : pro Δ 17 ( proΔ17 ) isolated guard cell. Values are means of three biological replicates ± SE. Lowercase letters represent significant differences between lines (Tukey–Kramer HSD, P

    Article Snippet: qRT-PCR analysis qRT-PCR analysis was performed using an Absolute Blue qPCR SYBR Green ROX Mix (AB-4162/B) kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Generated, Expressing, Quantitative RT-PCR, Isolation

    RIG-I binds U1 snRNA accumulated in the cytoplasm to mediate radiation-induced IFN-beta response A. RIG-I binds diverse non-coding RNA molecules, majority of which are snRNAs. Graphic representation indicating the distribution of non-coding and repetitive RNA molecules bound to RIG-I following exposure to IR as compared to total irradiated cellular RNA. Transcripts were mapped to reference genomes using RepeatMasker. See Methods for further details. B. qRT-PCR quantification of U1 RNA from purified RNA bound to ectopically expressing WT and K858A-K861A mutant RIG-I HEK293 cells exposed to IR (6 Gy) or left untreated. Cells were UV crosslinked at 150mJ/cm 2 48 hours post-IR treatment prior to cell lysis. U1 RNA levels were normalized to the geometric average of 3 housekeeping genes (18S rDNA, GAPDH, and β-actin). Fold change was determined relative to un-irradiated controls. C. U1 RNA levels quantified by qRT-PCR from total cellular and RIG-I pulldowns in RIG-I overexpressing HEK293 and HCT116 cells. U1 RNA levels were normalized to the geometric average of 3 housekeeping genes (18S rDNA, GAPDH, and β-actin). Fold change was determined relative to un-irradiated controls. Time course of cytosolic accumulation of U1 RNA measured by qRT-PCR from purified total cellular RNA following cellular fractionation of nuclear/mitochondrial and cytoplasmic fractions of HEK293 D. and HCT116 cells E. exposed to IR (6 Gy) or left untreated. F. The structure of the U1 snRNA illustrating the four stem loop (SL) regions. G. Relative IFN-beta luciferase reporter activity of HEK293 cells following a 24 hour stimulation with synthetic oligonucleotides corresponding to U1 RNA stem loop (SL) regions I to IV or a combination of SL I + II and SL II + III. H. IFN-b levels in culture supernatant from ICR RIG-I +/+ and RIG-I −/− primary MEFs 24 hours post-stimulation with the same set of synthetic U1 oligonucleotides used in G. The amount of U1 synthetic oligonucleotides used in all stimulation experiments was 1μg. P values were determined using unpaired Student's t -test. Error bars are SEM. *** P

    Journal: Oncotarget

    Article Title: Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs

    doi: 10.18632/oncotarget.8420

    Figure Lengend Snippet: RIG-I binds U1 snRNA accumulated in the cytoplasm to mediate radiation-induced IFN-beta response A. RIG-I binds diverse non-coding RNA molecules, majority of which are snRNAs. Graphic representation indicating the distribution of non-coding and repetitive RNA molecules bound to RIG-I following exposure to IR as compared to total irradiated cellular RNA. Transcripts were mapped to reference genomes using RepeatMasker. See Methods for further details. B. qRT-PCR quantification of U1 RNA from purified RNA bound to ectopically expressing WT and K858A-K861A mutant RIG-I HEK293 cells exposed to IR (6 Gy) or left untreated. Cells were UV crosslinked at 150mJ/cm 2 48 hours post-IR treatment prior to cell lysis. U1 RNA levels were normalized to the geometric average of 3 housekeeping genes (18S rDNA, GAPDH, and β-actin). Fold change was determined relative to un-irradiated controls. C. U1 RNA levels quantified by qRT-PCR from total cellular and RIG-I pulldowns in RIG-I overexpressing HEK293 and HCT116 cells. U1 RNA levels were normalized to the geometric average of 3 housekeeping genes (18S rDNA, GAPDH, and β-actin). Fold change was determined relative to un-irradiated controls. Time course of cytosolic accumulation of U1 RNA measured by qRT-PCR from purified total cellular RNA following cellular fractionation of nuclear/mitochondrial and cytoplasmic fractions of HEK293 D. and HCT116 cells E. exposed to IR (6 Gy) or left untreated. F. The structure of the U1 snRNA illustrating the four stem loop (SL) regions. G. Relative IFN-beta luciferase reporter activity of HEK293 cells following a 24 hour stimulation with synthetic oligonucleotides corresponding to U1 RNA stem loop (SL) regions I to IV or a combination of SL I + II and SL II + III. H. IFN-b levels in culture supernatant from ICR RIG-I +/+ and RIG-I −/− primary MEFs 24 hours post-stimulation with the same set of synthetic U1 oligonucleotides used in G. The amount of U1 synthetic oligonucleotides used in all stimulation experiments was 1μg. P values were determined using unpaired Student's t -test. Error bars are SEM. *** P

    Article Snippet: qRT-PCR analysis 1 μg total RNA was subjected to DNase I treatment in a 30 μL reaction volume using DNase I, RNase-free (Thermo Scientific) following the manufacturer's protocol. cDNA was synthesized from 10 μL of the DNase treated RNA using the High-Capacity cDNA Reverse Transcription Kit (LifeTechnologies) following the manufacturer's protocol.

    Techniques: Irradiation, Quantitative RT-PCR, Purification, Expressing, Mutagenesis, Lysis, Cell Fractionation, Luciferase, Activity Assay

    MAVS is necessary for ionizing radiation-induced Type I interferon signaling A. Proposed mechanism of MAVS-dependent activation of Type I IFN signaling in the cellular response to IR. B. Transcriptional profiling of C57BL/6 wild-type (WT) and MAVS −/− primary MEFs demonstrating MAVS-dependent expression of Type I IFN-stimulated genes (ISGs) 48 hours following exposure to IR (6 Gy). Heatmap displays differences in gene expression values between WT and MAVS −/− MEFs; red indicates high expression and blue low expression. Inset shows qRT-PCR validation of Usp18 , Ifit3 , Stat1 , Ddx58 , and Cdkn1a gene expression values in WT and MAVS −/− MEFs after IR treatment. C. Top-ranked cellular pathways (top) and functions (bottom) (Ingenuity Pathway Analysis) activated by IR in WT MEFs. Pie-chart displays the relative abundance of each functional category among all significant functions ( P

    Journal: Oncotarget

    Article Title: Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs

    doi: 10.18632/oncotarget.8420

    Figure Lengend Snippet: MAVS is necessary for ionizing radiation-induced Type I interferon signaling A. Proposed mechanism of MAVS-dependent activation of Type I IFN signaling in the cellular response to IR. B. Transcriptional profiling of C57BL/6 wild-type (WT) and MAVS −/− primary MEFs demonstrating MAVS-dependent expression of Type I IFN-stimulated genes (ISGs) 48 hours following exposure to IR (6 Gy). Heatmap displays differences in gene expression values between WT and MAVS −/− MEFs; red indicates high expression and blue low expression. Inset shows qRT-PCR validation of Usp18 , Ifit3 , Stat1 , Ddx58 , and Cdkn1a gene expression values in WT and MAVS −/− MEFs after IR treatment. C. Top-ranked cellular pathways (top) and functions (bottom) (Ingenuity Pathway Analysis) activated by IR in WT MEFs. Pie-chart displays the relative abundance of each functional category among all significant functions ( P

    Article Snippet: qRT-PCR analysis 1 μg total RNA was subjected to DNase I treatment in a 30 μL reaction volume using DNase I, RNase-free (Thermo Scientific) following the manufacturer's protocol. cDNA was synthesized from 10 μL of the DNase treated RNA using the High-Capacity cDNA Reverse Transcription Kit (LifeTechnologies) following the manufacturer's protocol.

    Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Functional Assay

    Effects of HWB treatment on the expression of flavonoid biosynthesis-related genes and the occurrence of red lenticel discoloration on mango fruit cv. Shelly. (A) qRT-PCR profile of differentially expressed genes Ugft3, PAL, CFIL and CHSï , which are related to the flavonoid biosynthesis process, naringenin-chalcone synthase activity, and the phenylpropanoid biosynthesis pathway. (B) level of lenticel discoloration of HWB-treated and control fruits, and (C) lenticel discoloration symptoms on mango fruits, cv. Shelly following HWB treatment. qRT-PCR values were normalized to the values obtained in samples from untreated mango fruits at 0 h. Expression data are means of two replicates. Lenticel discoloration was evaluated following 2 weeks of storage at 12°C [ 9 ]. Average values followed by different letters differ significantly at P

    Journal: BMC Genomics

    Article Title: De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment

    doi: 10.1186/1471-2164-15-957

    Figure Lengend Snippet: Effects of HWB treatment on the expression of flavonoid biosynthesis-related genes and the occurrence of red lenticel discoloration on mango fruit cv. Shelly. (A) qRT-PCR profile of differentially expressed genes Ugft3, PAL, CFIL and CHSï , which are related to the flavonoid biosynthesis process, naringenin-chalcone synthase activity, and the phenylpropanoid biosynthesis pathway. (B) level of lenticel discoloration of HWB-treated and control fruits, and (C) lenticel discoloration symptoms on mango fruits, cv. Shelly following HWB treatment. qRT-PCR values were normalized to the values obtained in samples from untreated mango fruits at 0 h. Expression data are means of two replicates. Lenticel discoloration was evaluated following 2 weeks of storage at 12°C [ 9 ]. Average values followed by different letters differ significantly at P

    Article Snippet: Analysis by qRT-PCR Single-stranded cDNA was synthesized from 1 μg of total RNA by means of the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Quantitative RT-PCR, Activity Assay

    Shelly. Effect of HWB on differential expression of chlorophyll and anthocyanin accumulation-related genes and color development in mango cv. Shelly. (A, B) qRT-PCR gene-expression profiles of genes related to (A) chlorophyll accumulation ( Thl1ch , LHCIIb, Oxepch and PIRC ) and (B) anthocyanin synthesis ( 85A2 and Anthocyanin5 ). The expression profile comprises data taken from samples of mango tissues sampled from cv. Shelly at four different time points after HWB treatment. (C) Changes in color index after 16 days of storage at 12°C followed by 8 days at 20°C. Vertical bars indicate SD of five replicates. qRT-PCR values were normalized to the values obtained with untreated mango fruit samples at 0 h. Expression data are the means of two replicates.

    Journal: BMC Genomics

    Article Title: De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment

    doi: 10.1186/1471-2164-15-957

    Figure Lengend Snippet: Shelly. Effect of HWB on differential expression of chlorophyll and anthocyanin accumulation-related genes and color development in mango cv. Shelly. (A, B) qRT-PCR gene-expression profiles of genes related to (A) chlorophyll accumulation ( Thl1ch , LHCIIb, Oxepch and PIRC ) and (B) anthocyanin synthesis ( 85A2 and Anthocyanin5 ). The expression profile comprises data taken from samples of mango tissues sampled from cv. Shelly at four different time points after HWB treatment. (C) Changes in color index after 16 days of storage at 12°C followed by 8 days at 20°C. Vertical bars indicate SD of five replicates. qRT-PCR values were normalized to the values obtained with untreated mango fruit samples at 0 h. Expression data are the means of two replicates.

    Article Snippet: Analysis by qRT-PCR Single-stranded cDNA was synthesized from 1 μg of total RNA by means of the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Quantitative RT-PCR

    Validation of RNA-seq results by means of qRT-PCR. Ten differentially expressed genes (two from each of clusters 1 to 5) were examined by RNA-seq and qRT-PCR at four different time points after HWB treatment: A , B (cluster 1); C , D (cluster 2); E , F (cluster 3); G , H (cluster 4); and I , J (cluster 5). Values were normalized to the values obtained with untreated mango fruit samples at 0 h and the proportional fold-change (FC) was calculated. Expression data are means of two replicates.

    Journal: BMC Genomics

    Article Title: De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment

    doi: 10.1186/1471-2164-15-957

    Figure Lengend Snippet: Validation of RNA-seq results by means of qRT-PCR. Ten differentially expressed genes (two from each of clusters 1 to 5) were examined by RNA-seq and qRT-PCR at four different time points after HWB treatment: A , B (cluster 1); C , D (cluster 2); E , F (cluster 3); G , H (cluster 4); and I , J (cluster 5). Values were normalized to the values obtained with untreated mango fruit samples at 0 h and the proportional fold-change (FC) was calculated. Expression data are means of two replicates.

    Article Snippet: Analysis by qRT-PCR Single-stranded cDNA was synthesized from 1 μg of total RNA by means of the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

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

    Differential expressions of genes modulating the mechanism of resistance to A. alternata in naturally infected mango fruits. (A) Effect of HWB on alternaria black spot (ABS) symptom development on mango cvs. Palmer, Kent, Tommy Atkins, Keitt, Lily and Shelly. (B) ABS symptom development on naturally infected fruits cv. Keitt following HWB treatment. (C) qRT-PCR differential expression profiling of genes Syn121 , glutardoxin , IT1K2 and AOS of cv. Shelly. Fruit peel tissues were sampled at four different time points after HWB treatment. RNA was extracted and served as a template for cDNA followed by qRT-PCR analysis of the genes of interest. Proportional increases in relative expression values were normalized against the samples of untreated mango fruits at 0 h. Expression data are means of two replicates. ABS-covered area was evaluated after 4 weeks of storage at 12°C. Average values followed by different letters differ significantly at P

    Journal: BMC Genomics

    Article Title: De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment

    doi: 10.1186/1471-2164-15-957

    Figure Lengend Snippet: Differential expressions of genes modulating the mechanism of resistance to A. alternata in naturally infected mango fruits. (A) Effect of HWB on alternaria black spot (ABS) symptom development on mango cvs. Palmer, Kent, Tommy Atkins, Keitt, Lily and Shelly. (B) ABS symptom development on naturally infected fruits cv. Keitt following HWB treatment. (C) qRT-PCR differential expression profiling of genes Syn121 , glutardoxin , IT1K2 and AOS of cv. Shelly. Fruit peel tissues were sampled at four different time points after HWB treatment. RNA was extracted and served as a template for cDNA followed by qRT-PCR analysis of the genes of interest. Proportional increases in relative expression values were normalized against the samples of untreated mango fruits at 0 h. Expression data are means of two replicates. ABS-covered area was evaluated after 4 weeks of storage at 12°C. Average values followed by different letters differ significantly at P

    Article Snippet: Analysis by qRT-PCR Single-stranded cDNA was synthesized from 1 μg of total RNA by means of the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Infection, Quantitative RT-PCR, Expressing